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Spineless Wonders: Welfare and Use of Invertebrates in the Laboratory and Classroom
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Volume 52 Number 2
JOURNAL
In This Issue:
Spineless Wonders:
Welfare and Use of Invertebrates
in the Laboratory and Classroom
Volume 52 • Number 2 • 2011 • pp 121-232
ILAR Council
Stephen W. Barthold, Chairman
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University of California, Davis
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Leticia V. Medina
Animal Welfare and Compliance
Abbott Laboratories
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Association for Assessment
and Accreditation of Laboratory
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Myrtle Davis
National Cancer Institute
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James G. Fox
Division of Comparative Medicine
Massachusetts Institute of Technology
Cambridge, MA
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Joseph W. Kemnitz
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University of Wisconsin
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Judy A. MacArthur Clark
Animals Scientific Procedures Inspectorate
London, UK
Daniel S. Marsman
Personal Health/Feminine Care Safety
Procter & Gamble
Mason, OH
Martha K. McClintock
Departments of Psychology and
Comparative Human Development
The University of Chicago
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Timo Olavi Nevalainen
National Laboratory Animal Center
University of Eastern Finland
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Steven M. Niemi
Center for Comparative Medicine
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Innovative Medicine
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Stephen A. Smith
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Virginia Polytechnic Institute and State University
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Volume 52, Number 2, 2011
Copyright 2011 by the Institute for Laboratory Animal Research. All rights reserved.
Printed in the United States of America on acid-free paper.
2011
Volume 52, Number 2
Spineless Wonders: Welfare and Use of Invertebrates
in the Laboratory and Classroom
121
Introduction: Laboratory Invertebrates: Only Spineless,
or Spineless and Painless?
Paul L.R. Andrews
126
Invertebrate Models for Biomedical Research, Testing, and Education
Susan E. Wilson-Sanders
153
Culture and Maintenance of Selected Invertebrates in the Laboratory
and Classroom
Stephen A. Smith, Joseph M. Scimeca, and Mary E. Mainous
165
Invertebrate Resources on the Internet
Stephen A. Smith
175
Pain and Suffering in Invertebrates?
Robert W. Elwood
185
Nociceptive Behavior and Physiology of Molluscs: Animal Welfare
Implications
Robyn J. Crook and Edgar T. Walters
196
Anesthesia, Analgesia, and Euthanasia of Invertebrates
John E. Cooper
205
Philosophical Background of Attitudes toward and Treatment
of Invertebrates
Jennifer A. Mather
213
IACUC Challenges in Invertebrate Research
Chris Harvey-Clark
Online only in the ILAR e-Journal
Effects of Isoflurane Anesthesia on the Cardiovascular Function of
the C57BL /6 Mouse
Christakis Constantinides, Richard Mean, and Ben J. Janssen
221
Thanks to Institutional Supporters
223
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Introduction
Laboratory Invertebrates: Only Spineless, or Spineless and Painless?
Paul L.R. Andrews
All animals are equal, but some animals are more equal
than others.
– George Orwell, Animal Farm
T
he above well-known quotation from Animal Farm in
some ways illustrates humans’ ambivalent view of the
relative status of invertebrates and vertebrates in a
number of settings: in the research laboratory (differing legal
protection), in wildlife conservation (e.g., the appeal of beetles vs. giant pandas), in the home (e.g., swatting flies vs.
the humane disposal of mammalian vermin), and in cuisine
(e.g., methods for cooking crustaceans vs. chicken). Even
the terms “spineless” and “lacking a backbone” are used
pejoratively.
Invertebrates are clearly considered by many to be at the
“lower end” of a scale of creatures that puts humans at the
extreme “upper end,” although the units on the y-axis of this
graph are a matter for debate. And within the Vertebrate and
Invertebrate subphyla, terminology implying some sort of
ranking is often used even in academic publications—fish,
which account for about half of vertebrate species, are referred to as “lower” vertebrates (e.g., Sneddon 2004) and
Octopus vulgaris (a cephalopod) as an “advanced” invertebrate (e.g., Wells 1978). But Packard (1972) makes a compelling case that the “lower” vertebrate and “advanced”
invertebrate are more than a match for each other in evolutionary competition.
The demarcation in the way invertebrates are viewed,
and as a consequence treated, extends to the laboratory.
While this issue of the ILAR Journal is very welcome and
may mark a turning point, it is notable that this single issue
covers about 95% of animal species (the more than 1 million
invertebrate species) whereas previous issues have been devoted to amphibians, fish, and birds (ILAR 2007, 2009,
2010). This reflects the relative paucity of information in the
area of invertebrate welfare and the relative youth of this
Paul L.R. Andrews, BSc, PhD, is Professor of Comparative Physiology in
the Division of Biomedical Sciences at St. George’s University of London
in the United Kingdom.
Address correspondence and reprint requests to Prof. Paul L.R. Andrews,
Division of Biomedical Sciences, St. George’s University of London, Cranmer
Terrace, London, SW17 0RE, UK or email [email protected].
Volume 52, Number 2
2011
topic as an area for research, with little study in major areas
critical for research, such as criteria for general anesthesia.
Legislation also reflects the invertebrate/vertebrate divide. The United Kingdom enacted one of the first pieces of
national legislation covering animal experimentation, the
Cruelty to Animals Act of 1876 (Tansey 1998). This Act of
Parliament permitted “the advancement of new discovery of
physiological knowledge by experiments calculated to give
pain” but it applied only to nonhuman vertebrates. It is not
unreasonable to suggest that the exclusion of invertebrates
indicates that, in contrast to vertebrates, they were considered to have a lesser (if any) perception of pain. Smyth
(1978) comments that “invertebrates look far less like us,” so
humans may find little to recognize in common with “us” in
their appearance or behavior and therefore find it harder to
empathize or connect at any level.
The 1876 UK legislation also reflects the species that
were commonly used in physiological studies at the time
(dogs, cats, rabbits, and frogs). In 1986 the legislation was
revised and became the Animals (Scientific Procedures) Act;
that revision did not include invertebrates but an amendment
in 1993 gave O. vulgaris the same legal protection as vertebrates with respect to experimental procedures (however, to
date there have been no studies on O. vulgaris under the authority of the Act).
In the European Union the October 2010 revised Directive (2010/63/EU)1 “on the protection of animals used for
scientific purposes” covers “live cephalopods” under Article
1, 3b (however, decapod crustacea—e.g., crabs, lobsters—
were included in drafts of the new EU legislation but not in
the adopted directive). Member states are required to transpose it into national legislation by November 2012 and apply
it by January 2013. Some of the challenges that will need to
be addressed in the European Union to comply with this directive are considered in the articles in this issue.
Debating and solving the ethical and welfare issues that
are often taken for granted when dealing with laboratory
vertebrates in general and mammals in particular for “advanced
invertebrates” will provide a useful learning process that will
inform best practice when considering invertebrates that lack
1Available online (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=
OJ:L:2010:276:0033:0079:EN:PDF); this and other websites cited in this
Introduction were accessed between February 22 and April 11, 2011.
121
legal protection. The difficulties likely to be encountered
should not be underestimated. Even Russell and Burch
(1959, 6) in their classic Principles of Humane Experimental
Technique avoided the issue: “The higher invertebrates perhaps deserve a review to themselves, but they raise many
problems which would gravely complicate an account which
can otherwise be quite general and confident.”
The papers in this special issue cover four major aspects
of invertebrates in the laboratory: (1) the use of invertebrates
in biomedical and related research, (2) the culture and maintenance of invertebrates, (3) evidence for pain and suffering
and their alleviation, and (4) attitudes and their influence on
regulation and oversight. Each of these will be reviewed
briefly before concluding this introductory overview with
some comments about future directions.
Use of Invertebrates in Biomedical and
Related Research
…[T]he solution of a physiological or pathological problem often depends solely on the appropriate choice of the
animal for the experiment so as to make the result clear
and searching.
– Claude Bernard (1949/1865)
There is little doubt that invertebrate species have made major contributions to biomedical research even if judged only
by their contribution to Nobel Prizes in the last 50 years, as
illustrated in the following examples2:
•
•
•
•
•
Caenorhabditis elegans: genetic regulation of organ development and programmed cell death (Brenner, Horvitz,
and Sulston; 2002);
sea urchin and clam eggs: key regulators of the cell cycle
(Hartwell, Hunt, and Nurse; 2001);
Aplysia: signal transduction in the nervous system
(Carlsson, Greengard, and Kandel; 2000);
honeybee: organization and elicitation of individual
and social behavior patterns (von Frisch, Lorenz, and
Tinbergen; 1973);
squid: ionic mechanism involved in excitation and inhibition in the peripheral and central portions of the nerve
cell membrane (Eccles, Hodgkin, and Huxley; 1963).
Wilson-Sanders (2011) provides a comprehensive overview
of the diverse species of invertebrates used in biomedical
and related research, with tables summarizing their utility in
studies of developmental biology, genetics, and diseases.
While the emphasis is on Drosophila melanogaster and
C. elegans primarily because of their tractability for genetic
and molecular studies, it would be unwise to neglect the potential of the diversity of over a million species of invertebrates to contribute to biomedical research as model
2Information about these Nobel Prizes is available online at http://nobelprize.
org/nobel_prizes/medicine/laureates.
122
organisms to reveal fundamental biological processes including those involved in disease. Among the reasons to consider invertebrates for use in research are their
(1) simpler system than that of vertebrates. This is the
argument often applied to studying nervous systems
(Usherwood and Newth 1975) such as that of Aplysia
californica and using species such as the honeybee as
behavioral models.
(2) unique or larger structure than that of vertebrates. For
example, the squid giant axon (originally mistaken for a
blood vessel and rediscovered by Young in 1933) and
giant synapse revealed fundamental insights into the operation of neurones (see Bullock and Horridge 1965 for
references). The brain-controlled skin chromatophore
system of cephalopods provides another example of
a unique biological system (Hanlon and Messenger
2008).
(3) properties not readily exhibited by vertebrates. Pupation
in insects provided important insights into the Hox (homeotic) family of genes fundamental to the organization
of body plans. The striking capacity for tissue regeneration is seen in many invertebrates but particularly in
echinoderms, as exemplified by arm regeneration in brittlestars (Amphiura filiformis; Bannister et al. 2005) and
regeneration of the gut in sea cucumbers (Holothuria
glaberrima; Mashanov et al. 2010). It is likely that
interest in these properties will increase because of the
growth of research in tissue engineering for medical
applications.
Several drivers are likely to increase the use of invertebrates in research, including the possibility of “replacing” vertebrate models with invertebrates (“relative substitution”;
Russell and Burch 1959), although this requires an assessment
of sentience so that an animal with “higher” sentience is replaced by one with “lower” (e.g., how would an ethical committee approach the replacement of a trout by an octopus to
answer the same biomedical problem?); the relatively low cost
of some invertebrates; and different regulatory and ethical
considerations (but see below). However, the overriding justification should be a scientific one based on a “cost-benefit”
analysis—that is, an assessment of the “cost” to the animal
and the benefit, in the broadest sense, of doing the research.
Culture and Maintenance of Invertebrates
The use of invertebrates in research immediately raises the
question of their sourcing and maintenance, as discussed by
Smith and colleagues (2011). This is a crucially important
aspect of the laboratory use of invertebrates as most research
requires a supply of “standardized” animals throughout the
year and journals require increasingly detailed information
about the animals used in a reported study (see below). Sourcing and maintenance (including the correct environmental
conditions to ensure optimal health, welfare, and, if required,
ILAR Journal
reproduction) may be relatively simple for D. melanogaster
and C. elegans, but for marine species obtained from the
wild they may be more of a challenge. Advances have been
made in the culture of many aquatic invertebrates including
cuttlefish (Sepia officinalis and S. pharaonis) and squid (Sepioteuthis lessoniana), but laboratory studies on O. vulgaris
and Eledone cirrhosa usually involve wild-caught animals
(e.g., Malham et al. 2002).
The inclusion of cephalopods in the revised EU legislation (as mentioned above) will necessitate the development
of guidelines covering all aspects of their provision, maintenance, and welfare. Furthermore, bearing in mind the differences between the squid, cuttlefish, octopus, and nautilus, it
is likely that each species will need its own set of guidelines.
It will also be necessary to develop humane methods for
handling (e.g., atraumatic weighing of cephalopods to monitor welfare may not be that simple and even in familiar mammalian species such as mice different handling techniques
have a major impact on stress and anxiety; Hurst and West
2010) and for anesthesia and euthanasia, together with criteria for anesthesia and identification of pain and distress.
Evidence of Pain and Suffering and
Methods for Their Alleviation
Elwood (2011), Crook and Walters (2011), and Cooper
(2011) review different aspects of these difficult and somewhat controversial topics, an understanding of which is
essential for both ensuring animal welfare during experimentation and minimizing suffering. Elwood (2011) and
Crook and Walters (2011) draw attention to the difference
between nociception and pain perception and the survival
advantage of the ability to detect, avoid, and learn from noxious stimuli that have the potential to damage tissue. The
general hierarchical organization of the central nervous system is well established in vertebrates, but it is appropriate to
exercise caution about applying preconceived notions to invertebrate nervous systems.
Defining Nociception and Pain
Nociceptors can be identified by a growing range of molecular markers (e.g., those in the transient receptor potential cation channel subfamily V member 1 [TRPV1], or capsaicin,
receptor family) and more classically by recording from the
afferent nerve with careful characterization of stimulus intensity-response relationships. It is worth recalling that the
behavioral consequences triggered by nociceptor activation
are encoded in the primary afferent signal transmitted from
the tissue to the central nervous system. In vertebrates responses to activation of nociceptors range from localized
responses (e.g., edema, vasodilatation) in the affected tissue
via axon collaterals if present, to reflex responses mediated
via the spinal cord (e.g., limb withdrawal reflex, scratch reflex) and brainstem resulting in a coordinated response often
involving several body systems. Complex endocrine reVolume 52, Number 2
2011
sponses to “stress” mediated via the hypothalamic-pituitaryadrenal axis require the more rostral projection of the
information encoded in the nociceptive afferent, and conscious
perception (feeling pain and the associated emotional aspects)
requires projection to the thalamus and cerebral cortex.
Humans tend to think of the effects of noxious mechanical, thermal (hot and cold), and chemical stimuli (including
pH) on the surface epithelium (“skin”), but both eating and
breathing (air or water) can expose the “interior” of the animal to noxious stimuli that may evoke reflex response such
as vomiting and coughing (both complex reflex motor responses mediated via the brain stem and widely present in
vertebrates) to remove the irritant, and, in the case of ingested toxins, the unpleasant sensation of nausea (requiring
cerebral cortical processing in humans) that is important in
the genesis of learned aversions (Stern et al. 2011). Researchers have reported ejection of ingested toxic food in the
sea anemone (Lindquist and Hay 1995) and gastropod Pleurobranchaea (McClellan 1983) and of gastric contents in the
squid Sepioteuthis sepioidea (Garcia-Franco 1992). Painful
sensations can also arise from noxious stimulation of the viscera (e.g., gut pain) and could be as much a cause of reduced
food intake in an invertebrate as in a vertebrate.
Assessing and Treating Pain/Nociception
in Invertebrates
How can scientists determine whether invertebrates “feel”
pain? In considering the issue of assessing pain in animals,
Bateson (1991) wrote, “We may feel confident about a mammal or even a bird. But what about a locust or an octopus?”
For vertebrates there is generally good knowledge of comparative brain neuroanatomy, allowing a degree of reverse
engineering based on knowledge of human brain pain pathways to determine whether similar pathways exist in other
vertebrates. Even among vertebrates, however, conclusions
about “higher” brain functions based on comparative neuroanatomy have been challenged when considering whether
fish feel pain (Rose 2002). Furthermore, as imaging techniques have improved it is becoming clear that even among
mammals there are differences between primates and nonprimates in the brain pathways involved in processing information from nociceptors (e.g., Craig 2002). Thus investigators
should not underestimate the difficulty in identifying functionally analogous pathways in invertebrates with fundamentally differently organized central nervous systems. It is
worth recalling that until the late 1980s there was a common
view that human neonates did not feel pain (Fitzgerald and
McIntosh 1989).
Molecular, neurophysiological, and behavioral studies
have provided evidence for responses to noxious mechanical
stimuli in the hermit anemone (Calliactis parasitica) and the
California sea slug and to noxious mechanical, thermal
(heat), and chemical stimuli in the medicinal leech (Hirudo
medicinalis), D. melanogaster, and C. elegans (St. John
Smith and Lewin 2009 for review).
123
Behavioral studies provide important insights into the
question of pain perception, but knowledge of the way information is processed is important and, together with molecular and physiological (biomarkers of stress such as heart rate,
endocrine, and metabolic changes) studies, will be key to
identifying endogenous pathways capable of modulating nociception, the transmitters of which provide targets for analgesics (e.g., opioids, cannabinoids, steroids).
Anesthetic and analgesic techniques for invertebrates are
relatively poorly developed in contrast to those for vertebrates (and especially mammals), although as Cooper (2011)
points out there may be more information in the world literature and a systematic approach is needed for collecting, assessing, and applying that information. There is certainly a
need for some consensus on criteria for general (surgical)
anesthesia in all invertebrates used in the laboratory and for
decapod crustacea and molluscs in particular. Anesthesia has
been used only for short-duration manipulation or surgical
interventions. Techniques for sustained general anesthesia
and maintenance of physiological systems would permit
in vivo neurophysiological or functional brain imaging
studies, and the lack of such methods limits knowledge of
the central processing of nociceptive inputs, especially in
cephalopods.3
Finally, although pain (usually assumed to be cutaneous)
is perhaps the most common focus from a welfare perspective, animals are susceptible to numerous other unpleasant
experiences such as anxiety, asphyxia, dyspnoea, fear, headache, itching, and photophobia. Researchers, animal care
staff, and IACUC members should be mindful of all such
stressors in considering the welfare of invertebrates in the
laboratory.
Attitudes and Their Influence on
Regulation and Oversight
“Slugs Displace Bunnies in the Lab” (Davis 2002). This
headline accompanied a news article reporting the possibility of replacing the Draize test using rabbits with an irritancy
test measuring defensive secretions from slug skin. Although
many would view this as a positive development, a spokesman for the British Union for the Abolition of Vivisection is
quoted as calling the use of any animal “morally unacceptable” (Davis 2002). This example illustrates the difficulty
not only in making judgments about the use of animal in research but also particularly in determining the “relative positions” of vertebrates and invertebrates. This is a complex
area and Mather (2011) tackles it by considering the philosophical basis for attitudes toward invertebrates. She describes the contractarian, utilitarian, and rights-based
approaches, using diverse examples ranging from the treatment of invertebrates in the kitchen, commercial fisheries,
3In
the European Union it is likely that the forthcoming legislation will
drive the development of a consensus view on many of these aspects for
cephalopods.
124
public aquariums, and the laboratory. She accords decapod
crustacea and cephalopods special consideration (as is the
case in several other articles in this special issue). Mather
(2011) concludes that “as invertebrates are better understood, people—whatever their value system—will come to
appreciate and take better care of them.”
It is to be hoped that greater understanding will translate
to the laboratory and this is likely to be facilitated by ethical
review and regulatory frameworks, which are reviewed in
the final article, by Harvey-Clark (2011). He presents two
IACUC case studies that clearly reveal the particular challenges of research using invertebrates and then provides a
useful resource to engage researchers and regulators in confronting some of the key issues.
Where Next?
Scientific journals have a key role to play in encouraging
adoption of best practice in the welfare of animals used in
research by ensuring that experiments on invertebrates
are properly reported. The recently published “ARRIVE”
guidelines for reporting animal research (Animal Research: Reporting In Vivo Experiments; Kilkenny et al.
2010, www.nc3rs.org.uk/ARRIVE) provide a clear list of
the essential elements that need to be reported for in vivo
experiments and, although primarily aimed at studies in
vertebrates, can be readily adapted for use in invertebrates
(especially decapod crustacea and cephalopods). Comprehensive reporting of methodological aspects is important to enable assessment of welfare and facilitate the
systematic gathering of information on ethics, experimental design, housing, husbandry, and adverse events induced by experimental procedures. Many of these aspects
are considered in the following articles and especially
that by Harvey-Clark (2011).
There seems to be some agreement that in laboratory
procedures involving invertebrates in general, and decapod
crustacea and cephalopods in particular, the “precautionary
principle” should operate at least until there is definitive evidence of their ability to suffer, recognizing that pain may be
only one component of suffering. The agreed special position of cephalopods (particularly octopuses and cuttlefish) is
already reflected in some national legislation (e.g., Canada).
In the European Union it is likely that over the next 2 years
such legislation will spur research to address a range of
issues—from optimal anesthetic and handling protocols
to recognition of signs of pain and distress analogous to
those developed for mammals (e.g., Morton and Griffiths
1985)—as cephalopods, at least, will have the same legal
protection as is afforded to vertebrates.
Understanding the functioning of phylogenetically ancient brains in highly evolved animals with a fundamentally
different organization (at least anatomically) from vertebrates represents a major intellectual challenge and may also
prompt reconsideration of some prevailing ideas of consciousness (see Edelman and Seth 2009 for discussion).
ILAR Journal
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125
Invertebrate Models for Biomedical Research, Testing, and Education
Susan E. Wilson-Sanders
Abstract
Invertebrate animals have been used as medicinals for 4,000
years and have served as models for research and teaching
since the late 1800s. Interest in invertebrate models has increased over the past several decades as the research community has responded to public concerns about the use of
vertebrate animals in research. As a result, invertebrates are
being evaluated and recognized as models for many diseases
and conditions. Their use has led to discoveries in almost
every area of biology and medicine—from embryonic development to aging processes. Species range from terrestrial
invertebrates such as nematodes and insects to freshwater
and marine life including planarians, crustaceans, molluscs,
and many others. The most often used models are the fruit
fly Drosophila melanogaster and the minuscule nematode
Caenorhabditis elegans. Topics in this article are categorized
by biologic system, process, or disease with discussion of
associated invertebrate models. Sections on bioactive products discovered from invertebrates follow the models section, and the article concludes with uses of invertebrates in
teaching. The models reviewed can serve as references for
scientists, researchers, veterinarians, institutional animal
care and use committees (IACUCs), and others interested in
alternatives to vertebrate animals.
Key Words: animal model; bioactive compound; instructional model; invertebrate
Introduction
History and Highlights of Invertebrate Use
in Research
I
nvertebrate models of human genetics and disease first appeared in the scientific literature in the late 19th century. A
search of the National Library of Medicine’s PubMed database for the dates 1800–2010 revealed a progressive increase
in research involving invertebrates. During 1800–1900, three
invertebrate articles appeared. William E. Castle, an animal
geneticist, was one of the first researchers to publish studies
based on invertebrates—he utilized the sea squirt (Ciona
intestinalis) as his research model and published his dissertation on this species in 1896.1 The remaining two articles for
the 19th century both appeared in the Journal of Physiology;
the first reported on the presence of hematoporphyrin in the
integument (Munn 1886), the second on respiratory exchange
in marine invertebrates (Vernon 1895).
Research with invertebrates increasingly appeared in the
scientific literature during the early 1900s, but it was not until the early 1940s that significant numbers of such papers
were published. During the period of 1923–1943, 16 papers
were based on invertebrate research as compared to nearly
14,000 during 1943–1963. Research with invertebrates
showed further expansion in the 1960s, with over 40,000 papers on invertebrates published from 1963 to 1973. Research
in the 21st century has continued to show the growing importance of invertebrates in biological and biomedical research:
in 2008–2010 PubMed showed 44,000 papers that used invertebrate species as models for studies of genetics and disease and for drug development and testing.
Another mark of the importance of invertebrates to biomedical research is the number of Nobel Prizes awarded to
researchers who have used them, whether as their primary
model or one of several animal species. Since the first Nobel
Prize for Medicine in 1901, 74 of the awards have been based
on animal research, and 18 of these included invertebrate
species.2 Drosophila was the model for Thomas H. Morgan’s
discoveries regarding the role of chromosomes in heredity;
he was awarded the Nobel Prize for Medicine in 1933 and
gave credit to Charles Woodworth and William Castle as pioneers of the Drosophila model.3 Caenorhabditis elegans’
genome was one of the first to be sequenced and the importance of this organism to scientific advancement is highlighted by three Nobel Prizes awarded in the 21st century.
The first, in 2002, was awarded to Sydney Brenner, Robert
Horvitz, and John Sulston for their work on similarities in
genetic and molecular mechanisms of organ development
and programmed cell death between humans and C. elegans.
Andrew Fire and Craig Mello won the 2006 Nobel Prize for
1Castle’s
Susan E. Wilson-Sanders, DVM, MS, DACLAM, is Director of University
Animal Care at the University of Arizona in Tucson.
Address correspondence and reprint requests to Dr. Susan E. WilsonSanders, Director, University Animal Care, University of Arizona, P.O. Box
245092, Tucson, AZ 85724-5092 or email [email protected].
126
papers are available on the website of the American Philosophical
Society (www.amphilsoc.org); this and other websites cited in this article
were accessed between November 9, 2010, and April 12, 2011.
2Information from www.animalresearch.info/en/medical/nobelprize.
3Information about this and other Nobel Prize laureates discussed in this
article is available online at http://nobelprize.org/nobel_prizes/medicine/
laureates.
ILAR Journal
their studies with C. elegans illustrating the conservation of
genes between species and elucidating the fundamental
mechanisms of gene regulation (Nass et al. 2008). Most recently, the 2008 Nobel Prize for Chemistry was shared by
Martin Chalfie, of Columbia University, for his work with
C. elegans utilizing the green fluorescent protein (GFP) to
facilitate his research on touch sensitivity and gene expression (Grandin 2009).
Applications of Invertebrates in Research
Biomedical research involving the use of animals has been
the cornerstone of medical progress for the past several centuries, but ethical concerns about the use of vertebrates,
which are more commonly understood to be sentient animals, have led researchers, veterinarians, and others in laboratory animal science to search for alternatives. Invertebrates
can serve as replacements for their vertebrate counterparts in
many areas of research, testing, and education.
A new area of focus for invertebrate research is drug development, including the discovery of bioactive products
from both terrestrial and marine invertebrates. Invertebrates
may also play a pivotal role in toxicity and efficacy testing of
new pharmaceuticals for both human and animal diseases,
sparing vertebrate animals from preliminary testing. As a result of animal rights proponents’ pressure on professional,
preprofessional, and K–12 schools, the use of live vertebrate
animals is rare in education. Invertebrates can serve as alternative teaching subjects, providing students with opportunities to observe behavior, anatomy, physiological principles,
pathology, results of genetic manipulation, and mechanisms
of drug actions.
To assist researchers, veterinarians, and institutional animal care and use committee (IACUC) members searching
for models of specific conditions and diseases, this article is
organized by biologic system, process, or disease with discussion of associated invertebrate models. Given the genetic
and molecular basis of many of mechanisms and diseases,
these topics are discussed in the systematic context with
additional models found in the tables (which list models not
discussed in the narrative, for reference and to illustrate the
breadth of invertebrate use).
Because of their long prominence as research models,
Drosophila and C. elegans are the first subjects of this discussion of invertebrate models.
The Fly and the Worm
Drosophila melanogaster
The fruit fly (Drosophila melanogaster) is one of the most
studied organisms in the animal kingdom. Cytogenetic research has led to the complete mapping and sequencing of its
chromosomes, enabling its use in an array of biological and
biomedical investigations (Gilbert 2008). Thanks to the vast
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2011
number of genetic and molecular tools available for use
with Drosophila, coupled with similarities in development
and behavior, the fruit fly has served as a unique and sensitive model for the study of human genetics and disease
(Beckingham et al. 2005). The power of Drosophila genetic
screens has led to the study of many mutant strains that have
helped elucidate visual and behavioral pathways, embryonic
patterning, and the development of models for numerous human diseases (Beckingham et al. 2005).
Several databases have been created to contain the genetic records for this tiny but exquisitely precious mine of
information. A comparison of one of these databases against
over 900 human disease genes (Drysdale and FlyBase Consortium 2008) showed that 714 human disease sequences
matched to 548 Drosophila genes, of which 153 could be
associated with mutant alleles—79 matched to human malignancies and 74 to human neurologic diseases (Reiter et al.
2001).
Drosophila modeling has also been used to express protein products found in human disease and to compare the
resulting pathologic condition in the fly to the human counterpart (Beckingham et al. 2005). This approach has yielded
positive results in three types of neurodegenerative disease:
Alzheimer’s, Parkinson’s, and polyglutamine diseases such
as Huntington’s chorea; for each, a fly model has been created and used in research (Bonini and Fortini 2003; Iijima
et al. 2004; Iijima and Iijima-Ando 2008).
Drosophila’s current and potential uses are expected to
transcend virtually every area of biologic study (Gilbert
2008) and to play a role in therapeutic trials for drug candidates targeting many human and animal diseases (Beckingham
et al. 2005; Gilbert 2008). Beckingham and colleagues (2005),
Gilbert (2008), and Korey (2007) provide excellent reviews
of Drosophila’s uses in biomedical research.
Caenorhabditis elegans
Caenorhabditis elegans models have many advantages
over vertebrate animals for use in biological and biomedical studies. These small worms are highly prolific reproducers with a short generation time, easily grown under
laboratory conditions, and inexpensive to care for (Nass
et al. 2008; Riddle 1997; Wood 1997). Additionally, C. elegans
is anatomically simple and has a fully mapped nervous
system (White et al. 1976). Humans and C. elegans have
virtually the same number of genes, and there are many
parallels in the ways that these divergent species operate
on genetic and molecular levels. As a result, C. elegans
has become an instrumental model for understanding the
molecular mechanisms involved in many human diseases
(Nass et al. 2008). It can serve as a model of both forward and
reverse genetics, with mutants, transgenics, and knockouts
easily created; and worms that express GFP enable in vivo
observation of cellular and metabolic processes (Jorgensen
and Mango 2002; Nass et al. 2008; Riddle 1997; Wicks et al.
2001).
127
Caenorhabditis elegans has also been used to study basic biological and physiological processes that are common
to all animals (Strange 2007). It has served as a model for
Parkinson’s, Alzheimer’s, and Huntington’s disease, diabetes, cancer, immune disorders, and the development and testing of therapeutic agents for these diseases (Artal-Sanz et al.
2006a; Faber et al. 1999; Link 2001; Nass et al. 2008; Pujol
et al. 2008). C. elegans may someday be the model of choice
for in vivo testing of new drugs, including high-throughput
screening technologies (Silverman et al. 2009).
Biological Models
Developmental Biology
Invertebrates have been used in the study of embryology
since the late 19th century, and several Nobel Prizes have
been awarded to scientists who utilized invertebrates in their
quest to understand developmental biology. Mechnikov published papers on the embryology of insects during the late
1860s and won the Nobel Prize in 1908. In 1995, the Nobel
Prize for Medicine was awarded to three researchers who
used Drosophila to evaluate genetic control of early embryonic development.
This section provides information on several of the main
developmental models; Table 1 lists additional models.
Drosophila melanogaster and Caenorhabditis elegans
A number of invertebrates have been used in developmental
biology, but the two primary organisms are Drosophila and
C. elegans, with Drosophila most often used. During the
past 20-plus years, genetic studies using Drosophila have
elucidated the regulatory mechanisms that control development of the embryo (Baker and Thummel 2007). Genetic
screens in Drosophila have led to the discovery of the signaling pathways Notch, Wingless, and Hedgehog and furthered
knowledge of vertebrate development and disease (Bier
2005). Genetic alteration of fly embryos has provided information about a variety of biological mechanisms (Shen et al.
2007). Planar cell polarity studies in Drosophila and other
arthropod embryos have shown that coordination of cell polarization occurs in the development and function of many
organs, particularly in epithelial cells such as the gut epithelium, which needs to move secretions (Simons and Mlodzik
2008). Drosophila models of planar cell polarity have focused on the development of hair patterns, the eye, and the
cochlea and have supported comparisons to mammalian development pathways for each.
Because of the close similarities between Drosophila
and vertebrate cardiogenesis and the conservation of key
genes, the fly’s heart serves as an excellent model of cardiac
development and disease (Medioni et al. 2009). Study of cardiac development in the fly has led to understanding of the
Table 1 Selected invertebrate models of developmental biologya
Model
Species used
References
Bone morphogenic proteins
Drosophila
Raftery and Sutherland 2003
Calcium signaling
Asterina pectinifera
Santella et al. 2008
Cilia regulation of development
pathways
Caenorhabditis elegans
Pedersen et al. 2008
Chromatin insulators
Drosophila
Gurudatta and Corces 2009
Developmental glycobiology
Drosophila
ten Hagen et al. 2009
Formation of the nervous system
Drosophila
Kulesa et al. 2009; Quan and Hassan 2005
Gene regulatory networks
Echinoidea
Peter and Davidson 2009
MicroRNA function in embryogenesis
C. elegans
Wienholds and Plasterk 2005
Nitrous oxide signaling during neural
development
Locusta, Schistocerca, Acheta,
Manduca, Drosophila
Bicker 2007
Pattern signaling and retinal
development
Drosophila
Baker 2007; Buscarlet and Stifani 2007
Pituitary patterning
Drosophila
Veitia and Salazar-Ciudad 2007
Regulatory switches
Drosophila
Borok et al. 2010
Semophorin in developing nervous
system
Caelifera (grasshopper)
Bonner and O’Connor 2000
Tubulogenesis
Drosophila
Kerman et al. 2006
aThese
128
models are provided for reference; discussion of other models is provided in the text.
ILAR Journal
molecular and cellular mechanisms that underlie morphogenesis and has elucidated the genetic control of cardiac
physiology (Medioni et al. 2009). The Drosophila heart
model may also play a role in efforts to identify unknown
genes and the regulatory networks that contribute to normal
heart development and function (Tao and Schulz 2007).
Other aspects of Drosophila anatomy and function have
proven useful in research. Because of the similarities between
Drosophila and mammalian mechanisms of hematopoiesis,
the fly serves as a model for vertebrate blood cell development (Crozatier and Meister 2007; Crozatier et al. 2007).
The developing Drosophila eye has been used to study the
Notch and tyrosine kinase signaling mechanisms, which
direct cell fates during development; recruitment of factors
(e.g., transcription factors for gene expression) is important
in animal development (Voas and Rebay 2004). The fly’s
excretory system has been used to study the development and
differentiation of the renal system across species (Denholm
and Skaer 2009).
In C. elegans the pharynx (foregut) has served as a key
model for the study of general organ development, with high
applicability to vertebrate embryology (Mango 2007). Research
on the nematode has clarified the mechanisms of neural cell
development by showing that the C. elegans gene sem-4 participates in the control of mesodermal and neuronal cell development (Basson and Horvitz 1996). The gene lin-9, which affects
signal transduction pathways that control gonadal development
in the nematode, has counterparts in many species, making C.
elegans a useful model for studies of developmental biology
and intercellular signaling mechanisms (Beitel et al. 2000).
Vulval development in C. elegans can be used as a model for
understanding the roles of chromatin remodeling in multiple
development pathways (Andersen et al. 2006). Additionally,
through study of the proteins and genes involved in this development, scientists may be able to recognize how complex proteins could become targets for cancer therapy (Andersen et al.
2006). C. elegans has been the primary model used in highthroughput genome scale analysis evaluating the involvement
of genes in tissue development (Ge et al. 2006).
Other Invertebrate Species
In recent years deuterostomes and other marine invertebrates, as well as insects, have been increasingly used both
to understand the evolution of these organisms and to shed
light on developmental processes in higher animals including humans (Arendt et al. 2008; Bicker 2005, 2007; Darling
et al. 2005; Holland and Gibson-Brown 2003; Isbister and
O’Connor 2000; Lowe 2008; Pourquie 2000; Swalla 2006;
Wessel et al. 2010). Grasshoppers (Dissosteira carolina and
others) have been used to study neural cell development,
specifically, the genetics and development of axons through
evaluation of grasshopper limb bud growth cones (Isbister
and O’Connor 2000). The molecule nitric oxide (NO) is
thought to play a role in the regulation of neuronal growth
and migration, and gastropod molluscs and embryonic
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2011
grasshoppers serve as models for elucidating NO signaling
pathways (Bicker 2005, 2007).
For over a century the sea squirt (Ciona intestinalis)
has been used as a model for studying animal development
(Holland and Gibson-Brown 2003; Passamaneck and Di
Gregorio 2005). This animal may also play an important role
in studies to determine how to solve comprehensive gene
networks in chordates and evaluate how these networks control development (Davidson 2007). The sea star, or starfish
(Asterias forbesii), has been utilized in developmental research related to reproductive processes. Basic techniques
(e.g., oocyte isolation, microinjection, and polymerase chain
reaction) have been developed so that new researchers can
easily work with these animals (Wessel et al. 2010). The genome of the starlet sea anemone (Nematostella vectensis)
has been the focus of a genome project; as a result, this
organism has been proposed as a model for molecular and
evolutionary biology (Darling et al. 2005). Somitogenesis, a
multistep patterning process in vertebrates, has been modeled in a variety of animal models from mice to protostomes
and deuterostomes; research with invertebrates has indicated
that the genetic machinery responsible for this segmentation is
conserved throughout the animal kingdom (Pourquie 2000).
Stem Cell Biology
Stem cell biology has its roots in research performed with
invertebrates: Stammzelle, the German word for stem cell,
was created based on research using crustacean primordial
germ cells in the 1890s by the German researcher Valentin
Haecker (Kohlmaier and Edgar 2008). Invertebrates continue to
be key models for understanding many of the characteristics
and genetics of stem cells, including their pluripotency and
ability to self-renew through proliferative growth (Kohlmaier
and Edgar 2008).
The stem cell niche has been evaluated through study of
Drosophila gonads. This research has furthered knowledge
of the structure of the niche and its ability to produce signaling pathways, which lead to stem cell self-renewal (Lin 2002;
Palasz and Kaminski 2009). The germline of C. elegans—
specifically, its distal tip cell, the foundation for the animal’s
stem cell niche—has also served as a model for stem cell
biology (Byrd and Kimble 2009; Hubbard 2007). Using genetic analysis of Drosophila muscle and satellite stem cells,
researchers have modeled the biology of vertebrate muscle
stem cells (Figeac et al. 2007). Planarians, uniquely, regenerate from their stem cell system and thus serve as a model
for gene and stem cell regulation (Agata 2003). Cnidarians
(Hydra, Nematostella) have been used to study stem cell signaling pathways and other mechanisms of stem cell biology
and function (Watanabe et al. 2009).
Endocrine Function and Metabolism
Drosophila, C. elegans, and marine invertebrates have been
useful in the study of endocrine and metabolic diseases.
129
Drosophila is an increasingly common model for understanding metabolism across species boundaries (Baker and
Thummel 2007). The ability to study the genetics of metabolic function in this small but sensitive model has provided
insights into the central regulatory pathways of vertebrates.
Study of the fly has also elucidated the pathogenesis of
human metabolic diseases such as diabetes and obesity.
Drosophila has served as an excellent model for the study of
diabetes, lipid metabolism, and other mechanisms of metabolism, including sterol adsorption and trafficking defects
that occur in Niemann-Pick type C disease (Baker and
Thummel 2007). Both diabetic and obese flies, as well as
genetically “lean” and hypoglycemic phenotypes, have been
created as models for human disease (Bharucha 2009).
Drosophila is not suitable for the study of all aspects of
human metabolic control; for example, the leptin signaling pathway is not present in the fly. But insulin signaling
is very similar in flies and humans, making Drosophila an
ideal model to study the ways insulin regulates metabolism
(Teleman 2009).
Transgenic flies have been developed to study the molecular endocrinology of neuroendocrine signaling and control (Dow 2007). Insulin receptor–like signaling pathways in
flies regulate a transcription factor known as DAF-16/FOXO,
a “master regulator” of many biological mechanisms (Lin
et al. 1997; Ogg et al. 1997). Both Drosophila and C. elegans
can serve as models for clarifying the mechanisms of this
transcription factor, which controls lifespan, metabolism,
and stress responses, and, in the worm, regulates the dauer
stage (when the animal goes into a state of hypometabolism)
(Mukhopadhyay et al. 2006). C. elegans’ dauer stage can be
used to model protein targets in the stress responses of higher
animals, and study of this stage may lead to recognition
of therapeutic targets for human diseases such as ischemia,
insulin resistance, neurodegenerative diseases, and cancer
(Lant and Storey 2010).
Caenorhabditis elegans has shown promise as a model
organism for studying AMPK (5′-AMP-activated protein kinase) signaling (Beale 2008), because it has demonstrated
evidence of having AMPK pathways. AMPK is often called
“the master metabolic switch” as it plays a key role in regulating metabolism, protein synthesis, and cell growth and in
mediating the actions of hormones (Beale 2008). Research
into the endocrine signaling pathways and hormone production control in C. elegans has yielded insights into similar
pathways in humans (Beckstead and Thummel 2006).
Hedgehog signaling studies in Drosophila, nematodes,
and mice have shown that this pathway inhibits the amount
of fat in the body; thus, manipulation of the pathway may be
useful in treating hyperlipidemia, obesity, and type 2 diabetes in humans (Suh et al. 2006, 2007). Studying the gene
Adipose, Suh and colleagues (2007) determined that both
mice (Adp) and flies (adp) heterozygous for the gene are
obese and insulin-resistant, indicating that this gene has an
antiadipogenic ability (Gilbert 2008). Other studies have
used the fly as a model for human fatty liver disease; the fly
oenocyte, which is comparable to the mammalian hepatocyte,
130
participates in lipid metabolism by producing enzymes that
lead to ketogenesis (Arquier and Leopold 2007; Downer
1985). In addition to serving as a model for lipid metabolism, the fly may be an effective model both in efforts to
discover more genes involved in obesity and diabetes and in
the screening of therapeutic agents developed for lipid-based
diseases (Gilbert 2008).
Other endocrine models include the silkmoth and the sea
squirt. The silkmoth (Bombyx mori) has an insulin-related
peptide gene that is similar to human preproinsulin (Yoshida
et al. 1998). The sea squirt (Ciona intestinalis) has many
analogues to hormones (e.g., gonadotropin-releasing hormone, insulin, and insulinlike growth factor [IGF]) found
in higher animals and thus may prove to be a useful model
for understanding the function of these hormones and for
the study of neuroendocrinology (Sherwood et al. 2006).
And Drosophila has insulinlike peptides that serve as hormones, neurotransmitters, and growth factors (Wu and
Brown 2006).
Immunology
Allorecognition and Adaptive Immune System
Allorecognition and its molecular basis have been studied in
ascidian urochordates such as the sea squirt and the star ascidian (Botryllus schlosseri) (Ben-Shlomo 2008). As they
most likely share common ancestors with vertebrates, research into their patterns of self-recognition have provided
insight into the development of the immune response of
vertebrates.
Even though urochordates are not recognized to have an
adaptive immune system, some genes in organisms such as
C. intestinalis are related to those in vertebrates and give rise
to adaptive immunity (Du Pasquier et al. 2004). Studies using urochordates and other invertebrate deuterostome model
systems have provided information on mechanisms of antigen receptor diversification and immune system development relevant to vertebrates (Eason et al. 2004). Gene
rearrangement studies in other species, such as lampreys and
molluscs, have also provided information about differences
and similarities between adaptive and innate immune systems (Flajnik and Du Pasquier 2004). McKitrick and De
Tomaso (2010) provide an excellent review of the molecular
mechanisms of allorecognition in B. schlosseri.
Both colonial and solitary free-living reef corals such as
Fungia scutaria show evidence of histocompatibility and
allorecognition. Sea anemones from the order Actiniaria
also have similar immune systems (Jokiel and Bigger 1994).
These animals, along with B. schlosseri, can be used to study
the evolution of the immune system and could serve as models for screening new therapeutics targeting cellular immunity and transplant rejection. The star ascidian has also been
suggested to serve as a model for maternal-fetus allorecognition issues, as the organism has a natural killer (NK) cell
similar to human uterine NK cells (Lightner et al. 2008).
ILAR Journal
Immunity and Response to Infection
Innate immunity is the invertebrate’s primary defense
against infectious organisms, and this system has similarities to that of vertebrates (Magor et al. 1999). The fly’s
sensing and signaling cascades during infection have stimulated the use of Drosophila as a model for innate immunity and response to infection (Ferrandon et al. 2007; Royet
et al. 2005). Similarly, C. elegans’ innate immunity has
been used to study immune defense and the role of cellular
stress in an organism’s response to infection (Millet and
Ewbank 2004). The nematode has also been used to model
the activation of genes in response to infection (GravatoNobre and Hodgkin 2005). Octopus maya has been suggested as a model for immune responsiveness because of
its ability to become infected by pathogenic organisms
(Van Heukelem 1977).
Macrophages
Invertebrates have played a key role in the history of the
macrophage. The term “macrophage” (phagocyte) was coined
by Ilya Ilyich Mechnikov, a comparative embryologist and
winner of the Nobel Prize in 1908. He observed in starfish
larvae a group of cells that had unusual characteristics—the
ability to move in tissue: after introducing small rose thorns
into the larvae, he noted the next morning that the thorns
were surrounded by the mobile cells. He further studied this
phenomenon using the freshwater flea (Daphnia magna),
exposing it to fungal spores: the spores were attacked and
isolated by the Daphnia macrophages.
Macrophage-like cells are present in many species of
invertebrates. Often they originate from mesenchymal,
endothelial, or fibroblastic cells that differentiate into phagocytes (Naito 2008). For example, Hydra, a member of the
phylum Cnidaria, has cells with phagocytic capability that
play a role in the animal’s ability to recognize “self” (Bosch
and David 1986; Kobayakawa and Koizumi 1997; Naito
2008) and, thus, make it a useful model of graft rejection.
Molluscs also have cells that can act as macrophages, but the
origin of these cells is mesenchymal, not hematopoietic, indicating divergent paths of differentiation (Naito 2008).
Neuroimmunology
Neuropeptides can transfer information from the nervous
system to the immune system, perhaps serving as regulators
of immune response (Stefano et al. 1991, 1996, 1998). Opioid peptides are present in the neural tissues of several molluscs, including the blue mussel (Mytilus edulis) (Stefano
and Leung 1982) and the garden snail (Helix aspersa)
(Marchand and Dubois 1986), and are involved in immune
processes. These animals can thus serve as models to explore connections between the immune system and neural
regulation (Liu 2008).
Volume 52, Number 2
2011
Infectious Disease
Viruses have been found in the genome of many species
including invertebrates (Becker 2000). The gypsy element,
which infects Drosophila, was the first retrovirus recognized
in invertebrates and may be an ancestral precursor of vertebrate retroviruses (Pelisson et al. 2002). The gene flamenco
modulates gypsy in Drosophila, and the interaction of the
two genes has provided an excellent model for the study of
the genetic relationships between virus and host (Bucheton
1995).
Drosophila has also served as a model for other hostparasite relationships—for example, as a host for the Burkholderia cepacia complex (Bcc), a group of bacteria that
contribute to severe health risks among humans with cystic
fibrosis (Castonguay-Vanier et al. 2010). Bcc studies performed in Drosophila showed similar virulence patterns to
those observed in mammals, indicating that the fly is a useful
alternative model for such studies (Castonguay-Vanier et al.
2010).
Virulence screening for agents with biological warfare
potential may be possible in Drosophila. Recently, genomewide virulence screens were performed for Francisella
novicida using Drosophila as the host. Researchers identified many similarities in gene function between flies and
mammals but also found that a considerable number of the
virulence factors that play a role in mammals do not in the
insect model (Ahlund et al. 2010).
Pathogenic fungi have been studied in worms and insects. Several major fungal pathogens (e.g., Aspergillus,
Candida, Cryptococcus) infect and kill roundworms, fruit
flies, and wax moths. Because the genes that modulate virulence in these invertebrates are remarkably similar to those
of humans, these three species have been evaluated as alternatives to mammalian models of fungal disease (Chamilos
et al. 2007). Researchers have specifically used C. elegans
to study bacterial and fungal virulence, pathogenicity, and
mechanisms of host defense against invaders (Fuchs and
Mylonakis 2006). Conservation of virulence mechanisms
between roundworms, fruit flies, and wax moths and higher
animals has led to the increased use of these and other invertebrates in virulence studies (O’Callaghan and Vergunst
2010).
Because of the similarities between human and invertebrate infections, insects are considered a model of choice for
studying opportunistic microorganisms. Insect models can
allow for rapid screening of potentially opportunistic infections while minimizing concerns about the ethics of vertebrate animal experimentation (Scully and Bidochka 2006).
O’Callagan and Vergunst (2010) provide a review of the use
of Drosophila and C. elegans as models for infectious
disease.
Other invertebrates used to model infectious disease
include Daphnia and marine shrimp species. Daphnia is recognized as a model system to study host-parasite interactions for diversity of parasites, from bacteria to helminths
(Ebert 2008). Study of viral diseases in marine shrimp has
131
led to discovery of RNA interference–based therapies that
may result in similar therapies for viral diseases of higher
animals, including humans (Krishnan et al. 2009).
Memory, Learning, and Behavior
The California sea slug (Aplysia californica; Glanzman
2006, 2008, 2009), opalescent sea slug (Hermissenda crassicornis; Alkon 1987), and pond snail (Lymnaea stagnalis;
Lukowiak et al. 1996)—all gastropod molluscs—have served
as models in studies of neuronal mechanisms of learning and
memory. Such studies have also used many cephalopod molluscs, especially the coleoid group, which includes octopuses, cuttlefishes, and squids.
The octopus has been an effective model in studies of
behavioral communication. Octopuses use body color and
tentacle positions to indicate their attitude toward approaching prey or other octopi (Pribram 1973)—their stance and
color are comparable to the facial expressions and body language of monkeys and humans. Octopi have also proven
useful in reversal learning experiments, with comparable results to similar studies in rats (Sutherland and Mackintosh
1971).
The chambered nautilus (Nautilus pompilius), one of the
most ancient cephalopods, has been used in Pavlovian conditioning studies (Crook and Basil 2008), and Aplysia and H.
crassicornis have been used to study classical and operant
conditioning (Baxter and Byrne 2006). Studies in Aplysia
have led to elucidation of the molecular mechanisms involved in all phases of implicit memory (Hawkins et al.
2006), and the mud flat crab (Chasmagnathus convexus) has
served as a model in similar studies (Romano et al. 2006).
Decapod crustaceans have been used to study aggressive behaviors (Barron and Robinson 2008).
Among insects, honeybees (Apis mellifera) have a long and
rich history as research models—Aristotle studied them
and recorded his observations of their behavior (Elekonich
and Roberts 2005). More recently, von Frisch (1967) studied
the bee’s behavior and communication through dance. Even
though the brain of the honeybee is less than 1 cubic millimeter in diameter, it is very accessible for study and can serve as
a model for many higher-order cognitive processes (Giurfa
2006, 2007). Investigators have studied learning, memory,
and sensory processing by focusing on honeybee patterns of
navigation and foraging, as bees follow several routes to and
from their nest to their preferred blossoms, requiring them
to recall memory sequences and respond to memory cues
(Chittka et al. 1999; Collett 2005; Menzel 1999; Menzel and
Giurfa 2006). Furthermore, the complete sequencing of the
honeybee genome makes this tiny creature an excellent research model (Menzel et al. 2006). The study of gene expression and of the endocrine, metabolic, and neural physiology
of bee colonies is revealing how these and other animals respond to their environment (Elekonich and Roberts 2005).
Scientists are also studying neural mechanisms of reward reinforcement in honeybees (Gil et al. 2007).
132
Ants have been used to study the molecular genetics of
social behavior and adaptation (Robinson et al. 1997) and to
discover associative links between long-term memory and
visual stimuli (Collett and Collett 2002). In the cricket (Gryllus
bimaculatus), the sensory system and ability to respond to
environmental stimuli have been compared to the complex responses of vertebrates to their environments (Jacobs
et al. 2008). Drosophila has served as a model for olfactory learning and memory, partly because of the ability to
chemically mutate genes in this organism (Glanzman 2005;
McGuire et al. 2005). With the ability to manipulate Drosophila genes and to model aggressive behavior and its genetic
basis (Robin et al. 2007), study of the fly may lead to understanding of the genetic and molecular basis of human emotions (Iliadi 2009). The fly also serves as a model of several
human cognitive disorders and may be useful in the evaluation of drug therapies for them (Skoulakis and Grammenoudi
2006).
C. elegans has been the subject of studies on the behavior and genetics of habituation, the use of long- and shortterm memory for learning (Giles and Rankin 2009), and the
neural and molecular mechanisms of behavior (Schafer
2005; Sengupta and Samuel 2009). Its dauer stage serves as
a model for the molecular mechanisms behind stress response behavior (Lant and Storey 2010).
Musculoskeletal Disease
The metabolism of proteins in the flight muscles of the tobacco hornworm (Manduca sexta) has been studied (Tischler
et al. 1990), and the species has been used in space flight
research to understand the effects of low gravity on muscles.
Studies using both Drosophila and C. elegans have enhanced
understanding of muscle formation and degeneration (Kim
et al. 2008); for example, research with C. elegans contributed to knowledge of regulatory muscle proteins and the
maintenance of muscle under certain physiological and
pathological conditions (Kim et al. 2008). Striated muscles
of the leech (Pontobdella muricata) have dystrophin-associated proteins that have striking similarities to those in humans; thus, leeches and other annelids may be useful in the
study of interaction sites for muscular dystrophy–associated
proteins (Royuela et al. 2001).
Neural and Neuromuscular Systems
and Disease
Drosophila and C. elegans
The fruit fly and the nematode are the primary invertebrate
models for many areas of neurobiological study.
Work with Drosophila has a long history and includes
the cloning of the first potassium channel; demonstration of
transient receptor potential (TRP) channels through cloning
of trp; discovery of the genes responsible for the biological
ILAR Journal
clock; and studies of courtship behavior, sleep patterns,
learning, alcoholism, and aggression (Foltenyi et al. 2007;
Gilbert 2008; Grosjean et al. 2008; Vosshall 2007). The fly
serves as a model for many specific disorders. For example,
there is a homologue or orthologue in Drosophila for most
of the approximately 300 genes that participate in human
retardation (Inlow and Restifo 2004), so the mysteries surrounding Fragile X retardation may be unraveled through
study of the fruit fly model (Pan and Broadie 2007). Drosophila models will also play a vital role in the identification
and evaluation of new therapies for neurological diseases,
providing a preliminary animal model before the use of
mammals (Marsh and Thompson 2004, 2006; Whitworth et al.
2006).
C. elegans is an important model for understanding the
pathophysiology and molecular mechanisms of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and
Huntington’s (Johnson et al. 2010; Troulinaki and Tavernarakis
2005).
Tauopathies
Drosophila and C. elegans have served as models for
Alzheimer’s disease and other tauopathies (Crowther et al.
2005; Luheshi et al. 2007; Wheeler et al. 2010). Alzheimer’s
is postulated to result from amyloid toxicity that initiates
aggregation of proteins into amyloid fibrils (Luheshi et al.
2007). In the fly model, Crowther and Luheshi found that
protein aggregation in fly brains leads to dysfunction of
neurons and neuronal degeneration, which progresses to
memory loss and shortened lifespan, hallmark symptoms
of Alzheimer’s (Crowther et al. 2005; Luheshi et al. 2007).
Transgenic C. elegans created to express human beta amyloid peptide (Abeta) develop intracellular deposits with the
classical Alzheimer’s amyloid fibrillar component, indicating the usefulness of this organism in the study of Alzheimer’s
(Link et al. 2001). The introduction of mutant human tau
into Aplysia neurons grown in culture induced neuropathologic lesions typical of Alzheimer’s, indicating that Aplysia
can serve as a model for this disease (Shemesh and Spira
2010).
Drosophila is contributing to knowledge of NiemannPick type C (NPC), a tauopathy in which an overabundance
of free cholesterol in the brain leads to neurodegeneration
(Patterson 2003; Vance 2006). The pathogenesis of NPC is
not well understood, but if either NPC1 or NPC2 is mutated
in the human, NPC is likely to result (Patterson 2003). To
determine whether the fly could serve as a model of NPC,
researchers reviewed the fly database (flybase.org) and identified potential NPC models (Fluegel et al. 2006; Huang et al.
2005). As in humans, the presence of npc1 is necessary for
sterol homeostasis; when it is mutated, the flies show molting defects. Study of Npc1a function in the fly has led researchers to hypothesize that NPC1 may play a vital role in
the transport of sterol to the endoplasmic reticulum and mitochondria (Huang et al. 2005). Drosophila also contains a
Volume 52, Number 2
2011
family of Npc2a genes whose mutation results in neurodegeneration (Huang et al. 2007). Similarities between NPC in
flies and humans suggest that the fruit fly can also serve as a
therapeutic test model for this devastating disease (Gilbert
2008).
Parkinson’s, Huntington’s, and Other
Neurological Diseases
The discovery of Drosophila homologues of Parkinson’sassociated loci has resulted in the use of pink1 and parkin fly
mutants as models for Parkinson’s disease (PD) (Greene
et al. 2003; Laurent 1999; Pesah et al. 2004). PD has also
been studied using C. elegans, which has not only dopamine
neurons, receptors, transporters, and the enzymes that catabolize dopamine (Nass et al. 2002, 2008; Nass and Blakely
2003; Sulston et al. 1975; Wintle and Van Tol 2001), but also
orthologues of most of the human Parkinson’s genes (Nass
et al. 2008). As a result, various genetic manipulations with
worms have produced models of the genetics and molecular
pathways of Parkinson’s. For example, C. elegans has been
used in human gene expression assays to identify genes associated with PD. The genes are overexpressed in dopamine
neurons; if they are PD-associated, the worms show signs of
neurodegeneration (Berkowitz et al. 2008). Mitochondrial
dysfunction plays a role in the pathology of PD (Korey
2007), and work with the C. elegans model has contributed
to understanding of intercompartmental proteostasis and its
role in cellular function (Kirstein-Miles and Morimoto
2010). Both Drosophila and C. elegans have been proposed
as alternatives to vertebrate animals in the screening of drugs
with therapeutic potential for PD (Pienaar et al. 2010).
Drosophila models show promise for elucidating many
other neurodegenerative diseases including Huntington’s
(HD) (Gilbert 2008) and neuronal ceroid lipofuscinoses
(NCL), neurodegenerative disorders associated with accumulations of cellular material and the formation of inclusions in lysosomes (Korey 2007). Research using Drosophila
homologues for HD and NCL has assisted efforts to understand the pathogenesis of both diseases (Korey 2007).
Fly homologues have also been found for several hereditary spastic paraplegias (HSPs), disorders that exhibit symptoms such as progressive weakness of the legs due to axonal
degeneration (Korey 2007). For example, Kennedy’s disease
results in progressive muscle atrophy and weakness in males
and is caused by an androgen receptor (AR) mutation. Research on flies on which an AR-like gene is overexpressed in
photoreceptor neurons may prove useful in the development
of therapeutic approaches for Kennedy’s disease (Matsumoto
et al. 2005).
Transgenic Drosophila models exist for Alzheimer’s,
Fragile X, HD, Kennedy’s, Machado-Joseph, NCL, spinocerebellar ataxia, and spinal and bulbar musculoatrophy
(Celotto and Palladino 2005). An unusual use of transgenic
technology in flies has been the production of a transgenic,
prion-induced neurodegenerative disease in Drosophila. The
133
resulting “mad fly” disease, characterized by locomotor dysfunction and shortened lifespan, is similar to GerstmannSträussler-Scheinker syndrome, an inherited prion disease in
humans (Chandran and Lewis 2007).
Human mitochondrial encephalomyopathy disorders include neuropathy, ataxia, retinitis pigmentosa, Leigh syndrome, and familial bilateral striatal necrosis, all of which
have components of neurological and muscular dysfunction
coupled with tissue degeneration (Korey 2007). A number of
fly mutants have phenotypes that can be directly related to
the symptoms observed in this complex set of disorders. For
example, flies with mt:ATPase6 mutation show shortened
lifespan, progressive degeneration of flight muscles, and
neural dysfunction (Celotto et al. 2006). Indicative of even
closer correlation with the human disease, these animals
have mitochondrial dysfunction and reduced ATP (adenosine triphosphate) production, both of which characterize the
human disease.
Recent reviews of Drosophila models of neurodegenerative diseases are available (Lu 2009; Lu and Vogel 2009).
Other Invertebrate Models
Because of their giant axons, fibers, and synapses, octopi
and squid are often used as research and teaching models for
neurobiology (Grant et al. 2006; Van Heukelem 1977). The
Yucatan octopus (O. maya), which can easily be grown under laboratory conditions, is used as both a teaching and research model for comparative psychology and neurobiology
(Van Heukelem 1977). Additionally, octopi and squids are
excellent models for neural electrophysiology, neurochemistry, and neurosecretion (Packard 1972; Sanders et al. 1975;
Young 1967, 1971). Research utilizing the long-finned squid
(Loligo pealei) in nerve conduction studies garnered Andrew
Huxley and Alan Hodgkin the 1963 Nobel Prize for Medicine. Because of its giant fiber system, Loligo has also been
proposed as a model for neurodegeneration and dementia
(Grant et al. 2006). The somatogastric nervous system of
decapod crustaceans (e.g., lobsters, crayfish, and crabs) can be
used for modeling the neuromodulator actions of vertebrates
(Stein 2009).
The brain of freshwater planarians (Platyhelminthes)
may appear to be very simple in comparison to that of humans, but these organisms have many neural genes and transcription factors that are homologous to those that cause
pathology in humans. Furthermore, planarians are known for
their ability to regenerate—even their central nervous system (CNS)—and researchers using gene silencing techniques
hope to elucidate the molecular mechanisms and genes that
enable the animal’s regeneration (Cebria 2007). Understanding this process may yield important information for treating
human CNS injuries and disease.
The highly sensitive auditory system of the cricket can
be an effective model in studies of the development of dendrites and their response to injury (Horch et al. 2009). The
cricket has also contributed to the understanding of adult
134
neurogenesis (the production of new neurons throughout
life), which occurs in most species including humans (Cayre
et al. 2007).
Pathophysiology
Aging and Healthspan
C. elegans has been used to study genetic regulation of
lifespan. Some C. elegans are endowed with multiple copies
of the gene sir-2.1 (counterparts occur in humans), which
works in combination with the transcription factor DAF-16
to produce greater longevity. The longevity is based on 143-3 proteins, which activate a pathway that increases resistance to both oxidative and genotoxic stress (Berdichevsky
et al. 2006). Evolution of lifespan and the biology of aging
have also been studied extensively in Drosophila (Grotewiel
et al. 2005).
Other models have been established in Drosophila and
C. elegans to evaluate insulin/IGF-1 signaling (IIS) pathways, the role of the intestine in germline signaling, and the
ablation of germline precursors in longevity—all of which
are involved in the regulation of lifespan (Mukhopadhyay
and Tissenbaum 2007). In one study, germline precursors
were ablated in C. elegans, which lived up to 60% longer
than their unaltered counterparts (Hsin and Kenyon 1999).
Because the IIS pathway participates in regulating lifespan in
both invertebrates and mammals, Drosophila and C. elegans
are effective models to study this pathway and the mechanisms through which it can increase lifespan (Giannakou and
Partridge 2007; Piper et al. 2008). The Yucatan octopus has
also been suggested as a model for healthspan and the effects
of aging (Van Heukelem 1977).
Many studies using invertebrates have focused on the
beneficial effects of reduced caloric intake on lifespan
(Kennedy et al. 2007; Masoro 2005). Similar studies in mice,
flies, and monkeys have shown rejuvenation of the immune
system when the animals were subjected to caloric restriction (Nikolich-Zugich and Messaoudi 2005), thus providing
the opportunity for extended life. Additional models of aging
and lifespan are shown in Table 2.
Apoptosis
The word apoptosis translated from the Greek means “falling or dropping off” and was used by the Greeks to refer to
petals and leaves falling from flowers and trees (Collins
English Dictionary 2009). It was not until 1842 that the German scientist Carl Vogt described the modern-day pathological process of apoptosis after studying the tadpole of the
midwife toad (Alytes obstetricans) (Vogt 1842). The term
then fell into disuse until 1965, when John Foxton Ross Kerr
of Australia recognized apoptosis, as distinguished from
traumatic cell death, while performing electron microscopy
of rat liver cells affected by acute injury (Kerr 1965). Kerr’s
ILAR Journal
Table 2 Other disease modelsa
Model
Species used
References
Age-related cardiac disease
Drosophila
Ocorr et al. 2007a,b
Aging
Bivalves, Drosophila,
Caenorhabditis elegans,
Macrostomum lignano
(flat worm)
Abele et al. 2009; Ballard 2005; Ballard et al. 2007;
Berryman et al. 2008; Brys et al. 2007; Campisi
and Vijg 2009; Grotewiel et al. 2005; Guarente
2007; Johnson 2008; Mouton et al. 2009;
Partridge 2008; Philipp and Abele 2010; Wolff
and Dillin 2006
Angiogenesis/vasculogenesis
Hirudo medicinalis
de Eguileor et al. 2004
Autophagy
Drosophila, C. elegans,
Platyhelminthes
Kang and Avery 2010; Kourtis and Tavernarakis
2009; Tettamanti et al. 2008
Axon guidance regulators
Drosophila
Duman-Scheel 2009
Caloric restriction/diet
C. elegans, Drosophila
Chen and Guarente 2007; Guarente 2007; Morck
and Pilon 2007; Partridge et al. 2005, 2008;
Piper and Bartke 2008; Pletcher et al. 2005
Ehlers-Danlos syndrome
Echinoderms
Szulgit 2007
Epilepsy
Drosophila, C. elegans
Baraban 2007
Freeze tolerance
Various species of insects
Sinclair and Renault 2010
Hypoxia
Drosophila, Daphnia magna,
C. elegans
Gorr et al. 2006; Romero et al. 2007
Lymphangioleiomyomatosis
Drosophila
Juvet et al. 2007
Mitochondria-associated
diseases
C. elegans
Ventura et al. 2006
Postural control
Clione (mollusc)
Deliagina and Orlovsky 2002; Deliagina et al. 2007
Sleep regulation
Drosophila
Cirelli 2009
Stem cell roles in cancer
Drosophila, C. elegans
Januschke and Gonzalez 2008; Nimmo and
Slack 2009
Testicular cancer
Drosophila
Browne et al. 2005
Tumor metastasis
Drosophila
Jang et al. 2007; Naora and Montell 2005
Tumor suppression
Drosophila
Vaccari and Bilder 2009
Wound healing
Hirudo medicinalis,
Drosophila, C. elegans
Grimaldi et al. 2006; Jane et al. 2005; Michaux et al.
2001
aThese
models are provided for reference; discussion of other models is provided in the text.
later work with colleagues Jeffrey Searle, Andrew Wyllie,
and Alastair Currie furthered understanding of the role of
apoptosis in both normal and disease processes (Kerr et al.
1972; O’Rourke and Ellem 2000).
Subsequent study of C. elegans was pivotal in the quest
for understanding the genetics of apoptosis. The 2002 Nobel
Prize in Medicine was awarded to three researchers—
Sydney Brenner, H. Robert Horvitz, and John E. Sulston—
for their work on genetic regulation of organ development
and programmed cell death, for which their primary model
was C. elegans. They were also able to determine that similar genes are present in humans to control apoptosis.
Pathways for apoptosis are conserved throughout most
of the animal kingdom, from invertebrates to humans, but C.
elegans and Drosophila remain models of choice, especially
Volume 52, Number 2
2011
for genetic, biochemical, and molecular-mechanistic studies
(Bao et al. 2005; He et al. 2009; Xu et al. 2009). Moths are
also useful in the study of apoptosis and in particular programmed cell death in skeletal muscles (Schwartz 2008).
Tumor necrosis factor (TNF), a cytokine involved in programmed cell death, has functional analogies in the earthworm Eisenia foetida. The worm counterpart to mammalian
TNF, while not homologous, has similarities in function based
in lecithinlike activity/domains (Beschin et al. 2004), so the
earthworm may be a useful model for studying apoptosis.
Cancer
Drosophila and C. elegans are long-established models for
the study of neoplastic diseases. Because the pathways of
135
gene functions in the two species have many similarities to
those in humans, the study of these organisms has provided
much insight into tumorigenesis in both humans and animals
(Gateff and Schneiderman 1967, 1969; Gilbert 2008; Kirienko
et al. 2010; Saito and van den Heuvel 2002).
Genetics. Many genetic mutations in the fly lead to uncontrolled cell division, neoplasia, and death (Gateff and
Schneiderman 1967, 1969; Gilbert 2008). As a result, Drosophila is a useful model for gene regulation, particularly tumor suppressor genes (TSGs) and oncogenes (Brumby and
Richardson 2005; Gilbert 2008; Menut et al. 2007). Similarly, C. elegans is also an excellent model for cancer including the study of apoptosis, cell cycle progression, growth
factor signaling, genome stability, and mechanisms of invasion and metastasis (Kirienko el al. 2010). Studies with C.
elegans have provided important clues about the function of
homologous oncogenes and TSGs in humans. Genomewide
RNA interference screens in C. elegans have facilitated the
identification of new cancer gene candidates and how they
function in the pathogenesis of cancer (Poulin et al. 2004).
Understanding how multiple genes function together to create a cascade of events provides opportunities for identifying
therapeutic agents that can target genes contributing to
cancer. C. elegans thus may serve as a key model in screening potential cancer therapeutic agents (Saito and van den
Heuvel 2002).
A specific genetic screen has been developed to delineate
neoplastic TSGs that provide control over cell polarity and
proliferation (Menut et al. 2007). Hippo, a kinase, modulates
a chain of events that lead to expression of genes involved
in cell proliferation and growth regulation (Badouel et al.
2009). Study of the Hippo pathway in flies has identified
close similarities to the same pathway in mammalian species
(Buttitta and Edgar 2007). In flies, mutations in hippo can
lead to epithelial cell proliferation in several tissues—and
play a role in managing apoptosis—so cancer researchers
can add Drosophila to their arsenal as a model for studying
control of cell proliferation (Gilbert 2008). Both Drosophila
and C. elegans have also served as models for evaluating the
role of FOXO genes in cancer; FOXO factors affect many
physiological processes, including differentiation of cells,
tumor suppression, cell-cycle arrest, and apoptosis (Arden
2008).
RUNX (runt-related) genes in humans regulate a number
of processes—control of cellular proliferation, maintenance
of stem cells, development of specific cell lineages, and regulation of cell differentiation; disruption of the genes leads
to pathology and often cancer. RUNX transcription factors
have been studied in Drosophila, C. elegans, and the purple
sea urchin (Strongylocentrotus purpuratus) (Braun and
Woollard 2009).
Drosophila is also being used to evaluate the roles of
axon guidance genes, such as Netrin and Deleted in Colorectal Cancer (dcc), for which the human counterparts are implicated in causative mechanisms of human cancer. These
models may prove useful in developing cancer therapeutic
agents (Duman-Scheel 2009).
136
Table 3 shows other invertebrate models for genetic
studies.
Molecular and Cellular Biology. Asymmetric cell division (the generation two different daughter cells from a single cell) plays a role in the development of cancer. The
mechanisms of such division and its potential to lead to tumorigenesis have been modeled in both C. elegans and Drosophila (Chartier et al. 2010), but the embryo of C. elegans has
served as the primary model for understanding asymmetric
cell division in cancer—in particular, determining stem cell
function and tumorigenesis in humans (Hyenne et al. 2010).
Molecular control of cell migration and tumor metastasis
has been modeled in Drosophila (Jang et al. 2007). For example, a model for ovarian cancer metastasis has been evaluated in Drosophila based on the migration of ovarian border
cells (Naora and Montell 2005). Human ovarian cells and fly
border cells have many similarities: both are controlled by
steroid hormones; during development, each can show characteristics of epithelial and mesenchymal cells; and both can
migrate to other sites to form cell nests (Gilbert 2008; Naora
and Montell 2005). Several genes and proteins have been
identified in border cell migration in flies. The proteins,
which also occur in women, have been tested in vitro on
ovarian cancer cells and the cytopathology observed. Unlike
cancer cells, border cell migration represents a normal process in flies—it ends without harm to the animal (Gilbert
2008; Naora and Montell 2005). Further studies in flies may
assist in determining the factors that turn off cell migration
and may lead to an understanding of ovarian cancer metastasis and, ultimately, methods to prevent it. Border cell migration may also serve as a biological assay for the development
of treatments for ovarian cancer (Gilbert 2008).
Drosophila has also been used to understand the concept
of cell competition, whereby cells of different genotypes are
located next to each other and compete for proliferative advantage. In cancer, the abnormal cells dominate the normal;
thus, cell competition may play a role in cancer development
(Baker and Li 2008). Planarians also have found a niche in
cancer research. These tiny organisms have been studied to
understand the molecular biology and genetics of cancer as
well as the possible role of regeneration in causing or curing
cancer (Oviedo and Beane 2009).
Table 2 lists additional cancer models.
Substance Abuse
Drosophila and C. elegans have been used to study the genetic and behavioral mechanisms of cocaine, alcohol, and
nicotine addiction (Schafer 2004; Wolf and Heberlein 2003).
In addition, the pond snail has been used to study cocaine
addiction and therapies to treat or prevent it; changes in
learning and memory in the snail can be easily evaluated and
demonstration of impairment is readily observed (Carter et al.
2006). The honeybee is another model for studying cocaine
addiction. Bees fed low levels of cocaine show altered patterns in their foraging dance and removal of cocaine from
their diet results in withdrawal effects (Barron et al. 2009).
ILAR Journal
Table 3 Genetic modelsa
Model
Species used
References
Behavior
Drosophila
Jasinska and Freimer 2009; Mackay and
Anholt 2007
Chromosome speciation
Drosophila, mosquitoes
Ayala and Coluzzi 2005
Cocaine-related behaviors
Drosophila
Heberlein et al. 2009
Complex traits
Drosophila
Mackay and Anholt 2006
CT/CGRPb
Pecten maximus, Haliotis tuberculata,
Crassostrea gigas, Drosophila
Lafont et al. 2007
Gene perturbations
Caenorhabditis elegans
Borgwardt 2008
Gene regulation
C. elegans, Drosophila
Ercan and Lieb 2009; Large and Mathies
2007; Mendjan and Akhtar 2007
Genetic interaction networks
C. elegans
Lehner 2007
Meiosis
C. elegans
Colaiacovo 2006; Schvarzstein et al. 2010
Mitochondrial DNA
Mytilus edulis, M. galloprovincialis,
Venerupis philippinarum, Lampsilis,
Inversidens japanensis
Breton et al. 2007
Muscle development
Drosophila
Maqbool and Jagla 2007
Myoblast fusion
Drosophila
Richardson et al. 2008
Noncoding RNA
Drosophila
Deng and Meller 2006
p53
C. elegans, Drosophila
Lu and Abrams 2006
RNA silencing
Drosophila
Kavi et al. 2005
Sleep
C. elegans, Drosophila
Andretic et al. 2008
Spindle assembly and
regulation
Drosophila
Buchman and Tsai 2007; Doubilet and
McKim 2007
Telomere protection
Drosophila
Cenci et al. 2005
Transposable elements
Drosophila
Le Rouzic and Deceliere 2005
Tumor susceptibility gene
(TSG) 101
Drosophila
Herz and Bergmann 2009
Wnt
Drosophila
Bejsovec 2006
aThese
models are provided for reference; discussion of other models is provided in the text.
calcitonin/calcitonin gene-related peptide
bCT/CGRP,
Ethanol response has been studied in several invertebrates. Research on ethanol sensitivity and tolerance in
Drosophila revealed genes that are directly linked to the behavioral responses of the inebriated flies (Berger et al. 2008).
Work with intoxicated Drosophila has shed light on the specific neurons that mediate observed behaviors (Scholz 2009).
The honeybee has also been proposed as a model of alcoholism and its effects. After consuming concentrations of
ethanol up to 20% of their diet (Abramson et al. 2000), the
honeybee’s behavior changes in ways similar to those observed in vertebrates, with effects on locomotion and learning. The social bee may also be appropriate to study alcohol
influence on language, social interaction, development, and
learning (Abramson et al. 2000). C. elegans has also served
as a model of alcoholism (Dolganiuc and Szabo 2009).
Volume 52, Number 2
2011
Toxicology
Pharmaceutical research requires the detection of adverse reactions to new drugs as early in development as possible. A
number of in vitro tests are available (e.g., cell culture, tissueslice) but do not always translate to animal model systems or
relate to clinical experience. Invertebrates can be used as models for many toxicological studies and can bridge the gap between in vitro models and vertebrate animal studies. Close
similarities to vertebrate response, rapid reproduction rate,
cheap cost, and ease of housing and care have made invertebrates key organisms in toxicologic screens (Avanesian et al.
2009). The literature shows numerous examples of the use of
invertebrates in toxicity evaluations; the most commonly used
organisms are C. elegans, Drosophila, and the water flea.
137
The nematode has proven to be an excellent model for
use in toxicology studies, drug development, and research
on environmental toxicology due to its mapped genome and
simple nervous system (Williams et al. 2000). Because of its
genetic and cellular similarities to humans, C. elegans is an
important model for high-throughput screening of therapeutic agents for human diseases (Nass et al. 2008). It can be
used in both LD50 (lethal dose, 50%) and behavioral paradigms (Dhawan et al. 1999), and it shows results comparable
to those of mouse systems (Williams et al. 2000). The worm
has been used for evaluations of genetic and environmental
toxicology, neurotoxicolgy, and high-throughput experiments to screen for molecular and genetic targets of chemical toxicity. Neurotoxicity can also be modeled in Drosophila
and various species of cockroaches (Peterson et al. 2008).
All of these organisms provide simple and inexpensive alternatives to mammals for evaluating the toxicity of new pharmaceuticals (Artal-Sanz et al. 2006; Leung et al. 2008).
Both the sludge worm (Tubifex tubifex) and D. magna can
also be used as models in preliminary toxicology screening,
including LD50 evaluations (Devillers and Devillers 2009).
Because of Daphnia’s sensitivity to toxicants, it is also used in
water quality monitoring to identify the presence of contaminants (Martins el al. 2007). In addition, the mussel has been
used to study organophosphate toxicity and proposed as an
additional biomarker for pollution (Brown et al. 2004).
Drosophila has proved to be a useful model in drug-feeding
experiments to evaluate new antiepileptic drugs (Ackermann
et al. 2008). Drug delivery, however, is a major challenge
when using Drosophila as it is very difficult to standardize
the amount of drug consumed in a fly’s diet (Avanesian et al.
2009); other options, such as microinjection in the abdomen,
have been proposed (Dzitoyeva et al. 2003). In spite of the
drug delivery challenge, a number of studies have used
Drosophila in toxicological evaluations (Ackermann et al.
2008; Avanesian et al. 2009; Gupta et al. 2007b; Patnaik and
Tripathy 1992).
Endocrine-active chemicals are present in the environment and some therapeutic agents have endocrine effects; in
both cases, these compounds disrupt normal endocrine function in mammals. Changes in sperm counts, breast cancer,
congenital abnormalities of the genitalia, and other pathologic conditions in humans are linked to these chemicals and
therapeutic agents. Invertebrates can be used to study endocrine effects of drugs and environmental contaminants
(Avanesian et al. 2009; Duft et al. 2007; Gupta et al. 2007b;
Patnaik and Tripathy 1992; Tatarazako and Oda 2007). For
example, researchers have observed adverse reproductive effects in Drosophila adults and cell lines exposed to a variety
of insecticides (Gupta et al. 2007b; Patnaik and Tripathy
1992). Similarly, Avanesian and colleagues (2009) studied
methotrexate toxicity in flies and found ovarian impairment
comparable to that observed in mammalian models. The
freshwater mud snail (Potamopyrgus antipodarum) and
Daphnia have also served as models for the assessment of
environmental chemicals (Duft et al. 2007; Tatarazako and
Oda 2007). Many gender-related differences in toxicant
138
effects have been noted in humans, and these effects can be
modeled in many different invertebrates including insects,
nematodes, crustaceans, molluscs, corals, and echinoderms
(McClellan-Green et al. 2007).
Environmental impacts on the lysosomal-autophagic
system have been studied in bivalve molluscs (Moore et al.
2006). Environmental metal contamination can be monitored
in various insects, as metal accumulates in chitinous exoskeleton and is incorporated into insects’ internal issues
(Hare 1992). Additionally, many aquatic species serve as
biomarkers for organic xenobiotic and metal contamination
in both fresh and saltwater (Rainbow 2007; Raisuddin et al.
2007; Sarkar et al. 2006).
“The Five Senses”
Hearing
The Johnston’s organ, located in the antenna of Drosophila,
is the counterpart of the mammalian ear. Similarities in the
genes involved in development of the hearing structures of
flies and mammals have led to speculation that study of fly
hearing may provide insights into deafness in humans
(Boekhoff-Falk 2005). The role and function of TRP channels, present in both flies and vertebrates, have been evaluated in flies to provide a model system to study vertebrate inner
ear disorders that affect hearing and balance (Cuajungco
et al. 2007). The creation of mechanical models has enabled
modeling of auditory transducer dynamics, which will be
used to test auditory performance in both vertebrates and
Drosophila (Nadrowski and Gopfert 2009).
Olfaction
There are many similarities—structural, functional, and
physiologic—between the olfactory systems of vertebrates
and insects (Kay and Stopfer 2006), making insects excellent models for the study of olfaction. Vertebrates use their
noses to smell, whereas insects have antennae; however,
both structures have specialized epithelium lined with ciliated olfactory receptor neurons (ORNs) that respond to
odorants (Hallem et al. 2006; Kay and Stopfer 2006; Malnic
et al. 1999; Rospars et al. 2003). From the ORNs, both vertebrates and insects send neural processes to the brain—the
insect’s antennal lobe and the vertebrate’s olfactory bulb
(Kay and Stopfer 2006; Laurent 1999). Investigators have
studied insect olfactory systems to understand interactions
between animals and their environment (de Bruyne and
Baker 2008). Other studies have focused on odor processing
and the capacity to detect odor blends using moths and honeybees (Lei and Vickers 2008), and C. elegans and Drosophila have been models in olfactory signaling research
(Kaupp 2010; Nakagawa and Vosshall 2009).
Decapod crustaceans can serve as olfactory system models because the cellular and morphological organization of
their olfactory system has many similarities to the human
ILAR Journal
olfactory pathway. In many decapod crustaceans, neurogenesis occurs in the olfactory system throughout life, so these
animals are being used to study lifelong neurogenesis, with
the aim of understanding how it occurs (Sandeman and
Sandeman 2003; Schmidt 2007). This research may elucidate methods to stimulate neurogenesis in humans to treat
neurological diseases or injuries.
All organisms are exposed to an enormous diversity of
chemicals in the environment, and understanding the way the
nervous system recognizes and responds to these chemical signals is challenging. The chemosensory system of insects has
become an important area of research, and Drosophila is the
primary model under study (Benton 2008). Crustaceans are also
effective models for the study of chemoreception, which is
based in their olfactory system (Derby and Sorensen 2008).
Taste and Satiation
Researchers study Drosophila to understand taste perception
and the neural circuits (in particular one called hugin) that
affect feeding behavior. An organism’s decision, whether
human or fly, is basically to eat or not to eat; the mechanisms
in the brain that prompt this decision are similar (Amrein
and Thorne 2005; Melcher et al. 2007).
Touch and Temperature Sensing
Responses to mechanical forces, including touch, are poorly
understood at the molecular level. Touch-sensitive mutants
of C. elegans have been created and the defective genes studied to help identify sensory components that affect the cells
that sense gentle touch (Bounoutas and Chalfie 2007).
All animals have the ability to detect changes in environmental temperature based on the presence of neuronal
and molecular substrates that affect thermosensation. The
three primary models for studies of thermosensation are the
mouse, C. elegans, and Drosophila (McKemy 2007). Investigators have also studied thermosensing in C. elegans to understand the molecular and cellular basis for neural plasticity
(Mori et al. 2007).
Vision
In the 1930s, horseshoe crabs (Limulus polyphemus) were
used by Haldan K. Hartline and C.H. Graham as their model
for studying the optic nerve, and in 1967 Hartline, Ragnar
Granit, and George Wald were awarded the Nobel Prize in
Medicine for their research into visual processes of the eye.
Hartline’s Limulus model continues to serve as an excellent
model for vision research because of its complex ocularneural network. Limuli have large, easily accessible retinal
neurons, allowing for electrophysiological study. Study of
the horseshoe crab has provided insight into the operation of
human vision, particularly adaptation to light and lateral inhibition (Liu and Passaglia 2009).
Volume 52, Number 2
2011
Octopuses have also been used in vision research. Their
sucker chemotactile systems have been compared to the
mammalian eye (Packard 1972; Sanders et al. 1975; Young
1967, 1971).
Other Models
Space Biology
Invertebrates are useful in studying the effects of ionizing radiation, both on earth and in space. C. elegans has been effectively
used in microbeam irradiation studies to evaluate bystander effects (Bertucci et al. 2009), to study the long-term effects of
radiation exposure in space travel, and to evaluate the gravitational effects of space travel on muscle gene expression (Zhao
et al. 2005). It has also been used to study natural space radiation exposure, and the resulting mutants have been genetically
evaluated (Nelson et al. 1994). Tardigrades (water bears, moss
piglets) have also served as models for open space research,
because they have the ability to survive desiccation, extreme
cold, and radiation, all of which occur in space. Additional invertebrate species showing potential for use in outer space biological experiments are the sleeping chironomid (Polypedilum
vanderplanki), the brine shrimp (Artemia salina and A. franciscana), and several types of rotifers, which are commonly
called “wheel animals” (Jonsson 2007).
Symbiosis
Throughout nature, symbiotic relationships between organisms contribute to survival and the ability to flourish. Even
humans are dependent on lowly microorganisms, which contribute to nutrition and defense. The relationship between host
and microbe has been modeled through invertebrates, which
have a diverse set of associated microorganisms (Chaston
and Goodrich-Blair 2010). The primary model systems in
such research are insects and nematodes. The former include
various species of termites (Hongoh et al. 2005; Ohkuma
2008; Yang et al. 2005), the honeycomb moth (Galleria mellonella; Gouge and Snyder 2006; Walsh and Webster 2003),
the tobacco hornworm (Manduca sexta; van der Hoeven et al.
2008), the Asian gypsy moth (Lymantria dispar; Broderick
et al. 2004), and Drosophila (Ryu et al. 2008). The nematode
Steinernema carpocapsae and its association with the bacterium Xenorhabdus nematophila have been used as a model
for symbiotic relationships (Goodrich-Blair 2007).
Bioactive Products
Biomaterials and Biomimetics
Baculovirus
Baculovirus-insect cell expression systems have enabled
the production of recombinant proteins for use in research
139
(Jarvis 2003), making it possible for investigators with basic molecular biology background to produce their protein
of choice very simply. Moths and butterflies are the most
common species used for the cell culture system (Jarvis
2003).
Luciferase
Bioluminescence, the ability of a living organism to produce light, results from a biochemical reaction where oxygenation of luciferin, a substrate, occurs through the action
of the enzyme luciferase (Day et al. 2004). Many organisms can produce bioluminescence, but the animal most
studied and used for biomedical research applications is
the firefly or lightning beetle (Photinus pyralis). Light in
beetles is produced in “lanterns,” organs containing photocytes that are layered between two rows of cells. Uric acid
crystals in the cell layers reflect the light produced by
the photocytes (Fraga 2008; Hastings and Wilson 1976;
Hastings 1983, 1989a,b). During the late 1800s, Raphael
Dubois, a French physiologist, studied the biochemical
properties of bioluminescence in these insects and created
a luminescent solution by crushing their abdomens and
mixing the crushed organs in cold water (Harvey 1957;
McCapra 1982).
Luciferases have been incorporated into many in vitro
molecular assays to allow evaluation of gene expression in
transformed cell lines (Contag et al. 1998, 2000; de Wet et al.
1987; Lim et al. 2009; Sherf and Wood 1994; Takakuwa
et al. 1997; Wood et al. 1989; Wood 1995; Zhang et al. 1994,
2008). In addition to the firefly and other beetles, luciferases
have been isolated from the sea pansy (Renilla reniformis)
and jellyfish (Aequorea victoria) and used as reporters
in mammalian cells and in other types of animal studies
(Contag et al. 2000). Cloning of image reporters, such as the
luc gene, is allowing researchers to study transcriptional
regulation, signal transduction, protein-protein interactions,
tumor transformation, cell trafficking, and targeted drug actions in living animals without invasive techniques (Gross
and Piwnica-Worms 2005).
Silk Products
For centuries silk fibers were the primary source of suture
materials, but in recent years synthetic materials have dominated the market. Studies of silkworm fibers have shown
biocompatibility between silk and the commonly used biomaterials polylactic acid and collagen (Altman et al. 2003).
As a result, silk and silklike fibrous proteins from the silkworm (B. mori), the golden orb web spider (Nephila clavipes), diadem spider (Araneus diadematus), and other insects
are being considered for use in biomedical applications such
as tissue scaffolding for joint repair (Altman et al. 2003).
The design of silk-inspired polymers and proteins and their
uses in bioengineering and biotechnology are reviewed by
Hardy and Scheibel (2009).
140
Biomimetics
Biomimetics is the study of a living organism to create a
device, either medical or nonmedical, by applying information gained from the organism. Invertebrates have been useful models in this area of applied research. For example, the
study of marine ragworms (Nereis virens and N. diversicolor) supported the development of a new endoscope based
on the ragworm’s ability to move in slippery substrates, similar to mucus in the gastrointestinal tract (Hesselberg 2007).
Drug Discovery
Invertebrates have been used for many centuries for their
medicinal properties. In the western hemisphere, the leech
(Hirudo medicinalis) was used for bloodletting of patients
with many different disorders and maggots were (and sometimes still are) used for cleaning wounds. In the East, the
Chinese valued the sea cucumber for its ability to cure many
human diseases (Kelly 2005).
A wide range of invertebrate species—from insects to
marine life—serve as reservoirs of bioactive compounds,
but marine life accounts for the largest number: over 14,000
pharmacologically active compounds have been identified from
marine plants and animals (Adrian 2007), 961 of them in 2007
alone (Blunt et al. 2009). Bis(indole) and tris(indole) alkaloids
are among the most commonly isolated compounds and show
high biological activity, with potential as pharmaceutical agents
(Gupta et al. 2007a). These alkaloids have a wide range of effects including antimicrobial (bacterial, fungal, viral), antiparricidal, anticancer, anti-inflammatory, antiproliferative, and
antiserotonin activity, and RNA and DNA synthesis inhibition.
In addition to the discovery of pharmacologically active compounds, invertebrates such as snails and sea anemones are being used in the creation of antisera used in humans (Redwan
2009). Excellent reviews are available of the many species
studied, compounds identified, and their biological activities
(Blunt et al. 2009; Gupta et al. 2007a; Kelly 2005).
While marine invertebrates are proving to be outstanding
sources of pharmaceuticals, the harvesting of marine life,
especially by third-world countries to sell species to pharmaceutical companies, is decimating native populations. There
is significant concern among conservationists that such overexploitation of marine animals is going to result in the extinction of many (Duckworth et al. 2003; Lawrence et al.
2010). Fortunately, many countries are initiating aquaculture
programs for the cultivation of marine invertebrates to spare
wild populations while providing new means of revenue for
their societies (Kelly 2005). As research and development
with bioactive compounds move forward, global measures
to ensure species protection and welfare will be critical.
Anticoagulants
Tick anticoagulant peptide and other natural anticoagulants
have been isolated from hematophagous invertebrates and
ILAR Journal
testing of these products in mammalian animal models of
thrombosis and atherosclerosis has shown that they have potent anticoagulant properties (Fioravanti et al. 1993; Ragosta
et al. 1994; Schaffer et al. 1991; Schwartz et al. 1996; Sitko
et al. 1992). Ticks and similar organisms may thus serve as
sources of natural inhibitors in the design of improved anticoagulants (Corral-Rodriguez et al. 2009) and antithrombotics
(Koh and Kini 2009). Evaluation of hirudin, a natural thrombin inhibitor present in the blood-sucking leech, for use in the
management of thromboembolic diseases (Markwardt 2002)
has resulted in the creation of two recombinant versions of
hirudin (Lepirudin and Desirudin) that are now on the market
for use in humans. Lepirudin has found a second niche as an
effective treatment for angina (Redwan 2009).
Sulfated fucans and galactans (homopolysaccharides) isolated from marine invertebrates have powerful natural pharmacological actions that can be therapeutically effective in humans
(Pomin 2009) as anticoagulants (Farias et al. 2000; Mourao and
Pereira 1999; Mourao 2004; Pereira et al. 1999), antithrombotics (Berteau and Mulloy 2003; Mourao and Pereira 1999;
Mourao 2004), and anti-inflammatories (Berteau and Mulloy
2003). The main source organisms for fucans and galactans are
the sea cucumber, sea urchin, and ascidians (Pomin 2009).
Antimicrobials
In 2005, Salzet reported the identification of over 30 neuropeptide-derived antimicrobials from such diverse invertebrate
species as shrimp, fly maggots, mosquitoes, scorpions, horseshoe crabs, sea cucumbers, and numerous other marine invertebrates (Lawrence et al. 2010; Salzet 2005). Chemokine
binding proteins (CBPs), lectins that have been isolated from
the sea worms Chaetopterus variopedatus and Laxus oneistus,
have potential as antivirals as research has shown that they can
inhibit HIV infection in cells and prevent virus transmission
from infected to unaffected T cells (Balzarini 2006). HIVinhibitory compounds have also been isolated from soft corrals
(Lobophytum sp.) from the Philippines (Rashid et al. 2000).
Sulfated fucans and galactans from marine invertebrates can
also serve as antivirals (Harrop et al. 1992).
Other antimicrobial peptides isolated from invertebrates
have other bioactive properties (Bulet et al. 2004). Cecropin A,
identified in the silkmoth (Hyalophora cecropia) and the mosquito (Anopheles gambiae), has antibacterial and antifungal
properties and lyses yeast cells. Stomoxyn, from the stable fly
(Stomoxys calcitrans), is toxic to bacteria and fungi and has
lytic effects on trypanosomes. Similar peptides have been isolated from fire ants (Pachycondylas goeldii), termites (P. spiniger), and the spiders Oxyopes kitabensis and Cupiennius salei.
Two bioactive peptides with multiple uses have been identified
in the stalked sea squirt (Styela clava) (Bulet et al. 2004).
development as cancer therapeutics (Adrian 2007; Jimeno
2002). C-nucleosides from the Caribbean sponge (e.g.,
Cryptotethya crypta) served as the chemical model for synthesis of Cytarabine, which is used in the treatment of leukemia and lymphoma (Schwartsmann et al. 2003). Compounds
in clinical development include didemnins, Kahalalide F,
hemiasterlin, dolastatins, cemadotin, soblidotin, bryostatins, ecteinascidin-743, and aplidine (Rawat et al. 2006;
Schwartsmann et al. 2003). Many of these compounds are
still being evaluated clinically; others (e.g., bryostatin 1)
have been removed from trials because of severe side effects
(Singh et al. 2008). Bonnard and colleagues (2010) recently
reported the discovery of antitumor promoters in two types
of Comorian soft corals; they are under investigation for potential use in cancer therapy. Sulfated fucans and galactans
from marine invertebrates have also been recognized to have
antimetastatic properties (Coombe et al. 1987).
Immune Protectors
Parasite colonization can provide protection from immunemediated diseases. Studies in mice colonized with helminths
showed that the animals were protected when challenged
with colitis, asthma, encephalitis, and diabetes (Elliott et al.
2007). Similarly, clinical trials have shown that helminth exposure can reduce the symptoms of ulcerative colitis and
Crohn’s disease in human patients by altering immune response. The effects on the immune response may result from
the induction of regulatory T cell activity (Weinstock et al.
2005). Induced helminth infections may some day be used to
treat many inflammatory and immune-mediated diseases
(McKay 2009).
Pain
Cone snail (Conus sp.) venom, known as conotoxin, can alleviate pain and prevent or treat epilepsy. Based on the number of species of cone snails and the number of conopeptides
each can produce, it is expected that 70,000 different conotoxins will be characterized and tested for therapeutic
potential (Ekberg et al. 2008). Evaluation of conotoxins in
mammalian animal models has led to numerous applications, including use as antinociceptives, antiepileptics, neuroprotectives, and cardioprotectives. Conopeptides may also
find use as therapeutic agents for cancer and neuromuscular
and psychiatric disorders (Han et al. 2008). The drug Prialt,
a derivative of conotoxin, has been approved for and is being
used to treat intractable pain (Lee et al. 2010); it has a potency 800 times that of morphine (Xia et al. 2010).
Sunscreens and Antioxidants
Cancer
Over the past 10 years, a number of compounds originating
from marine invertebrates have entered preclinical and clinical
Volume 52, Number 2
2011
A wide variety of marine organisms can be used to obtain
effective sunscreens and antioxidants. Mycosporinelike
amino acids (MAAs), prevalent in corals, have the ability to
141
absorb UV rays and serve as natural sunscreens in marine
animals (Dunlap et al. 1999); synthetic sunscreens have been
developed based on these MAAs (Dunlap et al. 1995, 1999;
Karentz et al. 1991).
Marine invertebrates, either independently or in symbiotic relationship with bacteria, also produce potent antioxidants (Dunlap et al. 1999). These antioxidants have potential
biomedical applications, ranging from use as food supplements to cosmetic additives and chemopreventives in oxidative stress-related disease.
Harvest of Bioactive Products from Sponges
Marine sponges contain a circular proteoglycan called
“spongican” that is involved in species-specific cell adhesion, resulting in cell aggregation (Fernandez-Busquets and
Burger 2003). There are many similarities between the responses of cell aggregation in sponges and processes in humans. For example, both human platelets and sponge cells
respond similarly to stimuli that either inhibit or accelerate
aggregation (Philip et al. 1992). Because of the cell-aggregation response, sponges can be used to study both inflammation and anti-inflammatory compounds and may serve as
models in anti-inflammatory drug development (Dunham
et al. 1985).
Spongicans may also be useful in the study and treatment of specific human diseases; for example, a derivative of
spongican has been shown to block replication of HIV, indicating potential as a treatment for HIV infection (MacKenzie
et al. 2000). Sponges may also serve as a model to evaluate
treatment for Alzheimer’s and other amyloid disorders, as
spongicans affect amyloid fibrils by causing aggregation,
thus helping block the characteristic lesions of Alzheimer’s
(McLaurin et al. 1999).
Spongin is a protein that makes up the fibrous skeleton of
sponges and may have potential uses in the treatment of osteoarthritis and other degenerative bone diseases (Kim et al.
2009). Spiculogenesis creates sponge skeleton and has been
studied to find new approaches to treat dental and bone disease. It is initiated by the enzyme silicatein, beginning the
process of creating silica nanoparticles that fuse in layers
around a central protein filament of silicatein and silintaphin-1, which serves as the scaffolding protein (Muller et al.
2009). This process has led to the development of synthetic biomaterials containing recombinant silicatein and
silintaphin-1, which have been used to induce biosilicamediated regeneration for tooth and bone defects. The assembly of silica nanoparticles by the action of silicatein and
silintaphin-1 results in the synthesis of light waves; these
nanoparticles could serve as an alternative to fiberoptics in
biomedical applications (Muller et al. 2009).
Sponges of the genus Spongosorites contain several
bis(indole) alkaloids that have potent antifungal properties
and produce moderate cytotoxicity in cancer cell lines (Oh
et al. 2006). Stevensine, an alkaloid metabolite with antitumor
properties, is produced by the sponge Axinella corrugata,
142
which is being laboratory raised to create sufficient quantities of Stevensine without affecting natural-living specimens
(Duckworth et al. 2003).
Invertebrate Models in Teaching
For most of the history of biological, medical, veterinary,
and agricultural teaching, vertebrate species have been the
models of choice because of their close similarities to humans and other target species. For example, veterinary colleges have used vertebrates to help students understand
physiology, pharmacology, and pathology and practice surgery and clinical care. Similarly, colleges of medicine have
used turtles, rats, dogs, and other vertebrates to teach medical students the fundamentals of physiology, pharmacology,
and other aspects of medicine. But fewer vertebrate animals
are being used in teaching as pressure from animal rights
activists has led many medical and veterinary programs to
reduce or even eliminate the use of animals, despite students’
expressed concerns about the lack of hands-on experience
with living animals.
In K–12 teaching, many animals commonly used in the
past have been extensively harvested or, among wild populations, decimated by disease. Reduced numbers of some species have led to the animals’ being listed as endangered,
which means that these animals cannot be taken from the
wild. As a result of all of these factors, fewer vertebrate animals are available or used for study by K–12, college, and
professional students.
Invertebrate species can serve as substitutes for vertebrates in some, but not all, educational experiences for students. The wide range of animals available for study, the vast
populations available either in the wild or from laboratory
suppliers, their similarities to (and differences from) vertebrates, the ease of keeping them, and the low cost of acquisition and maintenance—all contribute to their value as
experimental subjects (Deyrup-Olsen and Linder 1991).
A number of books and scientific articles document the
varied uses of invertebrates in teaching; Deyrup-Olsen and
Linder (1991) provide a concise review of the uses of invertebrates in teaching physiology. Additionally, a number of
examples of the use of invertebrates in teaching K–12 and
college-level students are available on the Internet. Following are some of the many examples of the use of invertebrates in teaching (with websites as available; also see Smith
2011, in this issue):
•
•
•
In lieu of the frog sciatic nerve preparation for demonstrating action potentials, recordings from cockroaches
(Periplaneta sp.) can be substituted.
Crayfish (Procambarus sp. and Pasifastacus sp.) are effective for modeling the effects of changing environmental temperature on metabolic rate (Casterlin and Reynolds
1977).
Circulation and the heart can be observed in Mercenaria
clams (Florey 1968a; Greenberg 1965) and crayfish
(Florey 1968b).
ILAR Journal
•
•
•
•
•
•
•
•
•
•
Aplysia is an effective model for teaching physiology
and neuroscience.
Mussels are useful for demonstrating muscle function
(Hoyle 1968).
Mussel and clam gills can be used to study the function
of cilia, including the effects of environmental toxicants on cilia (Deyrup-Olsen and Linder 1991; Hoar and
Hickman 1983).
The tobacco hornworm (Manduca sexta) can be used to
observe the active transport of ions in the midgut, serving as an alternative to frog skin preparations (DeyrupOlsen and Linder 1991).
Bryn Mawr College has developed the “Serendip” program (http://serendip.brynmawr.edu), providing a hands-on
guide to teaching middle and high school biology. The
classes include Invertebrate Diversity, for which students
purchase earthworms, snails, and arthropods from pet
stores and use them for class activities.
Oklahoma State University offers a class called Laboratory of Comparative Psychology and Behavioral Biology, using honeybees, houseflies, carpenter ants, and
crabs, to teach students about the neuronal mechanisms
of learning and memory (http://psychology.okstate.edu/
faculty/abramson).
The University of Southern California teaches a monthlong short course in Antarctica, where students study invertebrates to learn about the adaptations of animals,
including humans, in extreme environments (http://
antarctica.usc.edu).
Lafayette College has included invertebrate research
opportunities for undergraduates in its curriculum for a
number of years (Sherma and Fried 1987).
Johns Hopkins University School of Medicine uses C.
elegans as a molecular model to teach undergraduate students about congenital mysasthenic syndromes (Kaas
et al. 2010).
And the University of Arizona offers research projects
for students to study biological processes and disease
using C. elegans, black flies, mosquitoes, cockroaches,
shrimp, and others.
Because of the growing culture of invertebrates for food,
medicinal purposes, and pets, a number of veterinary colleges offer invertebrate classes to teach students how to
provide care for invertebrates (e.g., North Carolina State
University’s Invertebrate Medicine; www.cvm.ncsu.edu/
conted/invert.html). In addition, veterinary schools are teaching conservation medicine (Veterinary Examiner 2009), particularly targeting marine life. Marine invertebrates, so
necessary for their biologically active properties, will open
new avenues of practice for veterinarians, allowing the veterinary profession to provide additional contributions to environmental protection and animal welfare.
Many of the invertebrate species and models discussed
in this article have also been used in teaching to help students learn about basic biological processes. Advanced students can also use invertebrates to learn about genetics,
Volume 52, Number 2
2011
developmental biology, cancer, and other areas of study.
However, there remain teaching activities, such as learning and practicing surgical techniques or working with
livestock in animal science or wildlife biology curricula,
for which vertebrate animals will remain the models of
choice.
Conclusions
There is a long and illustrious history of invertebrates as
models for research, testing, and education. Use of Drosophila for genetic studies was established in the early 20th
century, and the fly has since become one of the most prolific models for mammalian disease. Similarly, C. elegans’
parallels with mammalian genetics and molecular biology
have made the worm a vital model for understanding the
molecular mechanisms involved in disease. While Drosophila and C. elegans remain the two most studied organisms, there is no paucity of information about other
invertebrates. Many terrestrial and marine organisms serve
as models for human disease and provide nonvertebrate
alternatives for preliminary toxicology and efficacy studies,
as evidenced by the examples in this article and its extensive bibliography, coupled with the 300,000-plus articles
on invertebrate use available through PubMed and other
databases.
Similarly, invertebrates have many uses in education, offering a wealth of educational opportunities throughout the
spectrum from K–12 classes to professional and graduate
curriculums without having to rely on vertebrate species.
However, vertebrate animals in teaching are still necessary,
so that students at all levels can learn from hands-on encounters with the animals they will work with during their careers
or with which they will share their lives as companions and
friends. And professional students must have the opportunity
to observe and practice techniques in living animals so that
they can ethically practice their professions—for the welfare
of both humans and animals.
For all uses of animals, IACUCs, veterinarians, members
of the scientific community, governmental regulators, and
the public must balance ethical concerns about the use of
animals with the needs of society. There is also an ethical
responsibility to question the use of higher animals—in research, testing, and teaching programs and in the evaluation
of protocols. Invertebrate models provide rich opportunities
for learning about and practicing ethical animal care and use
by meeting the first and most critical principle of Russell and
Burch’s Three Rs—replacement.
Acknowledgments
I thank Cassandra Carman for her assistance with the writing
management system and making copies of the 1500-plus articles we started with. I also thank my coworkers for their
patience for the many hours it took to complete the research
and article, leaving them many additional responsibilities.
143
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ILAR Journal
Culture and Maintenance of Selected Invertebrates in the
Laboratory and Classroom
Stephen A. Smith, Joseph M. Scimeca, and Mary E. Mainous
Abstract
Invertebrate species have been used for many years in the
laboratory and teaching environment. We discuss some of
the most commonly maintained invertebrates—the nematode (Caenorhabditis elegans), the California sea hare (Aplysia californica), the fruit fly (Drosophila melanogaster),
terrestrial hermit crabs, the horseshoe crab (Limulus polyphemus), and cephalopods—and briefly describe general techniques for culturing them in captivity. The aim of this article
is to give potential users an idea of the materials, methods,
and effort required to maintain each type of organism in a
laboratory or classroom setting.
Key Words: Aplysia; Caenorhabditis elegans; cephalopod;
fruit fly (Drosophila melanogaster); hermit crab; horseshoe
crab (Limulus polyphemus); invertebrate care; invertebrate
culture
comparative anatomy, developmental biology, genetics,
behavior, evolution, ecology, and conservation. Public
aquariums have displayed numerous species of invertebrates—sponges, coral, anemones, octopus, jellyfish, starfish, sea urchins, and crustaceans—and zoos have built ant
colonies, bee hives, and butterfly houses and assembled diverse insect and arachnid collections.
In this article we discuss some of the commonly maintained invertebrate species—the nematode (Caenorhabditis
elegans), the California sea hare (Aplysia californica), the
fruit fly (Drosophila melanogaster), terrestrial hermit crabs
(Coenobita spp.), the horseshoe crab (Limulus polyphemus),
and cephalopods (octopuses, squid, and cuttlefish)—and describe general techniques for culturing and maintaining them in
captivity. As this is only a brief introduction to these animals,
readers are encouraged to acquire more detailed information by
both consulting the literature cited in this review and conducting a thorough reference search for the latest information about
these species and their culture and maintenance.
Introduction
N
umerous species of invertebrates are used in a variety of research, teaching, and public display activities. These species have been adapted for captivity
over the years for their aesthetic value, ease of culturing, and
utility for a multitude of teaching and research purposes.
In the research laboratory, invertebrates have served as
models for cancer, aging, immunology, endocrinology, toxicology, developmental biology, tissue regeneration, genetics, molecular biology, learning and memory, and biomimetics
(Wilson-Sanders 2011, in this issue). In the primary and secondary school classroom they have been used to demonstrate
Stephen A. Smith, DVM, PhD, is a professor of aquatic medicine/fish health
and of wildlife and exotic animal medicine in the Department of Biomedical
Sciences and Pathobiology at the Virginia-Maryland Regional College of
Veterinary Medicine of the Virginia Polytechnic Institute and State
University in Blacksburg. Joseph M. Scimeca, DVM, PhD, is Director of
the Laboratory Animal Program at Southern Illinois University in
Carbondale. Mary E. Mainous, MS, is a laboratory specialist at the VirginiaMaryland Regional College of Veterinary Medicine.
Address correspondence and reprint requests to Dr. Stephen A. Smith,
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland
Regional College of Veterinary Medicine, Phase II, Duck Pond Drive,
Virginia Polytechnic Institute and State University, Blacksburg, VA 240610442 or email [email protected].
Volume 52, Number 2
2011
Caenorhabditis elegans
Caenorhabditis elegans is a free-living nematode that has
been used extensively for research in developmental biology,
neurobiology, behavioral biology, and cell death (Hope
1999; Riddle et al. 1997; Wood 1988) and, because it is
transparent, in anatomical studies. The genome of C. elegans
was the first to be completely sequenced for a multicellular
organism. Because the nematode has only 959 cells and the
position of each cell is consistent from worm to worm, researchers now know specifically which genes encode the
development of each individual cell (Brenner 1974; Strange
2006) and how alteration of a gene will affect a particular
cell’s development (Hall and Altun 2007).
Anatomy and Biology
Caenorhabditis elegans is an unsegmented nematode that is
about 1 mm in length. Its life cycle is completed in about 5
days at room temperature (22–23°C) if sufficient food is available. The worms are either male or, more often, hermaphroditic
and able to self-fertilize (Byerly et al. 1976; Stiernagle 1999).
The offspring of self-fertilized worms are usually genetically
identical to each other and to the parent. The parent nematode
153
lays eggs that hatch in about 12 hours and go through four
larval stages (L1-L4) before becoming adults. However, if
food is scarce, the L2 larvae enter a dauer stage, an inactive
alternative form that can live without eating or reproducing.
The organism can survive in this stage up to several months
until food becomes available, when it enters the L4 larval
stage for up to 15 days before becoming an adult.
Culturing C. elegans
The preferred food source for monoxenically culturing C.
elegans is a bacterial lawn of Escherichia coli OP50
(Stiernagle 2006). Five ml of Luria-Bertani broth (10 g tryptone, 5 g yeast extract, and 10 g sodium chloride in 1 L of
distilled water, dH2O1) is inoculated with a single colony of
E. coli OP50 and incubated for 1 to 2 days at room temperature without aeration (i.e., without shaking). A small sample
(100 µl) of this culture is then spread onto a standard (60 mm)
agar plate of nematode growth medium, taking care not
to spread the bacteria all the way to the edges of the plate
(Stiernagle 2006); the remainder of the liquid culture can be
used for the next 1 to 2 weeks if stored at 4°C. Plates are incubated for 1 to 2 days until a bacterial lawn is visible; lawns
can be stored at 4°C up to 1 week, but must be warmed to
room temperature before use. Bacterial lawns are then inoculated with C. elegans by using a sterile needle or platinum wire
to transfer 10 worms from an old plate, placing the worms near
the center of the new plate. Worms can also be transferred by
cutting a small chunk of agar from an old plate (using a sterile
spatula) and placing it near the center of the new plate. This
“chunking” method can transfer hundreds of worms and is
commonly used to maintain a worm colony (Figure 1).
Plates with worms are maintained at between 16°C and
25°C and observed daily. Worms are transferred to new
plates when the bacterial lawn has been consumed, usually
about every 3 to 5 days depending on temperature (the higher
the temperature the faster the lawn consumption). A recently
developed chemically defined medium (C. elegans Maintenance Medium, CeMM) allows for a more standardized and
systematic manipulation of the nutrients that the nematode
receives (Szewczyk et al. 2003).
Liquid axenic cultivation of C. elegans enables automated culturing and experimentation with large-scale growth
and screening. The nematode can also be grown in liquid
media using E. coli OP50 as a food source, but this should be
done for only one generation as keeping worms in liquid can
lead to dauer formation.
For long-term storage, worms are initially frozen in liquid nitrogen or gradually cooled to −80°C (Stiernagle 2006)
and then transferred to a 15% glycerol liquid freezing medium in cryovials for indefinite storage. They can be thawed
as needed, but recovery rates are low, and unused portions of
vials should not be refrozen.
1Abbreviations that appear >3x throughout this article: dH O, distilled
2
water; ppt, parts per thousand
154
Figure 1 A nematode (Caenorhabditis elegans) being transferred,
under a dissecting microscope (only the base shown), from a culture plate using a platinum wire technique. The nematode itself is
too small to be seen, but five “chunks” are clearly visible on the
plate from a previous transfer of C. elegans to the plate. (Photograph courtesy of the laboratory of Diya Banerjee, Virginia Tech)
Health Challenges and Treatment
Bacterial and fungal contaminants are the only potential
problems with maintaining stocks of C. elegans. Contamination does not usually harm the worms but it can make it difficult to score phenotypes and complete transfers. Fungal
contamination can be removed by repeated transfer of worms
to fresh E. coli OP50 lawns, and bacterial contamination can
be removed by treating the plate with a 5% sodium hypochlorite solution (Stiernagle 2006), which kills the bacteria on the plate without affecting worms in the egg stage.
After treatment, the eggs are washed with dH2O and seeded
onto a fresh lawn of E. coli OP50. Contaminated plates,
plates whose lawns have been consumed, and plates with
surplus worms should be placed in a biohazard bag and autoclaved for disposal.
Aplysia californica
The California sea hare (Aplysia californica) is an opisthobranch mollusc in the order Anaspidea. Aplysia has long
been an important animal model for developmental and neurological studies as well as for three different types of learning: habituation, sensitization, and classical conditioning
(Capo et al. 2009). The organism’s large and relatively few
neurons facilitate the study of neuronal architecture, physiology, and control of instinctive and learned behaviors. Research on Aplysia has also led to a greater understanding of
memory formation and developmental biology.2
2Unless
otherwise indicated, information presented here is from the
University of Miami/NIH National Resource for Aplysia at the Rosenstiel
School of Marine and Atmospheric Science; available online (http://aplysia.
miami.edu). This and other websites cited in this article were accessed on
March 1, 2011.
ILAR Journal
Anatomy and Biology
Aplysia californica is an herbivorous marine gastropod that
is found naturally in the Pacific Ocean off the coast of
California. Unlike other gastropods, it does not have a large
external shell but only a small vestigial shell that protects the
heart and other internal organs; this lack of an obvious shell
gives the animal a slug-like appearance. Aplysia has two
large ventrally placed tentacles and, on top of the anterior
portion of its head, two sensory tentacles called rhinophores,
which have tactile and chemoreceptor functions.
Aplysia breathes by directing water over the gills using
modified flaps, called parapodia, on its back or mantle. After
the water has passed over the gills, it is expelled through a
posteriorly directed tube called a siphon. When disturbed,
the animal retracts its siphon into the mantle cavity, but can
modify this behavior through experience. Aplysia also has an
ink gland just under the shell and releases a purple ink when
threatened. The mottled reddish-brown color of A. californica
and the color of its ink are derived from pigments in the
algae the animal consumes.
Aplysia is hermaphroditic but does not self-fertilize. Under natural conditions it alternates being male and female on
different days during the spring and summer mating season.
The animal lays several thousand eggs in long string masses
about an hour after mating. The eggs hatch 7 to 8 days later
(Hirscha and Peretza 1984; Kriegstein et al. 1974) and enter
the initial free-swimming veliger phase for about 12 days,
during which the veligers become larvae. The larval phase
lasts until 30 days posthatch, and then the animal enters the
juvenile phase. Aplysia reach adulthood at about 94 days and
can live for an additional 6 to 10 months (Hirscha and Peretza
1984; Kriegstein et al. 1974). Adults weigh 500–1000 g
(Gerdes and Fieber 2006; Michael C. Schmale, Rosenstiel
School of Marine and Atmospheric Science, University of
Miami, personal communication, November 24, 2010).
Figure 2 California sea hares (Aplysia californica) feeding on cultured red algae (Agardhiella spp.). Fresh, chilled seawater is entering the tanks at the top of the photograph and draining via the
bottom of the tanks to remove detritus and fecal material in a flowthrough, nonrecirculating system. (Photograph courtesy of Michael
C. Schmale, National Resource for Aplysia)
fluorescent light (Schmale, personal communication, November 24, 2010).3
Drosophila melanogaster
The fruit fly (Drosophila melanogaster) has been used as a
model research organism for almost a century (Ashburn et
al. 2005; Lachaise 1988; Rubin 2000), originally in studies
of genetic inheritance. Today it remains the classic organism
for genetic research, but more recent studies have focused on
molecular and developmental biology, especially embryonic
development although there is also interest in the development of adult structures in the pupal stage (Bate and Arias
1993; Goldstein and Fyrberg 1994; Markow and O’Grady
2006).
Housing and Diet
Aplysia may be housed in glass, fiberglass, or plastic tanks
that provide for ease of cleaning and adequate circulation
and/or filtration of water (Figure 2). Water quality is critical for maintaining healthy cultures of Aplysia. They require either natural seawater or artificial seawater at a
salinity of 30 to 36 parts per thousand (ppt1) and a water
temperature of 13–16°C. The pH of the water should be
between 8.0 and 8.6 as is typical for most marine environments, and the oxygen content should be at least 95% or
8–9 mg/l.
Aplysia californica should be fed to satiation every 3
days. Its recommended diet in captivity is marine macroalgaes, principally red algae (e.g., Gracilaria spp., Agardhiella
spp., and Laurencia spp.), which must be cultured separately
from the Aplysia. Red algae are grown in nutrient (nitrate
and phosphate)-supplemented seawater at room temperature
with aeration and either direct natural light or full-spectrum
Volume 52, Number 2
2011
Anatomy and Biology
Drosophila, like other insects, have an exoskeleton to protect
their internal body parts, and their bodies are divided into
three segments: the head, the thorax, and the abdomen. The
head comprises compound eyes, a mouth, antennae, and
ocelli. The thorax is the middle segment, where the wings
and legs are attached, and the rearmost part of the fly is the
abdomen. The body is covered with small hairs, called sensilla, which act as sense organs and provide information
about touch, taste, smell, and sound to the fly’s nervous
system.
The typical life cycle of Drosophila lasts about 12 to 14
days (Matthews 1994). Flies are fertile and begin mating
3As
distinct from the other species discussed in this article, the scientific
literature does not include reports of diseases or treatments for Aplysia.
155
within 12 hours of eclosure (emergence of an adult insect
from a pupal case). Females can store sperm deposited internally by multiple males. Fertilized eggs are deposited directly on a food source. Within a day, they hatch and the
larva emerges. The first instar larva stage lasts about 24
hours, then the organism molts to a second instar larva and,
after about a day, the third instar larva stage, which lasts
about 3 days, after which the larva molts and forms a pupa.
The pupal stage lasts 5 to 7 days, during which the larva
undergoes metamorphosis to become an adult fly.
Drosophila are sexually dimorphic: females (about 2½
mm in length) tend to be larger than males and have a pointed,
banded abdomen; the abdomen of the male is more rounded
and generally darker than that of the female. Males also have
a darkened area on their front legs called the sex comb. Virgin
females are generally larger than mature females.
Drosophila are typically maintained in commercially available fly culture vials or small glass jars containing a commercial fly growth medium (Figure 3). The medium is mixed
with tap water and allowed to solidify and dry before flies
are added to the vial and the mouth of the container closed
with a cotton or foam plug (Dahmann 2010). Flies are incubated at room temperature (22–23°C) and then transferred as
necessary, for example to new vials containing fresh medium
as the old medium is consumed or in the case of overcrowding by adult flies.
to the culture medium at the bottom, at which point the foam
plug is removed and the new vial quickly placed so that the
two vials open into each other. Gentle tapping will prompt
the flies to fall from the old vial into the new. When all flies
are in the new vial, the two vials are separated and a foam
plug is quickly placed into the new vial.
If it is necessary to separate adult flies based on sex or
phenotypic characteristics, they must first be anesthetized,
using a commercially available anesthetic (e.g., chloroform
or ether), CO2, or chilling (Dahmann 2010). They can then
be separated using a dissecting microscope and transferred
onto a white card or into a Petri plate for observation and
separation. Chilled flies must be kept on a chilled glass Petri
plate (on ice) or they will start to wake up. A small paintbrush is an effective tool for gently separating the flies, after
which the desired populations (usually 5 to 10 flies) can be
placed for mating in vials containing fresh medium.
Genetic crosses require the collection of virgin females
(which must be collected within 8 hours of eclosure), which
are anesthetized and placed in separate vials for 2 to 3 days
of observation to verify their virginity (virgin females can
lay eggs but the eggs will not be fertile and will not hatch).
The virgins are then placed in a fresh vial with an equal number of males and allowed to mate. Breeding flies are removed
from the vial when the larvae begin to pupate. The adult offspring can then be anesthetized, examined, and enumerated
using a dissecting microscope. Surplus flies can be disposed
of by first anesthetizing them and then placing them in a jar
or vial containing 70% ethyl alcohol, isopropyl alcohol, or
mineral oil.
Culturing and Genetic Breeding
Health Challenges and Treatment
One method of propagating Drosophila is to transfer adult
flies from the old vial to a new one as the food source is
consumed. Gently tapping the old vial causes the flies to fall
There are few health challenges associated with Drosophila
cultures. Contamination with bacteria or fungus and infestation with mites are the most common problems. Bacterial
and fungal contaminations are prevented or eliminated by
using only freshly made fly medium and by frequently transferring flies to clean vials containing fresh food. Bacteria can
also be eliminated by adding a small amount (1%) of a 1:100
penicillin-streptomycin solution to the surface of the food
and allowing it to dry completely before flies are added.4
Mites are difficult to eradicate; in most cases cultures contaminated with mites must be destroyed to eliminate the
infestation.
Housing and Diet
Hermit Crabs
Figure 3 The fruit fly (Drosophila melanogaster) being cultured in
bottles with sponge plugs to prevent escape. Note the larvae (white),
pupae (dark), and empty pupal cases (transparent) on the sides of
the bottle where the larval stages undergo metamorphosis to the
adult fly. (Original photograph taken by Robert Cudmore)
156
Hermit crabs have been used for scientific research in the
fields of behavior, competition, and population studies, but
they are more often purchased as pets or display animals for
school classrooms (Fox 2000).
4Detailed
information about Drosophila culture media is available from the
Harvard Medical School Drosophila RNAi Screening Center (www.flyrnai.
org/DRSC-PRC.html).
ILAR Journal
There are two broad categories of hermit crabs: marine
(or aquatic) and terrestrial (Giwojna 2009; Nash 1976).
There are four families of aquatic hermit crabs: Diogenidae,
Paguridae, Pylochelidae, and Pylojacquesidae; and terrestrial hermit crabs are classified into two families: Coenobitidae and Parapaguridae. Of the more than 600 species of
hermit crabs, most are marine species; the most commonly
maintained are species of Clibanatus, Paguristes, and Calcinus. There is only one known species of true freshwater hermit crab, Clibanarius fonticola, which lives in a small lake
on Espiritu Santo Island, Vanuatu, in the South Pacific Ocean.
Land hermit crabs are more commonly used as laboratory
animals or kept as pets than marine hermit crabs, and the
most common types are various species of Coenobita (e.g.,
C. clypeatus, C. compressus, and C. rugosus).
Figure 4 A typical classroom habitat for terrestrial hermit crabs.
Note the wood bark substrate used for burrowing by the hermit crab.
Anatomy and Biology
Hermit crabs are not true crabs, as they lack a complete exoskeleton. Only the front portion of the animal’s body is covered with an exoskeleton; the rearmost portion is soft tissue
that is protected by a shell. Similar to other crabs, hermit
crabs have five pairs of legs, one of which has a large pinching claw. The innermost fifth pair of legs is small and serves
to help keep the crab inside its borrowed shell. Hermit crabs
molt their exoskeletons periodically as they develop, and
move to successively larger shells for protection of the soft
body as they grow in size.
Hermit crabs are most active nocturnally. They are also
social animals, preferring to be in groups of three or more
rather than singly housed. They live for up to 30 years in
their natural habitats and as long as 15 years in captivity.
Terrestrial Hermit Crabs
Housing and Husbandry
Both tropical and subtropical land hermit crabs are commonly kept in a glass aquarium with a ventilated lid and a
substrate of sand, wood chips, or coconut fiber (Figure 4).
The substrate should be deep enough that the crab can bury
itself when it needs to molt.
Land crabs require water, and their habitat (“crabitat”)
should contain two nonmetallic water dishes, one with fresh,
chlorine-free water and the other with saltwater. The water
dishes should be deep enough for the crab to bathe but not so
deep that it cannot get back out of the dish, and the water in
each bowl should be changed daily. The habitat should be
cleaned out weekly to remove waste and any food that the
crabs may have hidden, and dishes and “toys” should be
cleaned and rinsed well to completely remove any detergent
residue.
Land hermit crabs are omnivores and scavengers, and
may be fed fruits, vegetables, and meats as well as commercially available pelleted food. Fruits and vegetables should
be washed thoroughly to remove any pesticide or detergent
Volume 52, Number 2
2011
residue. Hermit crabs may also occasionally eat their own
exoskeleton after shedding.
Temperature and humidity levels are critical (Provenzano
1962). Land hermit crabs have modified gills that allow them
to breathe air, but these gills must be kept moist. Humidity in
the crabitat should be between 70% and 80%. It should never
be allowed to drop below 70%, and above 90% it can cause
unacceptable bacterial and fungal growth. A natural sponge in
a dish of freshwater can help to maintain humidity. Temperature can be maintained with an undertank heater.
Hermit crabs like “toys” such as driftwood and stones;
items made of metal should be avoided. Empty shells of varying sizes should be provided for the crabs to use after molting.
Health Challenges and Treatment
One of the primary problems with land hermit crabs is suboptimal husbandry. Environmental stressors such as incorrect temperature or humidity can cause a crab to discard its
shell and go naked, rendering it vulnerable to injury and infection. Crabs may also lose limbs due to aggression from
tank mates, traumatic injuries, or improper environmental
conditions, but with proper husbandry the limbs generally
regenerate when the crab molts.
Of particular concern is infestation by various species
of mites. To prevent or remove them, the aquarium and
substrate should be thoroughly cleaned and sterilized on
a regular basis (e.g., once a week). Infestations of dust
(Dermatophagoides sp.), grain (Acarus sp.), and house (Liponyssoides sp.) mites can be prevented by removing uneaten food daily and by cleaning the tank weekly. Substrate
and pieces of wood may be baked in an oven to sterilize
them, and a small vacuum should be used to remove all sand
and grit from the corners of the habitat.
Infestation by fungus gnats (Bradysia spp.) can also be a
problem for terrestrial hermit crabs. In addition to a thorough
cleaning of the habitat and replacement of the substrate,
fresh gnat traps made with a small jar containing apple cider
vinegar or a similar solution can be added to the crabitat.
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There are also predatory mites, Hypoaspis sp. (Cloyd 2010),
that attack gnats and other mites and are effective for eradicating such infestations on hermit crabs.
Marine Hermit Crabs
Marine hermit crabs breathe through gills and require a saltwater aquarium (Bookhout 1964; Dawirs 1979; Harms 1992;
Roberts 1971; Young and Hazlett 1978). The salinity of the
water should be between 30 and 40 ppt, with a temperature of
22–28oC. The aquarium should be large enough that the crabs
have space to roam, and there should also be rocks or corals
for the crabs to climb. As with land hermit crabs, extra shells
of various sizes should be provided to accommodate molting.
Aquatic species are omnivores and scavengers and will
eat detritus, algae, shed exoskeletons, and dead organic matter. There are also several commercially available pelleted
foods for marine hermit crabs.
Horseshoe Crab (Limulus polyphemus)
Horseshoe crabs have survived essentially unchanged for
more than 200 million years, with fossil records of members
of the family Limulidae dating back as far as 500 million years
(Shuster et al. 2003; Walls et al. 2002). They are the closest
living relatives of the ancient trilobites and are more closely
related to modern-day scorpions and spiders than to true crabs.
Today only four species of horseshoe crabs remain in various
regions of the world. The “American” horseshoe crab, Limulus polyphemus, occupies the Atlantic coast of North America
from Maine to the Yucatan peninsula (Shuster 1990). The
other three species—Tachypleus tridentatus, T. gigas, and
Carcinoscorpius rotundicauda—are found in coastal waters
of Asia from India to Japan to the Philippine Islands (Shuster
1990). Because limited information is available about the culture of these latter species, the remainder of this section concerns the North American species of horseshoe crab.
Limulus polyphemus is a unique marine invertebrate with
multiple commercial uses. Once an important resource for fertilizer and livestock feed, the horseshoe crab is now a bait in
commercial whelk and eel fisheries; a common exhibit animal
in public aquaria and classrooms to teach conservation and
environmental issues; a laboratory research animal model to
study the embryology, physiology, and function of marine invertebrates; and the primary source of Limulus amebocyte
lysate, which is widely used to detect endotoxins on or in
medical devices, implants, and vaccines (Berkson and Shuster
1999; Botton and Ropes 1987; Walls et al. 2002).
prosoma has a pair of laterally located compound eyes, a centrally located ocellus, a raised anterior-to-posterior midline
keel, and a hinge connecting the prosoma and opisthosoma.
The opisthosoma protects the soft internal organs and book
gills, and the animal uses the highly mobile telson to right
itself. Ventrally the horseshoe crab has a single, anterior pair
of modified chelicera, followed by five pairs of segmented
legs, and then posteriorly the brachial appendages. The latter
are attached to the underside of the opisthosoma and bear five
pairs of book gills that contain the gill leaflets used for respiration, osmoregulation, and propulsion during swimming.
Like most invertebrates, the horseshoe crab has an open circulatory system with hemolymph containing a copper-based
hemocyanin for oxygen transport (Bolling et al. 1976).
When horseshoe crabs in the wild reach sexual maturity
they mate during an annual spring migration to inshore spawning areas. Females may lay multiple clusters of 20,000 to
30,000 eggs on each spawning visit to the beach, after which
multiple males pass over the clusters and release sperm to fertilize the eggs. Horseshoe crabs also readily spawn in the laboratory—females release eggs on the bottom of the tank and,
as in the wild, males externally fertilize the eggs (Brown and
Clapper 1981; French 1979; Gonzalez-Uribe et al. 1991).
The small, green, round eggs have a relatively opaque
shell (chorion) and take approximately 1 month to hatch at
room temperature. A day or two before hatching, the outer
shell splits open, revealing a transparent membrane surrounding the developing larva. First-stage larvae resemble
adult horseshoe crabs except for the lack of a telson, which
shows up after the first molt. In the wild, juvenile horseshoe
crabs molt several times the first year and thereafter once
annually, approximately 16 to 18 times over the next 10 to
12 years as the animal eventually reaches sexual maturity.
(In the laboratory, where nutrition can be optimized and water temperatures can be held constant or elevated above natural cyclic temperatures, juvenile horseshoe crabs may have a
higher number of molts annually.) The lifespan of horseshoe
crabs is not well documented, but it is estimated at 18 to 22
years (Shuster et al. 2003).
During the molting process, the horseshoe crab’s external carapace splits along the cranial peripheral margin of the
prosoma, allowing the soft-bodied crab to crawl out between
the dorsal and ventral portions of the old outer shell. Until
the new shell hardens, the horseshoe crab is susceptible to
predation by horseshoe crabs and other marine species.
It is not possible to morphologically distinguish the sexes
until the horseshoe crab reaches the final (terminal) molt. Sexually mature males are generally smaller than adult females
and are readily identifiable by their first pair of legs, which are
modified during the last molt into large bulbous claspers for
grasping the female’s opisthosoma during mating.
Anatomy and Biology
The body of the horseshoe crab is dorsoventrally flattened
and divided into three sections: a frontal prosoma (cephalothorax) with an anterior flange; a hindbody opisthosoma (abdomen), and a posterior telson (tail). The dorsal surface of the
158
Husbandry
Horseshoe crabs have been maintained in captivity in a wide
range of systems from small glass aquaria to large fiberglass
ILAR Journal
tanks depending on the size and number of animals (Figure 5;
Smith 2006; Smith and Berkson 2005). As with all aquatic
organisms held in captivity, adequate mechanical and biological filtration is essential for maintaining the appropriate
environmental quality.
Mechanical filtration is provided by flosslike material,
static sumps, sand filters, or other devices that filter out particulate matter, and biological filtration is provided by intank box filters, outside power filters, canister filters, external
trickle columns, or suspended bead columns, all of which
provide expanded surface area for bacterial growth, which in
turn removes nitrogenous waste products. A sand or crushed
coral substrate on the bottom of the tank or aquarium allows
for the natural burrowing activity of the horseshoe crab and
may even help during the animal’s molting process. However, depending on the type of filtration, a substrate may or
may not be appropriate as it increases the difficulty of cleaning the systems and may harbor harmful pathogens. Some
systems may also have protein skimmers, ultraviolet (UV)
filtration, and ozonation that reduce the presence or risk of
potentially harmful bacteria and other compounds in the water column.
Horseshoe crabs are extremely tolerant of a wide range
of environmental conditions, but it is nonetheless important
to conduct adequate water testing to evaluate various water
quality parameters on a regular basis (weekly or more often
depending on the number of horseshoe crabs or other biomass being maintained). Horseshoe crabs have been reported
to exist in natural waters ranging in temperatures from −5°C
to 35°C, and they can tolerate salinities ranging from 5 ppt to
35 ppt, although larval stages do not survive well at low salinities (Nolan and Smith 2009; Smith 2006; Smith and
Berkson 2005). In the laboratory, juvenile and adult horseshoe crabs have been maintained long-term with water
Figure 5 A simple recirculating aquaculture system for maintaining horseshoe crabs (Limulus polyphemus) in captivity. Note the
filtration unit at the far end of the fiberglass holding tank, the submersible pump in the tank for circulating water through the filter,
and the plastic screening on the bottom of the tank to assist the
horseshoe crab in righting itself (originally published in Smith and
Berkson 2005).
Volume 52, Number 2
2011
temperatures between 15°C and 21°C and salinity around 27
ppt (Brown and Clapper 1981; French 1979; Gonzalez-Uribe
et al. 1991; Laughlin 1982; Smith and Berkson 2005).
Both natural seawater and commercially prepared synthetic marine salts can be effective, but with a natural marine
water source appropriate disinfection is necessary before use
to reduce the risk of introducing any potential infectious or
parasitic pathogens into the system. With recirculation systems, 25–30% of the marine water should be replaced every
3 to 4 weeks to reduce the amount of accumulated nitrates
and to replenish any ions or minerals removed from the water by the crab’s osmoregulatory processes (Nolan and Smith
2009; Smith 2006).
Aeration of the system is important for both the animal’s
respiration and the nitrification process of the holding system. Most aquaculture systems use water movement or supplemental air stones to ensure adequate aeration.
Horseshoe crabs in the wild feed on a variety of bivalve
molluscs, marine snails, marine worms, and other benthic
invertebrates. In captivity, larval horseshoe crabs can be fed
newly hatched live brine shrimp, and juveniles and adults are
commonly fed dead fish, squid, small crabs, clams, and frozen brine shrimp (Botton 1984; Smith and Berkson 2005).
The horseshoe crab also readily consumes commercially
prepared artificial shrimp and fish diets but their nutritional
value for the horseshoe crab is not known (Smith 2006).
Health Challenges and Treatment
There are only a few descriptions of the diseases and syndromes that affect horseshoe crabs. Noninfectious challenges
in captive horseshoe crabs include water quality problems
(e.g., ammonia toxicity, pH extremes, gas supersaturation,
and high turbidity), developmental problems (e.g., molting
problems of the shell, legs, or telson), and traumatic injuries
caused during collection, transport, or overcrowding during
captivity (Nolan and Smith 2009; Smith 2006). Physical
trauma can result in puncture wounds, crushing, and fractures of the exoskeleton. Hemorrhage from traumatic lesions
often appears significant but is rarely fatal.
Infectious challenges include algae, fungus, colonial and
filamentous cyanobacteria, Gram-negative bacteria, and a
variety of parasites (Smith 2006; Nolan and Smith 2009).
Lesions of the shell due to external pathogens are probably
the most common problem seen in horseshoe crabs and are
usually evident in discoloration or erosion of the carapace.
Algal, fungal, and bacterial infections may colonize and
penetrate the carapace, eyes, and gill surfaces and go on to
become systemic, involving the deeper tissues of the organs,
gills, and circulatory sinuses and resulting in extensive tissue
necrosis and death. Specific bacteria species isolated from
external lesions of the horseshoe crab include Oscillatoria,
Leucothrix, Vibrio, Flavobacterium, Pseudomonas, and
Pasteurella.
In addition, a variety of internal and external parasites
have been reported, including protozoans, a larval digenetic
159
trematode, a few nematodes, and several turbellarid worms.
Probably the most significant parasites are the turbellarid
worms, which reside between the gill leaflets, on the ventral
surface of the carapace, or on the appendages. These ectoparasites lay stalked cocoons on the external surfaces of
the gill leaflets, causing superficial lesions on the surface of
the gill tissue.
The most noticeable organisms are the ectocommensals—bryozoans, sponges, barnacles, blue mussels, lady
slippers, snails, oysters, and whelks—that frequently occur
on the external surfaces of the exoskeleton but seldom cause
any harm to the horseshoe crab. Freshwater baths (3–12
min), acetic acid baths (3–5% for up to 1 hour), and formalin
baths (1–1.5 ppt for up to 12 hours) have been used to remove external parasites and ectocommensals from the carapace of the horseshoe crab (Bullis 1994; Landy and Leibovitz
1983; Nolan and Smith 2009).
The pharmacokinetic profiles of oxytetracycline and
itraconazole have been reported in the American horseshoe
crab (for a full discussion of anesthesia and analgesia in invertebrates, see Cooper 2011, in this issue). A single oral or
intravenous (i.v.) dose of oxytetracycline (25 mg/kg or 50
mg/kg) yielded pharmacokinetic data suggesting that i.v.
would be the route of choice for continued maintenance of
drug-serum concentrations (Nolan et al. 2007). Data from a
study involving i.v. administration of a single dose of itraconazole at 10 mg/kg suggested that such a dose would be
necessary every 24 hours to maintain an effective treatment
level (Allender et al. 2008). Fluconazole, injected i.v. into
the cardiac sinus at a dose of 2 mg/kg body weight every 4
days for 6 treatments, has been reported for the treatment of
Aspergillus niger fungal infections (Tim Tristan, Texas State
Aquarium, personal communication, November 22, 2010).
Unfortunately, most treatments for bacterial or fungal
infections are generally ineffective, as the horseshoe crabs
become lethargic and anorectic before eventually dying from
the infection. Humane and rapid euthanasia is possible with
the injection of 1 to 2 ml of pentobarbital solution directly
into the dorsal cardiac sinus (Smith 2006).
Cephalopods
Cephalopods (the name originates from the Greek meaning
“head foot”) are an ancient group of animals that has been
developing through the ages with many earlier species now
extinct. They have significant commercial value both for human and animal consumption and for basic and biomedical
research. Research has largely focused on neurobiology
and behavior, with studies on sensory perception, angular acceleration, vision, central and peripheral nerve conduction,
neurotransmitters, and cells associated with the skin function.5 Cephalopods have been successfully maintained and
cultured for over 30 years at the National Resource Center
for Cephalopods (NRCC), a laboratory supported by the National Institutes of Health’s National Center for Research
Resources. The squid (Sepioteuthis lessoniana) has been
cultured through seven successive generations (Walsh et al.
2002); the pharaoh cuttlefish (Sepia pharaonis) through five
consecutive generations in the laboratory (Minton et al.
2001) using closed, recirculating water filtration systems;
and S. officinalis through seven generations in a recirculating marine system (Forsythe et al. 1994). However, as explained under Husbandry below, cephalopods are not the
easiest of animals to maintain and care for unless the user is
both knowledgeable about species-specific aquatic environment needs and experienced with water quality parameters
essential for marine invertebrates.
Scientists believe ancestors of modern cephalopods (subclass Coleoidea) diverged from the primitive externally
shelled Nautiloidea very early, perhaps in the Ordovician period some 438 million years ago, before the existence of
mammals and fish on this planet. Cephalopods were once
one of the dominant life forms in the world’s oceans, but
today there are only about 800 living species.6 The class Cephalopoda (phylum Mollusca) has two very distinct subclasses: Coleoidea (octopuses, squid, and cuttlefish) and
Nautiloidea (nautilus). This section focuses on octopuses,
squid, and cuttlefish (nautilus can be raised in captivity but
the author [JMS] is most familiar with the Coleoidea).
Anatomy and Biology
Cephalopods have some unique anatomical features and
should be considered highly specialized animals that have
successfully evolved through the ages. Cuttlefish, octopuses,
and squids are semelparous, meaning they grow rapidly to
sexual maturity, spawn once, and die (Hanlon 1987). The
animals’ lifespan in laboratories, depending on temperature
and nutrition, is usually 1 year (the nautilus, in contrast, may
live up to 20 years; Scimeca 2006). Cephalopods (excluding
the nautilus) are primarily top predators and very active in
hunting their prey. They are not good community tank inhabitants and the various species are best housed separately.
Most cephalopods have only a shell remnant that is either greatly modified or completely absent. In the subclass
Coleoidea, the shell is internal and reduced in size. In the
order Sepioidea (the cuttlefishes and bottle-tailed squids),
the calcareous chambered shell is present internally, functioning as a buoyancy organ. Representatives of this subclass
include Sepia and Euprymna; S. officinalis is the common or
European cuttlefish. In the order Teuthoidea (the shallowwater and oceanic squids), the shell is reduced to a chitinous “pen” (or gladus) that lies dorsally in the body. The body
is elongated and usually finned, with eight suckered
arms and two long tentacles; the Atlantic brief squid (Lolliguncula brevis) belongs to this group. The order Octopoda
5Information
Resources on Amphibians, Fish and Reptiles Used in
Biomedical Research; available online (www.nal.usda.gov/awic/pubs/
amphib.htm).
160
6Information
from the Cephalopod Page (www.thecephalopodpage.org).
ILAR Journal
(the octopuses) have a shell that is markedly reduced and
split into two lateral rods, and a globular body with or without fins; the California two-spot octopus (Octopus bimaculoides) is an example of this group and the most popular
species among aquarists that keep octopuses.
Organisms in the other cephalopod subclass, Nautiloidea,
have a heavy, external, chambered shell that may be straight
or coiled; nephridia; two pairs of gills; and numerous nonsuckered appendages. There are six living species worldwide
in two genera, the most commonly known of which is Nautilus pompilius, the chambered nautilus.
The integumentary system is composed of a delicate
1-cell-layer-thick epidermis consisting of columnar epithelial cells with a microvillous border and a deeper dermis
containing chromatophores, iridophores, and leucophores
(Hanlon and Messenger 1996). Because of the fine nature of
the skin, utmost care and careful handling of cephalopods
are mandatory to prevent tears or other trauma to the skin
membrane.
The respiratory system of cephalopods consists of wellvascularized gills suspended in the mantle cavity. The water
flows over the gill lamellae in the opposite direction of blood
flow, producing a countercurrent system that maximizes the
exchange of gases (Wells and Wells 1982). Cephalopods are
the only molluscs with a closed circulatory system, comprising a complex arrangement of vessels and cardiovascular tissues, a single systemic heart, two branchial hearts, vena
cavae, and auricles, all of which have a contractile function
for movement of the hemolymph. The blue-colored hemolymph is similar to that of the horseshoe crab and other invertebrates in that the oxygen-carrying pigment is the
copper-based hemocyanin.
Cephalopods have a large and well-developed nervous
system that is the most evolved of all invertebrates. Octopuses, cuttlefish, and squids are often considered the most
“intelligent” invertebrate species in part because of their mating and social behaviors and their social recognition and display patterns (Boal 2006; Hanlon and Messenger 1996).
Husbandry
Creating a laboratory environment for cephalopods that promotes species-typical behavior, reproduction, and optimal
health conditions is challenging. There is a wide variety of
seawater systems suitable for maintaining these animals in
captivity and there is considerable variation in their design
to accommodate laboratory location and species (Figure 6).
Tanks and holding systems are highly variable and adaptable; both open and closed seawater systems are available.
Piping should be of PVC, not copper, which is toxic to all
cephalopods. With any type of support system, close monitoring is necessary to maintain optimal water quality because
animal densities in captivity are often higher than those in
the wild.
There is a wide range of suitable housing composition
and substrates depending on the proposed use (e.g., culture
Volume 52, Number 2
2011
Figure 6 Numerous octopuses (Octopus bimaculoides) in a fiberglass tank with short lengths of PVC piping for housing and enrichment. Note the live shrimp (upper left) provided for food.
or display) (Forsythe and Hanlon 1980; Forsythe et al. 1991;
Hanlon and Forsythe 1985; Yang et al. 1989), and several
companies custom design and produce tanks to suit different
needs. Fiberglass composite works well and tanks can be fitted with viewing windows (acrylic is strong but scratches
fairly easily). Large animal water troughs have also been
modified for use with cephalopods. Tanks with exposed
metal should be avoided.
Creating an environment suitable for cephalopods is essential to successful culture. The temperature range for temperate species of cephalopods is 15–22°C and for tropical
species 25–32°C. In general, salinity should be 27–36 ppt, pH
7.7–8.2, ammonia and nitrites <0.1 mg/l, and nitrates <20.0
mg/l (Hanlon 1987; Lee et al. 1994a; Oestmann et al. 1997;
Sherrill et al. 2000). Water outside these parameters will likely
cause cephalopods to become stressed and result in greater
susceptibility to disease. Therefore, routine monitoring of water quality cannot be overemphasized.
Water should be conditioned and adjusted for the correct
salinity, and, in the case of open water systems, filtered and
contaminants removed. Care should be taken when using potable municipal water, which may be treated with potentially
toxic chlorines and chloramines. Evaluating makeup water is
essential for the culture of cephalopods and sending water
out for analysis for metals, organics, hardness, and possible
toxins is well worth the time and effort. Such analysis enables more precise adjustment of the animal’s holding water
and thus results in a suitable environment that both supports
the animal’s health and minimizes variability in research
outcomes.
After analysis of the makeup water, water filtration
should occur as follows: from the holding tank water passes
through (1) a protein skimmer (or foam fractionator) that
strips dissolved organic compounds (this equipment is essential as cephalopods produce and often expel abundant ink
when alarmed or startled); (2) a mechanical filter that removes particles down to 100 µm; (3) a high-grade activated
carbon and a biological filter, where ammonia is eventually
broken down to nitrites and nitrates; and (4) a UV sterilization unit before the water is returned to the animal holding
161
tank. Experience in correct sizing of the biological filter will
help as some cephalopods produce large amounts of ammonia that need to be reduced to the relatively nontoxic nitrates.
Systems with anaerobic filters to remove nitrates reduce the
amount of water that needs to be exchanged on a regular
basis. Water conditioning and treatment equipment may well
take up more space than the holding tanks themselves.
All cephalopods actively catch and eat a variety of live
prey items (Boletzky and Hanlon 1983; Castro and Lee
1994; Lee 1994; Nixon 1987). Squids and cuttlefish maneuver so they can strike their prey with extension of their tentacles and rapid body propulsion. Once trapped in the
tentacles, the prey is drawn toward the mouth and bitten by
the chitinous beak or “mandibles” and swallowed. Cephalopods, depending on their growth stage, can be fed live mysid
shrimp, grass shrimp, or fish fry of a nonoily fish species.
Experience in the laboratory has shown that live or fresh
frozen prey produces better growth and survival than artificial diets, although careful evaluation of live or fresh frozen
food is important as it can sometimes transmit unwanted
pathogens.
Health Challenges and Treatment
Most health problems in cephalopods result from either poor
water quality or traumatic injuries. Wild-caught animals are
particularly prone to physical and mechanical damage. As
mentioned above, the epidermal microvillous skin layer is
easily injured, and cuts and abrasions of the skin from nets
and handling are a frequent cause of morbidity and eventual
mortality. Captive cuttlefish can jet across the tank and damage the skin, eventually leading to septicemia and death
(Scimeca and Oestmann 1995). Mantle lesions are typically
preceded by abnormal swimming behavior and commonly
lead to secondary bacterial infections that affect multiple organ systems and rapidly lead to death (Sherrill et al. 2000).
Furthermore, if the force of the animals’ propulsion against
the side of the tank is great, as is often the case with cuttlefish, the result may be fractures of the internal cuttlebone.
Bacterial infections and septicemias in cephalopods are
commonly reported to be caused by various species of the
bacterium Vibrio (Hanlon and Forsythe 1990; Scimeca
2006). Common infections secondary to abraded or traumatic
skin lesions have been summarized in different cephalopod
species (Scimeca 2006).
Cannibalism is well documented in captive octopuses,
cuttlefish, and squids (Budelmann 2010). Stocking densities
and limited food supplies have been suggested as possible
etiologies for this problem. Autophagy has been reported in
Octopus vulgaris and there is some evidence that it may be
due to a released substance or possibly a virus or bacteria
(Budelmann 2010).
Various anesthetic compounds are effective for cephalods; as with other animals, the age, sex, size, and species
should be considered closely before performing anesthesia.
Magnesium chloride (MgCl2) is the anesthetic agent of
162
choice for cephalopods because it is easy to obtain, inexpensive, stable, nontoxic, and easy to prepare (Messenger et al.
1985). A standard solution of 75 g of MgCl2 dissolved in 1 L
of dH2O and mixed with 1 L of seawater has worked well
(Scimeca and Forsythe 1999). This one to one (1:1) mixture
can be used for surgical anesthesia and some invasive clinical procedures, a 1:3 or 1:4 dilution is adequate for handling
and examinations, and a 1:10 dilution induces mild sedation
for stress reduction and shipping.
Preliminary evaluation of MgCl2 in five different genera
of cephalopods showed effective anesthesia in all individuals (Scimeca 2006). The site of action for MgCl2 is thought
to be the central nervous system because stimulation of the
fin nerve in anesthetized cuttlefish elicits a motor response.
Thus, it is believed that MgCl2 works at the postsynaptic
membrane of the nerve-muscle junction in crustaceans and
vertebrates (Scimeca 2006).
The MgCl2 preparation can also produce a surgical plane
of anesthesia in octopuses with the addition of an ethanol
“push” consisting of a 1% solution (by volume) of ethanol
(e.g., 10 ml of ethanol is added to 1 L of the 1:1 seawater/
MgCl2 mixture). And researchers have described the use of
ethanol for anesthetic induction and maintenance during an
excisional biopsy and surgical repair of a mycotic skin lesion
on a European cuttlefish (Harms et al. 2006). The authors
reported an induction time of less than 1 minute with 3%
ethanol (30 ml/l) and successful anesthetic maintenance at
1.5% ethanol in seawater.
Benzocaine has been used in the giant Pacific octopus
for effective anesthesia at concentrations above 1,000 mg/l
and also as a compound for euthanasia. This method of euthanasia was reported to be a relatively rapid and humane
method at 3,500 mg/l compared to other euthanasia practices
(Barord and Christie 2007).
Treatment with antibiotics such as enrofloxicin (Baytril),
gentocin, nitrofuran, and metronidazole has been reported
(for a review of suggested dosages and routes of administration, Scimeca 2006). Because cephalopod skin is just 1 cell
layer thick and the animals have a large surface area, care in
the dosage and time of exposure for water baths is essential—
it is easy to overdose these animals with water bath antibiotics. An alternative technique used with cuttlefish involves
injecting live shrimp with 10 mg/kg enrofloxicin and immediately feeding this medicated food to the cuttlefish. If the
cuttlefish are not eating, i.v. injection using a 25 g or smaller
needle of 5 mg/kg enrofloxicin into the muscular portion of
the cephalic vein every 8 to 12 hours is also effective (Gore
et al. 2005).
As is true for all species of laboratory animals, training
of staff and research personnel is essential for successfully
maintaining cephalopods in the research laboratory. Appropriate care and husbandry are imperative, as is clinical
observation, especially given the semelparous life cycle
challenges in the management of cephalopods’ 12- to 14month lifespan (Sherrill et al. 2000). Experience in the
necropsy and pathology of cephalopods is also a key component to success when problems arise related to colony health.
ILAR Journal
Aquarium personnel, research investigators, and pathologists need to work together in seeking answers related to
health problems or deaths of unknown etiology.
Conclusions
Invertebrates have been used for many years in various research, teaching, and public display settings. As with other
laboratory animals, the proper culturing and care of invertebrates in captivity entails specific housing, environmental,
nutritional, and management requirements. A thorough investigation of these requirements is always advisable together with implementation of the specific recommendations
to ensure the humane care and welfare of these organisms.
Acknowledgments
SAS thanks the laboratory personnel of Diya Banerjee of
Virginia Tech for providing the image of C. elegans transfer,
and Michael C. Schmale of the University of Miami for providing both the image and information concerning the culture of Aplysia. JMS thanks Philip Lee, Philip Turk, John
Forsythe, Paul DeMarco, and Roger Hanlon for their help
and experience with cephalopods.
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ILAR Journal
Invertebrate Resources on the Internet
Stephen A. Smith
Abstract
The use of invertebrates in research laboratories, classroom
teaching, and public displays has greatly increased over the
past 20 years, accompanied by a corresponding increase in
the amount of online information and literature about invertebrates. This brief overview of Internet resources is intended
to aid both novice and experienced individuals in the search
for such information.
Key Words: annelid; arthropod; cephalopod; Internet resource;
invertebrate; mollusc; platyhelminth
I
nvertebrates consist of over 30 phyla of organisms and account for more than 95% of all animal species on this planet.
They are extremely diverse organisms, ranging from simple sponges to complex cephalopods, and have played a major
role in exhibits or displays at most aquaria, zoos, and nature
centers worldwide for over a century. Invertebrates have also
been used for a variety of research purposes, from basic biological studies to investigations of mechanisms of cell signaling to experimental models of cancer and human disease. In
the classroom, invertebrates have been used for teaching zoology, anatomy, physiology, embryology, genetics, husbandry,
and conservation to primary, secondary, and college students.
The popular use of invertebrates coupled with the emergence of the Internet has spawned an array of online resources
for invertebrate species. Conservation of invertebrate groups
such as marine corals and freshwater molluscs is a major
theme of a number of websites, but only a few websites convey serious consideration of the welfare of invertebrates. As
concern increases for the care, welfare, and enrichment of
invertebrates cultured and/or maintained in captivity, this
will undoubtedly change in the near future.
The following compilation of Internet links is designed
to be a practical resource for all who use and care for invertebrates, from high school biology teachers to experienced
researchers. It is not meant to be an exhaustive listing as the
Stephen A. Smith, DVM, PhD, is a professor of Aquatic, Wildlife, and
Pocket Pet Medicine in the Department of Biomedical Sciences and
Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine,
Virginia Polytechnic Institute and State University in Blacksburg.
Address correspondence and reprint requests to Dr. Stephen A. Smith,
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland
Regional College of Veterinary Medicine, Virginia Polytechnic Institute and
State University, Duck Pond Drive, Blacksburg, VA 24060-0442 or email
[email protected].
Volume 52, Number 2
2011
exponential expansion of the World Wide Web simply does
not allow the publication of such a document. Instead, this
resource should be used as a guide for locating preliminary
information, beginning with established organizations that
have an interest in expanding knowledge about invertebrates
and/or ensuring their appropriate care and use in the laboratory, classroom, or display environment.
The list is organized by general phylum and selected species, with a descriptive title, web address, and brief synopsis
provided for each website. Specific information about taxonomy, natural history, husbandry, culture, welfare, anatomy
and histology, physiology, ontogeny, genetics, conservation,
toxicology, educational resources, listservs, and databases
must be derived from each group or species link.
The following list includes only those links that are considered to be of substantive value, and not sites of primarily
commercial interest. However, due to the ever-changing
world of the Internet, with new websites springing up every
day and other sites no longer functional, the accuracy and
consistency of the links cannot be guaranteed.1 In light of the
massive amount of information about invertebrates on the
Internet, it is possible that some useful websites or links are
inadvertently missing from this compilation.
General Information
The Invertebrate Phyla – Earthlife.net
www.earthlife.net/inverts/an-phyla.html
Brief overview of all invertebrate phyla with links to information on specific groups.
Invertebrate – Wikipedia
http://en.wikipedia.org/wiki/Invertebrate
Foundational information on the classification, phyla, and
life history of invertebrates.
The Invertebrate Animals
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/I/
Invertebrates.html
Introductory information on the origin and evolution of
invertebrates with descriptions of major taxa.
Invertebrates – Animal Kingdom
http://animalkingdom.net/category/invertebrates/
General website on invertebrates with links to other websites
for specific groups.
1All
the websites listed here were accessed on March 11, 2011.
165
The Tree of Life Web Project
http://tolweb.org/tree/phylogeny.html
General information about the biodiversity, characteristics, and phylogeny of different groups of invertebrate
organisms.
Invertebrate Zoology Online – Lander University
http://webs.lander.edu/rsfox/invertebrates/
Anatomical descriptions, systematics, and references for
over 100 invertebrate species used in zoology teaching
and research.
Invertebrates – Biology4kids.com
www.biology4kids.com/files/invert_main.html
Educational resource on invertebrate classification for primary and secondary teachers and students.
Invertebrate Lesson Plans – Discovery Education
www.discoveryeducation.com/teachers/free-lesson-plans/
invertebrates.cfm
Lesson plans, materials, suggested readings, and links for
teaching students in grades 6-8 about invertebrates.
Dissections Online – University of Buffalo
http://ublib.buffalo.edu/libraries/asl/guides/bio/dissections.
html
Links to dissection guides for a variety of invertebrates (earthworm, crayfish, grasshopper, tick, roundworm, starfish,
clam, squid, and sponge).
Invertebrate Printouts – Enchanted Learning.com
www.enchantedlearning.com/subjects/invertebrates/index.
shtml
Information and printouts on invertebrates for primary and
secondary education.
Invertebrates – Electronic Zoo/NetVet
http://netvet.wustl.edu/invert.htm
Links relating to Drosophila, bees, arthropods, worms, and
crustaceans.
Catalogue of invertebrate specimens and images from the
Hawaiian and Pacific areas including a comprehensive
checklist of all marine invertebrates native to the area.
Invertebrates – Australian Museum
http://australianmuseum.net.au/Invertebrate-Collections
Catalogue of the museum’s invertebrate collections, which
primarily focus on crustaceans, worms, insects, spiders,
and molluscs.
GBR Explorer – ReefEd
www.reefed.edu.au/home/explorer
Interactive online guide to the invertebrates of the Great Barrier
Reef.
Invertebrates as Indicators – US Environmental Protection
Agency
www.epa.gov/bioiweb1/html/invertebrate.html
Information about invertebrates as biological indicators of
watershed health.
Information Resources on the Care and Use of Invertebrates –
US Department of Agriculture
www.nal.usda.gov/awic/pubs/invertebrates.htm
Online compilation of references related to the care and use
of invertebrates in the laboratory, classroom, or display
environment (AWIC Resource Series No. 8, 2000).
Invertebrate Link – Joint Committee for the Conservation of
British Invertebrates
www.royensoc.co.uk/InvLink/Index.html
Forum to advance the conservation of invertebrates in
the United Kingdom through the exchange of information among organizations and statutory bodies for the
development of strategies, policies, and best practice
guidelines.
BugLife – The Invertebrate Conservation Trust
www.buglife.org.uk/
Information about the conservation of European invertebrates.
Riverwatch – Aquatic Invertebrates
www.riverwatch.ab.ca/how_to_monitor/macroinvertebrates.
cfm
Information on collecting, identification, behavior, habitat
preference, and pollution tolerance of aquatic invertebrates.
Basic Information on the Major
Invertebrate Phyla
Marine Species Identification Portal – ETI BioInformatics in
the KeyToNature program
http://species-identification.org/index.php
Information, images, and links for numerous species of
marine invertebrates.
Introduction to Porifera – University of California Museum
of Paleontology
www.ucmp.berkeley.edu/porifera/porifera.html
Introduction to the simplest phyla of invertebrates, with
examples of sponge types.
Invertebrates – Smithsonian Institute National Zoological
Park
http://nationalzoo.si.edu/Animals/Invertebrates/default.cfm
Interactive resource for general information on invertebrates.
World Porifera Database
www.marinespecies.org/porifera/
Database of sponges worldwide and part of the global initiative of the World Register of Marine Species (WoRMS)
to register all marine organisms.
Invertebrate Zoology – Bishop Museum
www.bishopmuseum.org/research/natsci/invert/
Sponges of Britain and Ireland
www.habitas.org.uk/marinelife/sponge_guide/
166
Porifera (Sponges)
ILAR Journal
Searchable database of publications on all aspects of sponge
classification and biology (based on the 1992 book
Sponges of the British Isles: A Colour Guide and Working Document, by Ackers, Moss, and Picton; revised in
2007 by Picton, Morrow, and van Soest).
Palaeos Metazoa: Porifera
www.palaeos.com/Invertebrates/Porifera/Porifera.htm
Introduction to the taxonomic anatomy of sponges.
Sponge Barcoding Project
www.spongebarcoding.org/
Database that compares DNA signature sequences to
conventional morphological taxonomic characters of
sponges.
Cnidaria (Corals, Anemones, Box Jellies,
Hydroids, and Jellyfish)
Introduction to Cnidaria – University of California Museum
of Paleontology
www.ucmp.berkeley.edu/cnidaria/cnidaria.html
Introduction to Cnidarians including basic characteristics
and examples of this phylum.
The Hydrozoan Society
www.ucmp.berkeley.edu/agc/HS/
List of scientists working on hydrozoans and links to other
websites on hydrozoans.
The Hydrozoan Directory – Natural History Museum,
Geneva
www.ville-ge.ch/mhng/hydrozoa/hydrozoa-directory.htm
Introduction to hydrozoans.
Synopsis of the Medusae of the World – National Marine
Biological Laboratory, Plymouth, UK
www.mba.ac.uk/nmbl/publications/jmba_40/jmba_40.htm
Links to a PDF collection describing the medusae of the
world.
The Scyphozoan – University of California, Merced
http://thescyphozoan.ucmerced.edu/tSAug07.html
Resource describing biodiversity in the Scyphozoa (true jellyfish) including their behavior, biogeography, ecology,
and evolution.
Biogeoinformatics of Hexacorals – Kansas Geological
Survey
www.kgs.ku.edu/Hexacoral/
Resource for methods and links related to the taxonomy, biogeography, habitat characteristics, and environmental
correlates of Hexacorallia (corals and sea anemones) and
allied taxa.
Hexacorallians of the World – Kansas Geological Survey
http://hercules.kgs.ku.edu/hexacoral/anemone2/index.cfm
Bibliographic references and a synonymy list for each taxon,
published distribution of each species, and images of
many species.
Volume 52, Number 2
2011
Octocoral Research Center – California Academy of Sciences
http://research.calacademy.org/redirect?url=http://
researcharchive.calacademy.org/research/izg/orc_home.
html
Information about soft corals, gorgonians, and sea pens, with
references, links, and images.
ReefBase – The WorldFish Center
www.reefbase.org/
Official database of the Global Coral Reef Monitoring
Network (GCRMN) and the International Coral Reef
Action Network (ICRAN), with consolidated knowledge about coral reefs to facilitate analyses and monitoring of coral reef health, understanding of the
relationship between human activities and coral reefs,
enhancement of the quality of life of reef-dependent
people, and informed decisions about coral reef use and
management.
Coral Reef Information System – US National Oceanic and
Atmospheric Administration
http://coris.noaa.gov/
Information from NOAA about coral reef research, monitoring, and management activities, with emphasis on the
US states, territories, and remote island areas.
Coral Reef Conservation Program – US National Oceanic
and Atmospheric Administration
http://coralreef.noaa.gov/
Expertise and information from a wide array of NOAA programs and offices to reduce harm to and restore the health
of coral reefs, including deep-sea corals, by addressing
national threats and local management priorities through
conservation activities.
EnviroLink – International Coral Reef Information
Network
www.envirolink.org/resource.html?itemid=2002111115030
70.477551&catid=3
General information about coral reefs, with coral reef outreach tools and resources such as fact sheets, a searchable directory of over 600 coral reef organizations,
images, and a list of teacher’s resources.
CORAL – Coral Reef Alliance
www.coral.org/
Educational resources to help local communities become environmental stewards for coral reefs.
National Coral Reef Institute (NCRI) – Nova Southeastern
University Oceanographic Center
www.nova.edu/ocean/ncri/
Information on the assessment, monitoring, and restoration
of coral reefs through research and education.
Remote Sensing of Coral Reefs – US National Aeronautics
and Space Administration
http://oceancolor.gsfc.nasa.gov/SeaWiFS/reefs/
Remotely sensed observations for mapping of coral reefs
around the globe.
167
Jellyfish – Wikipedia
http://en.wikipedia.org/wiki/Jellyfish
General information on the anatomy, life cycle, and classification of freshwater and marine jellyfish.
Jellywatch
www.jellywatch.org/
Reports of sightings of jellyfish and other marine life.
Freshwater Jellyfish
http://freshwaterjellyfish.org/
General information on the species, distribution, natural
history, and life cycle of freshwater jellyfish.
Ectoprocta (Bryozoans and Marine Mats)
The Bryozoa Homepage
http://bryozoa.net/
Introduction to bryozoans with an index to the described
taxa and an extensive list of links to other sites devoted
to bryozoans.
Bryozoa Introduction – Smithsonian Institute Marine
Station at Fort Pierce
www.sms.si.edu/irlspec/IntroBryozoa.htm
Information on species diversity, habitat, reproduction, and
development.
BryoZone Home – The Field Museum of Chicago
www.bryozone.com/index.php
Interactive global reference for the collection, preservation,
and dissemination of scientific knowledge on marine, estuarine, and freshwater bryozoans.
Freshwater Bryozoans – Wright State University
www.wright.edu/~tim.wood/index.html
Information about freshwater bryozoans with links to other
bryozoan websites and collections.
Platyhelminthes (Flatworms Including
Turbellarians, Trematodes, and Cestodes)
Introduction to the Playthelminthes – University of California
Museum of Paleontology
www.ucmp.berkeley.edu/platyhelminthes/platyhelminthes.
html
Brief overview of the diverse group of flatworms.
Marine Flatworms of the World
www.rzuser.uni-heidelberg.de/~bu6/index.html
General information, characteristics, and examples of polyclad flatworms; excellent source of quality images of
the marine flatworms of the world.
Planarian Resources on the Web
www2u.biglobe.ne.jp/~gen-yu/plaweb_e.html
General resource for information on planarians.
Global Cestode Database
http://129.237.138.100/PEETII/uconnPEETII.html
Multinational collaboration for taxonomic characterization
of the Cestoda.
168
Tapeworms.org
www.tapeworms.org
Links to workshops and databases for cestodes.
Annelids (Ragworms, Earthworms,
and Leeches)
Introduction to the Annelida – University of California Museum
of Paleontology
www.ucmp.berkeley.edu/annelida/annelida.html
Links to the history, evolution, systematics, and morphology
of this phylum.
Annelid Research Resources
www.annelida.net/res-res.html
Links to websites on annelid taxonomy, research, and
resources.
Center for Annelida Resources – Illinois Natural History
Survey
www.inhs.uiuc.edu/~mjwetzel/mjw.inhsCAR.html
Links to websites on annelid taxonomy, collections, and
other resources.
Earthworms – The Backyard Nature Website
www.backyardnature.net/earthwrm.htm
General information on the importance, anatomy, reproduction, and behavior of earthworms.
Echinodermata (Starfish, Brittle Stars, Sea
Urchins, and Sea Cucumbers)
Introduction to the Echinoderms – University of California
Museum of Paleontology
www.ucmp.berkeley.edu/echinodermata/echinodermata.
html
General information, characteristics, and examples of
echinoderms.
The CAS Echinoderm Web Page – California Academy of
Sciences
http://research.calacademy.org/redirect?url=http://researcharchive.calacademy.org/research/izg/echinoderm/
index.html
Comprehensive list of links to research, collections, and resources on echinoderms.
The Echinoid Directory – Natural History Museum of
London
www.nhm.ac.uk/palaeontology/echinoids/index.html
Information on the morphology, taxonomy, and classification of echinoids.
Virtual Echinoderm Newsletter – Smithsonian National
Museum of Natural History
http://invertebrates.si.edu/echinoderm/
Lists of researchers, publications, theses, and links to echinoderm information.
ILAR Journal
Sea Urchin Embryology (SUE) – Stanford University
www.stanford.edu/group/Urchin/contents.html
Comprehensive site for information on the anatomy, reproduction, development, culture, and research ideas using
sea urchins in the classroom, with lesson plans, images,
and supporting material.
Molluscs (Snails, Octopuses, Squid, Clams,
Scallops, Oysters, and Chitons)
The Mollusca – University of California Museum of
Paleontology
www.ucmp.berkeley.edu/taxa/inverts/mollusca/mollusca.php
General background information on the phylum.
Check List of European Marine Mollusca (CLEMAM)
www.somali.asso.fr/clemam/index.clemam.html
Taxonomically oriented database of the marine Mollusca of
Europe and adjacent areas.
Living World of Molluscs
www.weichtiere.at/english/mollusca/index.html
General information on the major groups of molluscs, with
additional information, links, and images of select
species.
Snail-world
www.snail-world.com/
General information on the evolution, species, anatomy,
feeding, and reproduction of snails.
Apple Snail (Ampullariidae)
http://applesnail.net/
Information (with links and images) about the anatomy, biology, care, ecology, and distribution of tropical and subtropical freshwater “apple” snails.
Conus Biodiversity – University of Washington
http://biology.burke.washington.edu/conus/
Information and systematics of the marine gastropod genus
Conus.
Molluscs – Museum of Malacology, Cismar, Germany
www.hausdernatur.de/hncadr.htm
Extensive list of general malacological web pages, societies,
journals, museums, and informal groups.
Molluscan Neuroscience Homepage – Georgia State University
www.squishybrain.org/drupal/
Links to resources related to gastropod neuroscience.
Bibliographia Nudibranchia – Nudibranch Systematic Index
– University of California
http://repositories.cdlib.org/ims/Nudibranch_Systematic_
Index_second_edition/
List of all names in the nudibranch bibliography.
Phylogeny of Nudibranchia – California Academy of Sciences
http://research.calacademy.org/redirect?url=http://
researcharchive.calacademy.org/research/izg/nudibranchs/
Descriptions of species, with natural groupings of sea slugs
based on studies of anatomy and supplemented with molecular studies.
Sea Slug Forum – Australian Museum
www.seaslugforum.net/aplycali.htm
Information about the behavior, anatomy, and care of nudibranchs, bubble shells, and sea hares; with links to websites that provide taxonomic lists, information, and
images of sea slugs based on geographic location.
Opisthobranchia of the World
www.medslugs.de/Opi/Opisthobranchia.htm
Links to opisthobranch images on the Web arranged alphabetically and taxonomically.
Freshwater Mollusk Websites – University of Minnesota
http://fwcb.cfans.umn.edu/personnel/staff/hove/Mussel.
web.sites.html
Comprehensive list of links to information and research on
freshwater mussels.
Sea Slugs of Hawaii
http://seaslugsofhawaii.com/index.html
Introductory material (with images, maps, and references for
species identified from Hawaiian Islands) and links to
numerous websites that provide images and information
on sea slugs based on geographic location.
The MUSSEL Project Website (MUSSELp) – University of
Alabama
http://mussel-project.ua.edu/
Revised classification of the Unionoida (freshwater mussels), with a database of recent unionoid species and
genera described to date.
Arthropods (Insects, Arachnids, and
Crustaceans)
Freshwater Mussel (Unionoida) Genera of the World – Illinois Natural History Survey
www.inhs.uiuc.edu/~ksc/MusselGenera.html
Image-based website illustrating all genera of freshwater
mussels (Unionoida).
Mollusk Bioliography Database – Illinois Natural History
Survey
http://ellipse.inhs.uiuc.edu:591/mollusk/default.html
Searchable database of freshwater mollusc literature.
Volume 52, Number 2
2011
Introduction to the Arthropoda – University of California
Museum of Paleontology
www.ucmp.berkeley.edu/arthropoda/arthropoda.html
General overview on the largest phylum of animals, the
Arthropoda.
Entomology on the Worldwide Web – Colorado State
University
www.colostate.edu/Depts/Entomology/
Comprehensive list of web links to various resources in
entomology including bibliographies, collections, databases, publications, and research activities.
169
Insects and Entomology – Iowa State University
www.ent.iastate.edu/List/
Index of web links to various resources in entomology including bibliographies, collections, databases, publications, and research institutions and societies.
Polydesmida
www.polydesmida.info/
General information about the millipede order Polydesmida,
with links to information on the millipedes of Australia and
multipedes of Tasmania.
Center for Insect Science Education Outreach – University
of Arizona
http://insected.arizona.edu/uli.htm
Information for “Using Live Insects in Elementary Classrooms for Early Lessons in Life,” with lesson plans and
fact sheets on species identification, natural history, collecting, and impacts of insects on the ecosystem.
World List of Marine, Freshwater and Terrestrial Isopod
Crustaceans – Smithsonian National Museum of Natural
History
http://invertebrates.si.edu/isopod/
Comprehensive list of research, collections, and bibliographic information on isopods.
BugGuide.Net
http://bugguide.net/node/view/15740
Online community of naturalists who share information, observations, and images of insects, spiders, and other related creatures.
The Chironomid Home Page – Museum of Zoology University of Michigan
http://insects.ummz.lsa.umich.edu/~ethanbr/chiro/index.
html
Extensive list of material related to identification, checklists,
culture, researchers, and references to the Chironomidae,
or nonbiting midges.
Fleas of the World – Brigham Young University
http://fleasoftheworld.byu.edu/index2.htm
General information on the morphology, systematics, phylogeny, and evolution of fleas.
Fleas (Siphonaptera) – Zoological Institute, St. Petersburg,
Russia
www.zin.ru/Animalia/Siphonaptera/index.htm
General information on the morphology, systematics, ecology, and fauna of fleas.
The Insect and Spider Collections of the World – Bishop
Museum
http://hbs.bishopmuseum.org/codens/codens-r-us.html
Comprehensive list and links to major insect and spider collections of the world.
The Myriapoda (millipedes and centipedes) of North America –
North Carolina State Museum of Natural Sciences
http://nadiplochilo.com/index.html
General information, images, and references for millipedes
and centipedes.
The Myriapoda (millipedes and centipedes) – East Carolina
University
www.myriapoda.org/index.html
Introductory information on millipedes and centipedes.
Centre International de Myriapodologie (CIM) – Musée
National d’Histoire Naturelle de Paris
www.mnhn.fr/assoc/myriapoda/INDEX.HTM
Comprehensive information on the Myriapoda and Onychophora worldwide.
170
Forum Flusskrebse
www.forum-flusskrebse.org/
German-based forum for general information on various
crayfish species.
Garnelen
www.garnele-online.de/
German reader-based online magazine covering the taxonomy, culture, care, and breeding of invertebrates, especially freshwater shrimp.
Global Crayfish Resources – Carnegie Museum of Natural
History
http://iz.carnegiemnh.org/crayfish/
Worldwide checklists, databases, identification keys, and
links for freshwater crayfish.
The Crayfish by T.H. Huxley
www2.biology.ualberta.ca/palmer/thh/crayfish.htm
HTML resource of the International Scientific Series (Appleton and Co., New York) publication on the common
or European crayfish.
ZooGene: A DNA Sequence Database for Calanoid Copepods
and Euphausiids
www.ZooGene.org/
International database of DNA type sequences for calanoid
copepods and euphausiids.
Information Resources on the Care and Use of Insects – US
Department of Agriculture
www.nal.usda.gov/awic/pubs/Labinsects/labinsects.htm
Compilation of references for the culture, rearing, and utilization of insects in the laboratory and for public display
(AWIC Resource Series No. 25, 2005).
Terrestrial Invertebrate Taxon Advisory Group – American
Zoo and Aquarium Association
www.titag.org/
Information about the conservation of invertebrates and the
management needs of facilities that exhibit invertebrates
for educational purposes, with husbandry information
from various institutions for several species of invertebrates (e.g., beetles, cockroaches, tarantulas).
Caresheets – Amateur Entomologists’ Society
www.amentsoc.org/insects/caresheets/
General husbandry guidelines for a variety of insects, arachnids, and other invertebrates.
ILAR Journal
Invertebrate Husbandry Guidelines – Australian Society of
Zoo Keeping
www.aszk.org.au/husbandry.invertebrate.ews
List of guidelines for invertebrates in general and specific
guidelines for mantids, grasshoppers, stick insects, and
several beetles and butterflies.
Invert Care
www.invertcare.com/
General husbandry guidelines for a variety of scorpions,
tarantulas, centipedes, millipedes, and other invertebrates.
Invertebrate Paleontology
Invertebrate Paleontology – Cleveland Museum of Natural
History
www.cmnh.org/site/researchandcollections/Invertebrate
Paleontology.aspx
Information about the collection, preservation, and interpretation of invertebrate fossils including sponges, bryozoans, corals, molluscs, arthropods, and brachiopods.
Invertebrate Paleontology – Harvard University (www.mcz.
harvard.edu/Departments/InvertPaleo/)
Invertebrate Paleontology – Florida Museum of Natural History (www.flmnh.ufl.edu/invertpaleo/search.asp)
Invertebrate Paleontology – Natural History Museum of Los
Angeles County (http://ip.nhm.org/)
Invertebrate Paleontology – Yale University (www.yale.edu/
ypmip/index.html)
Images of invertebrate fossils from various research
collections.
Information about Invertebrates
Commonly Used in Research, Teaching,
and Display
The Nematode (Caenorhabditis elegans)
Caenorhabditis elegans – Wikipedia
http://en.wikipedia.org/wiki/Caenorhabditis_elegans
General information on the biology, ecology, and laboratory
uses of C. elegans.
WormBook
www.wormbook.org/
Comprehensive collection of peer-reviewed chapters covering topics related to the biology, physiology, genetics,
and development of C. elegans; plus WormMethods, a
collection of protocols for maintaining C. elegans.
WormAtlas Homepage – Albert Einstein College of Medicine of Yeshiva University
www.wormatlas.org/
Database of behavioral and structural anatomy, with detailed
illustrated atlas of C. elegans anatomy.
Volume 52, Number 2
2011
WormBase Homepage
www.wormbase.org/
Information about the genetics, genomics, and biology of
C. elegans, with genetic, physical, and sequence maps.
Caenorhabditis Genetics Center – University of Minnesota
www.cbs.umn.edu/CGC/
Information about specific strains of C. elegans.
Caenorhabditis elegans server – Southwestern Medical Center University of Texas
http://elegans.swmed.edu/
General information and links to research with C. elegans,
with comprehensive list of researchers, servers, and
institutional and commercial laboratories working with
C. elegans.
C. elegans Project – Wellcome Trust Sanger Institute
www.sanger.ac.uk/Projects/C_elegans/
List of genome sequencing projects for C. elegans and C.
briggsae.
C. elegans Gene Index – Dana Farber Cancer Institute
http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.
pl?gudb=elegans
Inventory of genes and their variants of C. elegans, with information about the functional roles of these genes and
their products.
WormClassroom – University of Wisconsin-Madison
www.wormclassroom.org/
Education portal for uses of C. elegans in teaching and research; with sample curriculum, instructional materials,
and multimedia resources for teachers, and sections for
students.
The California Sea Hare (Aplysia californica)
California Sea Slug – Wikipedia
http://en.wikipedia.org/wiki/California_sea_slug
Foundational information on the biology and laboratory uses
of A. californica.
The National Resource for Aplysia – University of Miami
http://aplysia.miami.edu/
Commercial resource and informational site for A. californica,
with information about efforts to promote science education and community awareness of the relevance of research
on this species.
Learning in Aplysia
http://brembs.net/learning/aplysia/
Information about the neurobiology of learning and memory
in Aplysia.
The Fruit Fly (Drosophila melanogaster)
Drosophila melanogaster – Wikipedia
http://en.wikipedia.org/wiki/Drosophila_melanogaster
171
General information on the morphology, development, genetics, life cycle, and laboratory uses of the fruit fly,
D. melanogaster.
Drosophila – WWW Virtual Library
www.ceolas.org/VL/fly/
List of resources, centers, laboratories, suppliers, and protocols for research on D. melanogaster.
FlyBrain – University of Arizona
http://flybrain.neurobio.arizona.edu/
Online atlas and database of the central and peripheral nervous system of D. melanogaster, with information on
different anatomical structures, developmental stages,
and imaging techniques.
General teaching reference for D. melanogaster with a guide
to its care, maintenance, and genetic manipulation.
Fruit Fly – Western Kentucky University
bioweb.wku.edu/courses/Biol114/virgin_flies.asp
General secondary teaching reference for the development
of D. melanogaster.
J-fly
http://jfly.iam.u-tokyo.ac.jp/
Japanese-based data depository for Drosophila researchers,
with images, movies, manuals, and protocols.
Hermit Crabs
FlyBase – Consortium of four universities
http://flybase.org/
Database with descriptions of the genes and genome of the
fruit fly D. melanogaster.
Hermit crab – Wikipedia
http://en.wikipedia.org/wiki/Hermit_crab
General information on species, anatomy, and biology of
hermit crabs.
FlyView – Universität Münster
http://flyview.uni-muenster.de/
Image database of Drosophila development and genetics;
compatible with FlyBase for describing the genes and
genome of the fruit fly.
Hermit Crabs.org (www.hermitcrabs.org)
Hermit-Crabs.com (www.hermit-crabs.com)
Hermit Crab Habitat (www.hermitcrabhabitat.com)
Information about the care of terrestrial hermit crabs.
FlyMove – Universität Münster
http://flymove.uni-muenster.de/
Online resource for university students and teachers to study
the development of D. melanogaster, through a variety
of images, movies, and interactive media.
The Interactive Fly – Society for Developmental Biology
www.sdbonline.org/fly/aimain/1aahome.htm
Comprehensive online guide to D. melanogaster development, with numerous links to other Drosophila sites as
well as “The Atlas of Drosophila Development” originally published by Cold Spring Harbor Laboratory Press
(1993).
Drosophila Information Service – University of Oklahoma
www.ou.edu/journals/dis
Online archived journal devoted to descriptions of new mutants, techniques, research, and teaching exercises with
D. melanogaster.
Bloomington Drosophila Stock Center – Indiana University
flystocks.bio.indiana.edu/bloomhome.htm
Commercial source for obtaining specific strains of D. melanogaster for research.
Flies in Space – US National Aeronautics and Space
Administration
http://quest.nasa.gov/projects/flies/index.html#
Website designed for 5th–8th grade students with information about NASA’s life sciences research project focusing on D. melanogaster.
Drosophila – University of Arizona
http://biology.arizona.edu/sciconn/lessons2/Geiger/prelude.
htm
172
The Horseshoe Crab (Limulus polyphemus)
Horseshoe Crabs – Wikipedia
http://en.wikipedia.org/wiki/Horseshoe_crab
General information on the classification, anatomy, fisheries,
and blood use of the four living species of horseshoe crabs.
The Horseshoe Crab – The Ecological Research and Development Group (http://horseshoecrab.org/)
Horseshoe Crab – US National Oceanic and Atmospheric
Administration (www.ceoe.udel.edu/horseshoecrab/)
Horseshoe Crabs: “A Living Fossil” – Maryland Department
of Natural Resources (www.dnr.state.md.us/education/
horseshoecrab/)
Limulus polyphemus – Smithsonian Marine Station at Fort
Pierce (www.sms.si.edu/irlspec/limulu_polyph.htm)
General information on the natural history, biology, anatomy, conservation, ecology, habitat, medical uses, research,
fisheries management, and conservation of the Atlantic or
American horseshoe crab, L. polyphemus.
Horseshoe Crab (video) – Journal of Visualized
Experiments
www.jove.com/index/details.stp?id=958
Link to video journal (for biological research) entitled “Blood
Collection from the American Horseshoe Crab, Limulus polyphemus,” demonstrating bleeding of a horseshoe
crab (Armstrong and Conrad, 2008, J Vis Exp 20).
Horseshoe Crab
www.fao.org/docrep/field/003/AB736E/AB736E02.htm
General information on the biology, habitat, and culture of
the Asian horseshoe crab, Tachypleus tridentatus.
ILAR Journal
Cephalopods (Octopuses, Squid, Cuttlefish,
and Nautiluses)
Cephalopod – Wikipedia
http://en.wikipedia.org/wiki/Cephalopod
General information on the classification, distribution, anatomy,
life cycle, reproduction, and development of cephalopods.
Cephalopod – Tree of Life Web Project
http://tolweb.org/Cephalopoda
General information on the classification, characteristics,
and references of cephalopods.
The Cephalopod Page – Waikiki Aquarium and University of
Hawaii
www.thecephalopodpage.org/
General information, images, links, high school lesson plans,
references, and resources for octopuses, squid, cuttlefish,
and nautiluses.
CephBase – University of Texas Medical Branch and
Dalhousie University
www.cephbase.utmb.edu/ or www.cephbase.dal.ca/
Database-driven interactive website with taxonomic data, life
history, distribution, predator and prey data, images, videos,
references, and fisheries information for all species of cephalopods (octopus, squid, cuttlefish, and nautilus).
Cephalopods – Smithsonian Institute National Museum of
Natural History
http://invertebrates.si.edu/cephs/
Multimedia resource of videos that supplement traditional scientific publications on squids, octopods, and their relatives.
Cephalopods – Smithsonian Institution Research Information System (SIRIS)
http://siris-bibliographies.si.edu/ipac20/ipac.jsp?profile=
biball (keyword = cephalopod)
Comprehensive research database of over 4,500 publications
on cephalopods and cephalopod-related subjects.
Cephalopods of the World – Food and Agriculture Organization of the United Nations
www.fao.org/docrep/009/a0150e/a0150e00.HTM
Link to online publication, “An Annotated and Illustrated
Catalogue of Cephalopod Species Known to Date,” eds.
Jereb and Roper (FAO Species Catalogue for Fishery
Purposes 4, vol 1, 2005).
Cephalopods of the World – Food and Agriculture Organization of the United Nations
www.fao.org/docrep/009/ac479e/ac479e00.htm
Link to online publication, “An Annotated and Illustrated Catalogue of Species of Interest to Fisheries,” eds. Roper,
Sweeney, and Nauen (FAO Fisheries Synopsis 125, vol 3,
1984).
Cephalopod International Advisory Council
www.abdn.ac.uk/CIAC/
International resource for information on cephalopod biology, fisheries management, and research.
Volume 52, Number 2
2011
The EuroSquid World Wide Web Page – University of
Aberdeen
www.abdn.ac.uk/eurosquid/
General information, research projects, analysis of European
cephalopod stocks, references, abstracts, and squidrelated links.
National Resource Center for Cephalopods – University of
Texas Medical Branch
www.gulfbase.org/organization/view.php?oid=nrcc
Link to a program that historically provided squid, cuttlefish
and other cephalopods, as well as cells and tissues to researchers in the biomedical research community.
The Octopus News Magazine Online (TONMO)
www.tonmo.com/
Online forum for issues pertaining to the care, breeding, and
housing of octopuses, squids, and cuttlefish.
Squid-world – BioExpedition Publishing
www.squid-world.com/
General information on the evolution, species, anatomy,
feeding, and reproduction of squids.
Amazing Cuttlefish
www.users.on.net/~jamesmosby/cuttlefish/index.html
Information about the unique features, anatomy, and life
cycle of the cuttlefish.
Invertebrate Associations and Societies
American Association of Professional Apiculturists – www.
masterbeekeeper.org/aapa/
American Malacological Society – www.malacological.org/
index.php
American Society of Parasitologists – http://asp.usnl.edu/
American Tarantula Society – http://atshq.org/
Australian Coral Reef Society – www.australiancoralreefsociety.
org/
Coleopterists Society – www.coleopsoc.org/default.asp
Conchologists of America – www.conchologistsofamerica.
org/home/
Crustacean Society (Virginia Institute of Marine Science) –
http://web.vims.edu/tcs/?svr=www
Earthworm Society of Britain – www.earthwormsoc.org.uk/
Entomological Society of America – www.entsoc.org/
home
Entomological Society of Canada – www.esc-sec.org/
Freshwater Mollusk Conservation Society – http://mollusk
conservation.org/
International Association for Aquatic Animal Medicine (including invertebrates) – http://iaaam.org/
International Association for Neuropterology – www.
neuroptera.com/olinks.html
International Association of Astacology – http://iz.carnegie
mnh.org/crayfish/iaa/index.htm
International Bee Research Association – www.masterbee
keeper.org/aapa/
173
International Bryozoology Association – http://bryozoa.net/
iba/index.html
International Society of Arachnology – www.arachnology.org/
International Society for Invertebrate Morphology – http://
zoologi.snm.ku.dk/english/Forskning/Invertebrates/isim/
International Society for Invertebrate Neurobiology – www.
neurobiologie.fu-berlin.de/ISIN%20Info.htm
International Society for Reef Studies – www.coralreefs.org/
International Society of Invertebrate Reproduction and Development (ICIRD) – www.isird.org/index.html
International Society of Myriapodology and Onychophorology – www.mnhn.fr/assoc/myriapoda/index.htm
Lepidopterist Society – www.lepsoc.org/
Lobster Conservancy – www.lobsters.org/
National Shellfish Association – http://shellfish.org/
174
North American Dipterists Society – www.nadsdiptera.org/
Orthopterists’ Society – http://140.247.119.225/OrthSoc/
Royal Entomological Society – www.royensoc.co.uk/
Society for Invertebrate Pathology – www.sipweb.org/
Society of Nematologists – www.nematologists.org/
Systematic and Applied Acarology Society – www.nhm.
ac.uk/hosted_sites/acarology/saas/
Unitas Malacologica (International Society for Malacology) –
www.unitasmalacologica.org/index.html
World Dragonfly Association – http://ecoevo.uvigo.es/
WDA/dragonfly.htm
World Federation of Parasitologists – www.wfpnet.org/
page_regionalmembers.php
Xerces Society for Invertebrate Conservation – www.xerces.
org/
ILAR Journal
Pain and Suffering in Invertebrates?
Robert W. Elwood
Abstract
Defining Pain versus Nociception
All animals face hazards that cause tissue damage and most
have nociceptive reflex responses that protect them from
such damage. However, some taxa have also evolved the
capacity for pain experience, presumably to enhance longterm protection through behavior modification based on
memory of the unpleasant nature of pain. In this article I
review various criteria that might distinguish nociception
from pain. Because nociceptors are so taxonomically widespread, simply demonstrating their presence is not sufficient. Furthermore, investigation of the central nervous
system provides limited clues about the potential to experience pain. Opioids and other analgesics might indicate a
central modulation of responses but often peripheral effects
could explain the analgesia; thus reduction of responses by
analgesics and opioids does not allow clear discrimination
between nociception and pain. Physiological changes in
response to noxious stimuli or the threat of a noxious
stimulus might prove useful but, to date, application to invertebrates is limited. Behavior of the organism provides
the greatest insights. Rapid avoidance learning and prolonged memory indicate central processing rather than
simple reflex and are consistent with the experience of pain.
Complex, prolonged grooming or rubbing may demonstrate
an awareness of the specific site of stimulus application.
Tradeoffs with other motivational systems indicate central
processing, and an ability to use complex information suggests sufficient cognitive ability for the animal to have a
fitness benefit from a pain experience. Available data are
consistent with the idea of pain in some invertebrates and
go beyond the idea of just nociception but are not definitive.
In the absence of conclusive data, more humane care for
invertebrates is suggested.
ll species of animal are susceptible to a variety of naturally occurring hazards that can cause tissue damage.
Sharp objects, such as teeth or mandibles of predators,
or defensive thorns or spines in plants or animals, are common.
Chemicals, blunt objects, and thermal extremes may also cause
damage. Some plants (e.g., nettles) and animals (e.g., hymenoptera and coelenterates) have specialized structures that
are sharp, penetrate the tissues, and transfer noxious, potentially damaging chemicals. However, animals have mechanisms that enhance their ability to maintain the integrity of their
tissues through the detection of noxious stimuli and action to
get away from them and/or minimize their deleterious effects.
The sensory systems that respond to noxious stimuli
and mediate protective reflexes are termed nociceptors
(Sherrington 1906). Nociception is defined as “the neural
processes of encoding and processing noxious stimuli” (Loeser
and Treede 2008, 475) or the detection and reaction “to
stimuli that may compromise their integrity” (Besson and
Chaouch 1987, 67). Thus nociception is the perceptual
mechanism coupled with the organization of responses that
typically take the animal away from the stimulus or at least
are effective in terminating the perception. For example,
Drosophila larvae attacked by a parasitoid wasp respond by
rolling toward the stimulus, which causes the wasp’s ovipositor
to pull out and the wasp to leave (Hwang et al. 2007).
By contrast, the definition of pain in humans is “an unpleasant sensory and emotional experience associated with
actual or potential tissue damage, or described in terms of
such damage” (IASP 1979, 250). Various definitions have
been used with respect to animals—for example, “an aversive sensory experience caused by actual or potential injury
that elicits protective motor and vegetative reactions, results
in learned avoidance and may modify species-specific behaviors, including social behavior” (Zimmerman 1986, 1). A
shorter definition that excludes pain assessment criteria is
“an aversive sensation and feeling associated with actual or
potential tissue damage” (Broom 2001, 17).
It is clear that nociception is central to the concept of
pain, as without it the experience of pain is unlikely. However, simply observing a nociceptive ability does not demonstrate pain. Nociception per se is an involuntary rapid reflex
response and lacks the negative emotional response or feeling associated with pain (Bateson 1991; Broom 2001). Indeed, in humans the reflex response to touching something
hot precedes the experience of pain.
Key Words: behavior; discrimination learning; invertebrate; morphology; nociception; pain; physiology; stimulus
avoidance
Robert W. Elwood, PhD, is a professor of animal behavior at the School of
Biological Sciences of Queen’s University in Belfast, Northern Ireland.
Address correspondence and reprint requests to Prof. Robert W. Elwood,
School of Biological Sciences, Medical Biology Centre, Queen’s University
Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland or email
[email protected].
Volume 52, Number 2
2011
A
175
Although the distinction between nociception and pain is
widely accepted there are semantic issues that may cloud the
issue. The term “pain perception” is frequently used in pain
studies (e.g., Braithwaite 2010; Sneddon 2009) and nerve
fibers are said to “transmit pain” (Weary et al. 2006). Even in
studies that are overtly about nociception rather than pain,
nociception is described as “pain sensing” and ascending
tracts in the vertebrate spinal cord are said to carry “painful
sensory information” (Hwang et al. 2007) or “pain information” (Sneddon 2009). Larval Drosophila that lack a particular functional gene fail to roll away from thermal or
mechanical stimuli and the mutation has been called “painless” even though the gene function is in the activation of
transducer channels by which a neural signal results from
physical stimuli (i.e., a key part of nociception) (Tracey et al.
2003). The use of these terms blurs the critical distinction
between nociception and pain.
I prefer to use terms such as “pain experience” to denote
an internal awareness, coupled with a negative emotional
state or feeling, that results from perception of actual or
potential tissue damage. It is the damage that is perceived.
There is no “pain” to be perceived and the information carried to the brain in vertebrates is not in itself “painful.” The
pain results from a powerful, unpleasant emotion that is part
of, or coupled with, a strong motivation to terminate the experience that results from neural signals about tissue damage.
Clarity about the definitions of and distinctions between
nociception and pain is essential for determining whether
pain occurs in particular groups of animals.
Function and Evolution of Pain
As noted above, nociception provides a means of detecting
and escaping from a stimulus that might continue to cause
damage in the absence of action. Thus there are clear benefits in nociception and, presumably, they outweigh the costs
of developing, maintaining, and using the system. What further advantage is gained by having an additional system that
enables the experience of nociceptive inputs as an unpleasant emotion? It may be that the “emotional” component provides a long-lasting motivation that enables the animal to
better maintain its tissue integrity (Bateson 1991).
A nociceptive response may be organized as a reflex
(Sneddon et al. 2003) but may not be associated with a lasting memory and motivational change. Pain, on the other
hand, might induce a long-term memory and be coupled
with learning to avoid situations that gave rise to the original
pain experience (Bateson 1991). The greater the tissue damage in the original experience the greater may be the unpleasant emotional response and the greater the motivation
to avoid it in the future. Thus pain experience has a longerlasting effect and protects the animal from future damage in
a more effective manner than does nociception alone.
Nociceptive abilities are found in most of the major
animal phyla and thus are presumably a product of very early
evolution. They clearly predate the Cambrian “explosion,” a
176
period approximately 530–550 million years ago during
which the major modern phyla evolved (Budd and Telford
2009). The next step in the development of pain was probably a link between nociception (with the associated reflex
response) and a longer-term motivational change (with central processing and memory). But in which groups did this
development take place and when? Some researchers are
confident that it occurred only in vertebrates and that it is
evident in fish and also perhaps the more primitive lamprey
(Braithwaite 2010; Sneddon 2009). If correct then pain has
been around since the Cambrian explosion because that is
when many of the major invertebrate phyla evolved. But if it
is accepted that pain occurs in fish and lamprey then the
question is whether it is a novel development in the vertebrates or if the ability to experience pain predated the split of
the vertebrates from the ancestors of some other phyla and
could thus be present in some extant invertebrate groups?
Alternatively, if there is an advantage in experiencing pain, it
could be that it has evolved on more than one occasion
(Elwood et al. 2009).
How Can Pain Be Identified?
The interest in the potential of invertebrates to experience
pain lies in the quest to understand and improve welfare, as
humans generally seek to avoid causing pain or suffering to
animals (Broom 2007). If an animal responds to a noxious
stimulus in an adaptive fashion via a nociceptive reflex and
without any unpleasant experience, then welfare concerns
are diminished. The key objective of this article is to examine the evidence that some invertebrates may or may not experience pain.
Inferring feelings or mental states in animals is fraught
with difficulty (Dawkins 2006). A common approach is to
use argument by analogy (Dawkins 1980; Sherwin 2001): if
an animal responds to a potentially noxious stimulus in a
manner similar to that observed to the same stimulus in humans then it is reasonable to argue that the animal has had an
analogous experience (Sherwin 2001). However, Sherwin
(2001) notes differences in the acceptance of this argument
depending on the species rather than the behavior: observers
of a dog or primate writhing in response to an electric shock
accept that the animal is experiencing pain, whereas much
the same response in an invertebrate is often dismissed as
irrelevant to the question of pain. He suggests a more symmetrical approach when comparing vertebrates with invertebrates, with consistent acceptance or rejection of the
argument by analogy (Sherwin 2001). However, empathy for
invertebrates is typically low and some researchers believe
that it would be “inconvenient” if these animals were believed to feel pain (Kellert 1993).
Coupled with analogy, various criteria have been proposed
as collectively having the potential to demonstrate pain in mammals (Bateson 1991) and have been applied to pain in amphibians (Machin 1999; Stevens 2004), fish (Sneddon et al. 2003),
and various invertebrates (Broom 2007; Elwood et al. 2009;
ILAR Journal
Fiorito 1986; Sherwin 2001). In the following sections I consider criteria that are similar (but not identical) to those used
in previous work (Bateson 1991; Broom 2007; Sherwin
2001), assessing the presence of the following features:
•
•
•
•
•
•
•
•
suitable receptors;
a suitable central nervous system;
responsiveness to opioids, analgesics, and anesthetics;
physiological changes;
avoidance learning;
protective motor reactions;
tradeoffs between stimulus avoidance and other activities; and
cognitive ability and sentience.
Suitable Receptors
Sea anemones respond to mechanical stimuli and to the
stings of other anemones but not to thermal stimuli (Mather
2011, in this issue). Annelids have nociceptors that respond
to acid, capsaicin, and heat (although the sensitivity to acid
is lower than that seen in vertebrates; Smith and Lewin 2009)
and cells that respond to touch and pressure (Nicholls and
Baylor 1968). Nociceptors and nociceptive behavior have
been described in molluscs; for example, the snail (Cepaea
nemoralis) responds to a hot plate at >40°C by lifting the
anterior portion of its foot (Kavaliers et al. 1993). In Aplysia
californica (Castellucci et al. 1970; Smith and Lewin 2009),
once the stimulus threshold is reached, nociceptors increase
firing in line with subsequent increase in stimulus strength
and show maximal activity with the crushing or tearing of
tissues.
Nociceptive systems have been described in particular
detail in the nematode (Caenorhabditis elegans) and fruit
fly (D. melanogaster) (reviewed by Smith and Lewin 2009)
and molecular tools have been applied to elucidate the detailed development and functioning of their nociceptors
(Goodman 2003; Tobin and Bargmann 2004). Nociceptive
neurons of Drosophila larvae have multiple dendritic
branches with naked endings attached to epidermal cells
(Hwang et al. 2007); several classes of such multidendritic
(md) neurons have been described but not all are involved in
nociception. If all these md neurons are rendered inactive in
particular genetic mutants, the larvae fail to respond to noxious stimuli, showing that at least one is nociceptive (Tracey
et al. 2003). Selective silencing of particular classes of md
neurons, however, showed that one class serves as the primary nociceptive system and that the silencing of this class
of neurons eliminates the rolling response of larvae to thermal stimuli (Hwang et al. 2007). The nociceptive neurons
are also responsive to attacks by parasitic wasps attempting
to insert their ovipositor (as described above; Hwang et al.
2007), indicating that they are polymodal, as are those of
vertebrates (Goodman 2003).
The common theme of these studies is the ability of a
wide range of taxa to detect noxious stimuli and to translate
Volume 52, Number 2
2011
them into neuronal signaling (Tobin and Bargmann 2004).
The systems involved are complex but conserved across
markedly different taxa, as is evident from the use of Drosophila in drug discovery for application in vertebrates, especially humans (Manev and Dimitrijevic 2005). In addition,
these systems show adaptive, temporary, heightened, and
reduced sensitivity and these features are also conserved
across phyla (Babcock et al. 2009). One recent study, however, failed to detect nociceptors in decapod crustaceans and
also noted little ability to respond to noxious stimuli (Puri
and Faulkes 2010) despite organized responses to noxious
chemical and electrical stimuli noted in other studies (Barr
et al. 2007; Elwood et al. 2009; Elwood and Appel 2009).
Vertebrates have a variety of nociceptive fibers, some
myelinated and others not. By contrast, those in invertebrate
groups are only unmyelinated (Smith and Lewin 2009).
However, this distinction reveals little about any taxonomic
difference in pain experience because it is the unmyelinated
C fibers in mammals that are most prevalent and are important in the perception of stimuli that give rise to pain (Smith
and Lewin 2009). Because pain experience associated with
tissue damage typically depends on nociception, a lack of
nociceptors would suggest that the animal was insensitive to
noxious stimuli and could not experience pain. This argument was central in recent work in fish that demonstrated
nociceptors similar to those of mammals, allowing the conclusion that fish had apparatus that should be sufficient for
them to experience pain (Sneddon 2003). However, that
study was also right to state that the presence of nociceptors
per se does not demonstrate that pain is experienced.
A Suitable Central Nervous System
Because it is clear that the human brain is suitable for pain
experience there has been an assumption by some (Rose
2002) that only animals with structures very similar to those
of humans have the capacity to experience pain. For example, the possibility of fish experiencing pain has been dismissed because human pain is experienced in parts of the
cerebral cortex whereas fish lack this structure (Rose 2002).
If one accepts this argument then the possibility of pain being experienced by any invertebrate must be dismissed
because none has a central nervous system (CNS) built on
the vertebrate plan. However, according to the same logic it
could be suggested that because crustaceans or cephalopods
lack any of the visual system found in humans they must be
blind. This is not the case as both have a well-developed visual ability, each based on an entirely different CNS and receptors. Thus clearly the same function can arise in different
animal taxa using different morphology, and it appears to be
illogical to accept this reasoning for some experiences but to
dismiss it for pain (Elwood et al. 2009).
In his review of pain criteria Bateson (1991, 834) avoided
the idea that structures homologous to those of humans must
be present and instead suggested that there should be “structures analogous to the human cerebral cortex.” Because
177
many invertebrates have a remarkably complex brain structure,
albeit rather different from that of humans, some might have
structures analogous to the cortex (Smith 1991). For example, it has been suggested that specific brain areas in the
octopus are specialized for sensory analysis, memory, learning, and decision making and thus may be considered analogous to the human cerebral cortex (Wells 1978).
A second argument for rejecting a pain experience in invertebrates is that their brains might be too small. However,
the octopus brain is larger than that of most fish and reptiles
when regarded as a ratio of body weight (Smith 1991) and
even the brains of many decapod crustaceans (e.g., crabs,
lobsters, shrimp) are likely to be considerably larger than
those of many vertebrates when regarded in an absolute
comparison (Elwood et al. 2009). Broom (2007) notes that
brain size does not necessarily equate to complexity of function (Broom and Zanella 2004); indeed, the brains of some
invertebrates have a surprising complexity (Sandeman et al.
1992; Wells 1978), with clear functional separation of distinct areas, and thus might be sufficiently complex in function to enable pain experience (Broom 2007).
Responsiveness to Opioids, Analgesics, and
Local Anesthetics
Mammals have a system for regulating pain such that the
same tissue damage may result in very different responses
depending on the situation. For example, humans engaged in
sports often report little pain in response to tissue damage.
The physiological basis of this regulation is complex but in
part is dependent on endogenous opioids, release of which
reduces the pain experience. Injection of the opiate morphine
also reduces the pain experience and the opiate antagonist
naloxone reverses this effect. For these reasons the presence
of opioid receptors and responses to analgesics has been regarded as an indicator that animals experience pain (Bateson
1991; Roughan and Flecknall 2001; Sneddon et al. 2003).
A particularly persuasive approach is to offer the animal
a choice between water (or food) that does or does not contain an analgesic and to observe whether a preference develops for the analgesic when noxious stimuli are applied
(Colpaert et al. 1980). Danbury and colleagues (2000) found
that lame chickens consumed more feed containing an analgesic than did those that were not lame. As far as I am aware
this approach has not been tried with invertebrates, but various studies have applied analgesics and local anesthetics and
examined their effects on responses to noxious stimuli. For
example, in the crab (Chasmagnathus granulatus) electric
shock elicited a defensive threat display and the percentage
of animals that showed this response rose with the voltage
applied. Injection of morphine hydrochloride reduced the
crabs’ sensitivity to the shock in a dose-dependent manner
and naloxone injection inhibited the effects of morphine
(Lozada et al. 1988). Morphine also had inhibitory effects on
the escape tail-flick response to electric shock in mantis
shrimps (Squilla mantis) that was reversed by naloxone
178
(Maldonado and Miralto 1982), and researchers observed a
similar effect of opioids and naloxone in nematodes (Pryor
et al. 2007) and snails (Kavaliers et al. 1983).
This approach, however, is problematic because analgesics might produce a general reduction in responsiveness to
all stimuli. One way around this is to create a situation in
which analgesia might increase particular responses. In one
such study, fruit flies placed in a tube at the darker side of a
light gradient moved toward the light. If the center of the
tube was heated, however, the flies were inhibited from passing this section. The application of specific analgesics (agonists for GABAB that are effective analgesics in hot plate
tests in rats; Thomas et al. 1996) reduced this inhibition and
the flies passed through the heat to the lighter area (Manev
and Dimitrijevic 2005).
A recent study on the glass prawn (Palaemon elegans)
noted that the animal engaged in prolonged grooming of the
antennae and rubbed them against the side of the tank when
the antennae were treated with acetic acid or sodium hydroxide, but prior treatment with a local anesthetic (benzocaine)
reduced the rubbing and grooming (Barr et al. 2008). There
was no effect of benzocaine on the general locomotion of the
prawn so the reduction in the two behaviors was not simply
due to inactivity. However, the result with acid was not replicated in other decapod species (Puri and Faulkes 2010).
Both opioid analgesics and local anesthetics have effects
that appear similar to those observed in vertebrates. But
local anesthetics block sodium channels (Machin 2005)
and, in crayfish (Procambarus clarkia; Leech and Rechnitz
1993), prevent the conduction of impulses from nociceptors,
so it is the nociception that is reduced or eliminated. Furthermore, opioids may produce analgesia by acting on a modulatory system in the CNS (Tomsic and Maldonado 1990), but
they might also have a peripheral effect (Del Seppia et al.
2007; Pryor et al. 2007). In all of these cases the nociception
is or may be disrupted, so conclusions about the potential for
the animal to experience pain are limited.
Physiological Changes
Noxious stimuli applied to vertebrates typically result in
tachycardia, pupil dilation, and defecation. Changes in blood
flow, respiratory patterns, arteriole blood gases, electrolyte
imbalance, and endocrine changes are also common (Short
1998; Sneddon et al. 2003). The latter often involve corticosteroid release, which is used as a measure of stress (Stafford
and Mellor 2005).
There has been limited examination of similar responses
in invertebrates. Cephalopods are said to have an adrenal
system that releases adrenal hormones when the animal is
exposed to noxious, potentially painful stimuli, and noradrenaline and dopamine are released when the animal is
disturbed (Stefano et al. 2002). Crustaceans have a stress
hormone, the crustacean hypoglycemic hormone (CHH)
(Chang 2005; Lorenzon et al. 2004), that functions to convert glycogen to glucose in a manner analogous to that of
ILAR Journal
cortisol in vertebrates. Glucose rose substantially in edible
crabs when a claw was removed in a manner that caused a
wound but not when the crab was induced to autotomize the
claw (Patterson et al. 2007). However, this might be due to
the tissue damage per se rather than any negative emotional
state.1
There is a clear need for more studies on physiological
aspects of invertebrate responses to noxious stimuli. Of particular use would be an examination of physiological changes
during avoidance learning and during presentation of just the
conditioned stimuli (without the noxious event) to determine
whether features akin to anxiety are present.
Avoidance Learning
I have noted that nociception allows for an immediate escape
by use of a reflex response whereas pain enables motivational change and avoidance learning. The key function of
pain is thus to reduce damage over a relatively long term.
One would therefore expect to see evidence of rapid avoidance learning coupled with a long memory in an animal that
experiences pain.
Such evidence has been reported in Drosophila that
learned to associate an odor that preceded or overlapped
with an electric shock: after eight (Yarali et al. 2008) or
twelve trials (Tully and Quinn 1985) they avoided the odor
for up to 24 hours. Researchers have used this paradigm to
examine genetic influence on learning and memory and the
morphology involved in terms of brain region (de Belle
and Heisenberg 1994) and to create mutants for dissecting
biochemical pathways involved in learning and memory
(Sokolowski 2001). Curiously, Drosophila also learn to associate the odor if it occurs after the shock has ended and in
this case they show a mild preference for the odor, a feature
termed pain relief learning (Yarali et al. 2008).
Similarly, the crab C. granulatus associated a shock
with a particular location (Denti et al. 1988) after just a
single trial and retained the association for 3 (but not 24)
hours. Subsequent experiments involving multitrial training, however, showed retention after a 24-hour rest interval in a different environment from that used in training
(Fernandez-Duque et al. 1992).
Experiments with crayfish (P. clarkia) demonstrated an
association between a light and a shock given 10 seconds
later: the animals learned to respond by walking to a safe
area in which the shock was not delivered (Kawai et al.
2004). But the animal did this only if it was facing the area
to which it could walk to avoid the shock; if it was facing
away from the safe area it exhibited a tail-flick escape response, by which it moved away tail first. Despite repeated
pairings of light and shock, the animal did not learn to avoid
the shock by tail flicking in response to light. However, when
the animals that had experienced shocks while facing away
from the safe area were subsequently tested facing toward
the safe area they showed a very rapid avoidance of the shock
at the onset of the light. Thus they seemed to have learned
the association although they had not previously used it to
avoid the shock. This finding was explained by the specific
associations between cues and particular responses that are
also common in vertebrates.
Hermit crabs (Pagurus bernhardus) in their shell that
were shocked on the abdomen demonstrated a long-term behavioral change compared with crabs that were not shocked.
The shocked crabs were more likely to approach and enter a
newly offered empty shell (Elwood and Appel 2009) and,
compared to those not shocked, they moved into the new
shell more quickly, spent less time investigating it, and
inserted their chelipeds into its aperture less often before
moving in.2 This is consistent with the idea that shocked
crabs assessed their original shells as being of very poor
quality. Shocked crabs altered their behavior for up to a day
after the initial shock (the maximum time tested), indicating
a long-term shift in motivation about obtaining a new shell
after the aversive experience (Appel and Elwood 2009a).
Taken together, these studies on learning and motivational change show abilities in arthropods that seem to fit
this key criterion for pain experience.
Protective Motor Reactions
Protective motor reactions include reflex withdrawal from a
noxious stimulus, but this is a basic feature of nociception
and gives little indication of emotional state. Weary and
colleagues (2006) argued that prolonged rubbing denoted an
awareness of the site of the noxious stimulus and Sneddon
and colleagues (2003) noted that rainbow trout (Oncorhynchus mykiss) that had noxious chemicals injected into the lip
showed rubbing of the lip on the substrate, consistent with
the idea of pain.
Hermit crabs induced to evacuate their shells by electric
shock to the abdomen demonstrated sustained grooming by
use of claws on the abdomen (Appel and Elwood 2009a,b;
Elwood and Appel 2009), a response not seen when the crabs
are cracked out of their shell or evicted in a shell fight. Further, when either sodium hydroxide or acetic acid solution
was applied to one antenna of a glass prawn there was a significant increase in grooming of that antenna during which it
was pulled repeatedly through the animal’s small pincers
and mouth parts (Barr et al. 2008), and there was an increase
in rubbing of that antenna against the side of the tank. The
animal seemed to be aware of the specific location of the
noxious stimulus and directed its attention to the treated antenna. (However, work on three species of prawn found no
significant increase in directed grooming of treated antennae; Puri and Faulkes 2010.) In addition, when acetic acid
was applied to an eye of a glass prawn there was a marked
increase in grooming that involved both pincers moving
1Crabs
lacking a claw showed a higher level of glucose when an intact crab
was housed in the same tank, an effect that might be due to fear or increased
alertness (Patterson et al. 2007).
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2011
2Such a minimum of investigation of the new shell is otherwise characteristic
of crabs in shells of inadequate size (Elwood and Stewart 1985).
179
simultaneously and in very different and complex ways.
The grooming was directed specifically to that eye, again
demonstrating that the animal was aware of the location of
the noxious event (Barr et al., unpublished data). The complexity of these prolonged responses is beyond that expected
from a nociceptive reflex response and consistent with the
idea of pain.
Another protective motor response in arthropods is autotomy, in which an appendage is cast off from the body. In the
spider Argiope aurantia, legs may be autotomized when
damaged (Eisner and Camazine 1983). This was seen during
attempts by these spiders to capture ambush bugs (Phymata
fasciata), typically when the bug grasped a spider leg and
probed a joint with its proboscis (the venomous saliva is
painful to humans)—autotomy occurred within 5 seconds
(Eisner and Camazine 1983). Simple experimental penetration of the joint with a sterile pin did not cause autotomy,
indicating that the saliva had an effect. Eisner and Camazine
(1983) also injected bee and wasp venom, both of which induced autotomy. They found that when individual components of the venom were injected, some, but not all, produced
autotomy; effective components were histamine, serotonin,
phospholipase, and melittin, all of which induce pain in
humans; ineffective components were acetylcholine, bradykinin, hyaluridase, adrenaline, and dopamine. Acetylcholine
and bradykinin induce pain in humans but not autotomy in
spiders, and hyaluridase, adrenaline, and dopamine do not
induce pain in humans, suggesting a concordance between
pain effects in humans and autotomy in the spider.
Autotomy in crustaceans typically leaves a clean break
at a specific joint close to the main body, which immediately
seals to prevent loss of hemolymph. Cutting a membrane at
a joint distal to the autotomy plane, causing hemolymph loss,
elicits rapid autotomy (within a few seconds) of that appendage, preventing further loss of fluid (Patterson et al. 2007).
Crabs also autotomize limbs in situations that do not involve
hemolymph loss, for example if the whole animal is placed
on a hot plate (Fiorito 1986) or if the leg is subject to electric
shock or injected with acetic acid (Barr and Elwood, unpublished observations). The acetic acid treatment rapidly induces
autotomy in a dose-dependent manner and the results are
consistent with pain mediation of the autotomy response.
Tradeoffs between Stimulus Avoidance and
Other Activities
Bateson (1991) suggests that one criterion for pain should be
a relatively inelastic response (sensu Dawkins 1990), but a
response that is purely mediated by nociception is an inelastic
reflex—it should be the same regardless of other motivational
priorities. Thus an animal that is hungry or satiated would
likely exhibit the same reflex avoidance to a noxious stimulus, even if food is present. By contrast, pain is a negative
emotional state, typically coupled with a very high motivation to escape that state, and thus should be given a high priority and might appear to be inelastic. Thus it seems difficult
to discriminate pain from nociception by this criterion.
180
However, if variation in the response to noxious stimuli
is dependent on other motivational requirements then there
must be some higher-level interaction between competing
motivational systems (McFarland and Sibly 1975). In fish,
for example, those deprived of food are less likely to respond
to an electric shock in a feeding area than those that are not
food deprived (Millsopp and Laming 2008). And lame hens
stop limping in the period leading up to egg laying but limp
again after laying (Gentle 2001). Competition between different activities for expression or requirements is the essence
of motivational tradeoffs. Such competition is important for
pain research as it is a strong indicator that the response to
the noxious stimulus is not purely reflexive: tradeoffs clearly
involve some form of processing in which different needs
are weighed.
Tradeoffs were the subject of two experiments in which
hermit crabs were given shells with two small holes drilled
and electrodes inserted so the crab could be shocked on the
abdomen. When shocks of a single intensity were applied, at
a level that was hoped would not cause evacuation, some
crabs evacuated and were more likely to do so from a less
preferred shell species (Elwood and Appel 2009). Similarly,
when the shocks increased in intensity, crabs evacuated the
shell at a lower shock intensity if they were in a less preferred shell species (Appel and Elwood 2009b). Thus, the
animals’ response to the shock was determined in part by
their normal preference for particular species of shell. Further, they were much less likely to evacuate after being
shocked when the odor of a predator was present, suggesting
a tradeoff between shock avoidance and predator avoidance
(Wilson and Elwood, unpublished observations). These responses cannot be a reflex response as they required information from sources other than the noxious stimulus to have
an effect on the response.
This approach of determining what is “traded off”
against avoidance of the noxious stimulus may give insights
into an animal’s priorities (Dawkins 1990)—that is, what it
might “pay” to avoid the noxious stimulus in terms of lost
opportunities to satisfy other motivational demands. For example, among the shocked hermit crabs that evacuated their
shell some stayed near the shell and many got back into it;
others, however, walked away and even attempted to climb
the walls of the observation chamber (Appel and Elwood
2009a,b). This is remarkable because the shell is a vital resource and abandoning it indicates the aversive nature of
the shock.
Octopuses provide another example of tradeoff in their
avoidance of stinging sea anemones. Octopuses readily prey
on hermit crabs but experiments have shown that when the
crabs placed an anemone on their shell as protection the
octopuses dramatically changed their tactics. They tried a
variety of approaches such as moving below the anemone,
blowing jets of water at it, and using a single outstretched
arm. Thus they seemed to try to avoid the stings while attempting to maintain food intake, albeit with tactics that are
less efficient for food capture (MacLean 1983; Mather
2008).
ILAR Journal
High Cognitive Ability, Consciousness,
and Sentience
Several authors have considered a high cognitive ability coupled with consciousness or sentience a prerequisite for a
pain experience or at least have suggested that such abilities
make pain experience more likely. Bateson (1991, 832), for
example, suggests that “if the animal can be shown to be
conscious of what it is doing, then most people would conclude that it could experience pain.” Particular cognitive
abilities are also considered important in assessing the
welfare status of animals (e.g., Braithwaite 2010; Chandroo
et al. 2004; Duncan 1996; Duncan and Petherick 1991). At
the very least sentience probably involves awareness of internal and external stimuli (Chandroo et al. 2004; Duncan
1996), and “primary consciousness” involves the ability to
generate a mental scene in which diverse information is integrated for the purpose of integrating behavior (Chandroo
et al. 2004; Edelman and Tononi 2000). I consider here several examples of invertebrate abilities in integrating information from different sources to make “informed” decisions.
Spiders exhibit a variety of complex behaviors that appear to illustrate a capacity for information integration.
Jumping spiders, for example, are known to adjust hunting
methods depending on the type of prey and its ability to
escape (Bartos 2008). The hunting spider (Portia labiata),
when it hunts spitting spiders (Scytodes pallidus), which are
themselves predators of spiders and thus dangerous, gathers
information as to whether the spitting spider is carrying eggs
in the mouth and thus less dangerous; if so, the hunting
spider modifies its attack (Jackson et al. 2002). Furthermore,
when hunting prey in complex environments, Portia appears
to plan routes with detours that initially take it away from the
prey item to avoid obstructions (Tarsitano 2006). Such behavior suggests an ability to comprehend the complex spatial relationships between itself and the prey and possible
routes to a goal (Sherwin 2001).
Male giant cuttlefish (Sepia apama) exhibit a remarkable ability to change shape and color to switch between the
appearance of a female and that of a male in order to foil the
mate-guarding attempts of larger males. Norman and colleagues (1999) showed that small males that assumed the
body shape and patterns of a female were not attacked by
the larger mate-guarding male. When the larger male was
distracted by another large male intruder, the small males
changed body pattern and behavior to those of a male in mating display and successfully mated.
Squid use surprisingly complex color patterns for courtship and protection (Hanlon et al. 1994). A male can display
a courtship coloration on one side of the body toward a
female while at the same time displaying a completely different pattern on the other side to ward off an intruding male
(Mather 2004, 2008). And he can switch the sides of the
body showing the two displays as soon as the relative positions of the other two animals change.
Octopuses also show complex learning abilities (Edelman
et al. 2005). When confronted with a maze in which the
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2011
experimenter frequently changed the nature of the obstacles
octopuses readily solved the maze, apparently considering
the maze before proceeding (Moriyama and Gunji 1997), suggesting a level of ability that might signal consciousness.
A final example concerns hermit crabs, which when deciding to change shells systematically evaluate various components of potential new shells to determine whether they
offer a gain in terms of size, shape, and weight before moving in (Elwood and Stewart 1985). The evaluation continues
even after moving in and a crab may switch between the two
before making a final decision (Elwood 1995). Hermit crabs
that fight to take the shell from another hermit crab not only
evaluate the opponent’s shell (Dowds and Elwood 1983) but
also take in information about the opponent (e.g., its size and
power; Briffa and Elwood 2002; Dowds and Elwood 1985).
In addition, they monitor their own physiological state during the encounter in order to make effective fight decisions
(Briffa and Elwood 2000, 2002, 2005)—for example, when
lactate increases the attack rate slows and the attacker then
stops fighting (Briffa and Elwood 2002, 2005). Hermit crabs
can remember particular opponents for up to 4 days after an
encounter (Gherardi and Artema 2005).
It is clear from the above examples and others (Broom
2007; Mather 2008) that some invertebrates are capable of
integrating information from various sources, both internal
and external, to enable complex decisions. Also apparent is a
fine discrimination learning ability (Mather 2008), indicating a high cognitive ability. For example, honeybees can
learn a complex learning task in which they have to select
from previously unseen shapes on the basis of whether they
are symmetrical or not (Benard et al. 2006; Giurfa et al.
1996) and cephalopods appear particularly adept at a range
of learning tasks (Mather 2008).
Good discrimination learning may not necessarily indicate an ability to experience pain, but one might expect to
see such discrimination when pain is experienced—simple
nociceptive reflex avoidance results in an immediate withdrawal but does not imply any long-term motivational
change. To benefit from pain experience the animal needs
to be able to discriminate between the specific situation that
led to the pain and other situations that did not. Animals
that cannot make fine discriminations may avoid potentially
harmless or even useful situations or objects. Thus it seems
reasonable to speculate that the evolution of pain experience developed hand in hand with enhanced discrimination
learning.
Conclusion
It is clear that the various criteria I have described differ in
their usefulness in discriminating pain from nociception.
Because of the wide taxonomic occurrence of functional nociceptors, the demonstration of their presence does not indicate the capacity to experience pain, and investigation of the
central nervous system provides limited clues of what is or is
not suitable for pain experience. The use of opioids and other
181
analgesics might indicate a central modulation of responses,
but potential peripheral effects may explain the analgesia.
Physiological changes might prove useful but, to date, the
study of their appearance in invertebrates is limited and reveals little about their pain experience.
It is thus behavior that provides the greatest insights into
the likely experience of pain. Rapid avoidance learning, coupled with a prolonged memory, indicates central processing
and is consistent with pain, but it is more convincing after
one stimulus than after numerous repetitions. Complex, prolonged grooming or rubbing might indicate an awareness of
the specific site of stimulus application and seems to be more
than a reflex reaction. Tradeoffs with other motivational
systems indicate central processing and may be useful to determine what an animal will “pay” to avoid the noxious stimulus. An ability to use information from various sources
might indicate sufficient cognitive ability for the animal to
have a fitness benefit from a pain experience.
Evidence from behavioral studies is entirely consistent
with the idea that some invertebrates, particularly crustaceans and molluscs, experience pain. However, more studies
must use a variety of imaginative techniques to confirm that
invertebrates do indeed experience pain. Substantial research
on various taxa is necessary to assess which, if any, show (1)
rapid avoidance learning of noxious stimuli, (2) prolonged
responses directed to the specific site on their body where
the noxious stimulus was applied, and/or (3) tradeoffs between avoidance and other activities that would indicate central decision making rather than reflex reaction. Studies that
demonstrate marked physiological stress responses to conditioned stimuli that herald the imminent application of a noxious stimulus would also be helpful.
Clearly, a start has been made on some of these approaches
but much more is needed. Recently, Braithwaite (2010) was
confident enough to state that fish feel pain but invertebrates
do not. I do not share the confidence to make that discrimination. Neither do I feel confident in stating unequivocally that
some of them do feel pain, although it is clear that the responses described above cannot be explained just by nociceptive reflexes. While awaiting the results of further relevant
studies, perhaps all who use invertebrates should consider
the possibility that at least some might suffer pain and, as a
precaution, ensure humane care for these animals.
Acknowledgments
I thank Lisa Collins, Cameron Fletcher, and four anonymous
referees for many useful comments that helped to improve
this work.
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ILAR Journal
Nociceptive Behavior and Physiology of Molluscs: Animal Welfare Implications
Robyn J. Crook and Edgar T. Walters
Abstract
Nociceptive Biology and Evolution
Molluscs have proven to be invaluable models for basic
neuroscience research, yielding fundamental insights into a
range of biological processes involved in action potential
generation, synaptic transmission, learning, memory, and,
more recently, nociceptive biology. Evidence suggests that
nociceptive processes in primary nociceptors are highly conserved across diverse taxa, making molluscs attractive models for biomedical studies of mechanisms that may contribute
to pain in humans but also exposing them to procedures that
might produce painlike sensations. We review the physiology of nociceptors and behavioral responses to noxious
stimulation in several molluscan taxa, and discuss the possibility that nociception may result in painlike states in at
least some molluscs that possess more complex nervous systems. Few studies have directly addressed possible emotionlike concomitants of nociceptive responses in molluscs.
Because the definition of pain includes a subjective component that may be impossible to gauge in animals quite different from humans, firm conclusions about the possible
existence of pain in molluscs may be unattainable. Evolutionary divergence and differences in lifestyle, physiology,
and neuroanatomy suggest that painlike experiences in molluscs, if they exist, should differ from those in mammals. But
reports indicate that some molluscs exhibit motivational
states and cognitive capabilities that may be consistent with
a capacity for states with functional parallels to pain. We
therefore recommend that investigators attempt to minimize
the potential for nociceptor activation and painlike sensations in experimental invertebrates by reducing the number
of animals subjected to stressful manipulations and by
administering appropriate anesthetic agents whenever
practicable, welfare practices similar to those for vertebrate
subjects.
early all animals that have been studied display
marked behavioral responses to stimuli that cause
tissue damage—the ability to sense and respond to
noxious stimuli is an almost universal trait (Kavaliers 1988;
Smith and Lewin 2009; Sneddon 2004; Walters 1994). Nociception, defined as the detection of stimuli that are injurious
or would be if sustained or repeated, has clear adaptive advantages because it triggers withdrawal and escape during
injury or in the face of impending injury.
Key Words: Aplysia; cephalopod; ethics; invertebrate;
mollusc; nociception; pain; sensitization
Robyn J. Crook, PhD, is a postdoctoral fellow and Edgar T. Walters, PhD, a
professor in the Department of Integrative Biology and Pharmacology at the
University of Texas Medical School at Houston.
Address correspondence and reprint requests to Dr. Robyn J. Crook,
Department of Integrative Biology and Pharmacology, University of Texas
Medical School at Houston, 6431 Fannin Street, Houston, TX 77030 or
email [email protected].
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2011
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Nociception and Nociceptive Sensitization
The first stage of nociception occurs with the activation of
nociceptors, primary sensory neurons preferentially sensitive to
noxious stimuli or to stimuli that would become noxious
if prolonged (Sherrington 1906). Nociceptors were first
demonstrated in Chordata by Burgess and Perl (1967), in
Annelida by Nicholls and Baylor (1968), in Mollusca by
Walters and colleagues (1983a), in Nematoda by Kaplan and
Horvitz (1993), and in Arthropoda by Tracey and colleagues
(2003). Preliminary studies indicate that nociception in these
phyla involves many conserved sensory transduction processes
(Smith and Lewin 2009; Tobin and Bargmann 2004; Tracey
et al. 2003), although differences have also been found. It is
not yet known whether specialized nociceptors also occur in
other phyla, although this seems likely.
Many nociceptors exhibit a property rare among primary
sensory neurons: enhanced sensitivity produced by intense
stimulation of the sensory neuron’s peripheral terminals
(Campbell and Meyer 1983; Gold and Gebhart 2010; Hucho
and Levine 2007; Illich and Walters 1997; Light et al. 1992).
Enhancement of the sensitivity of a nociceptor, nociceptive
network, or behavioral response after noxious stimulation is
called nociceptive sensitization (Walters 1994; Woolf and
Walters 1991).
Nociceptive sensitization can be measured directly at the
neuronal and behavioral levels and has been investigated extensively in mammals because it is thought to represent concrete,
quantifiable effects that in humans are related to increased
pain sensitivity. As defined by the International Association
for the Study of Pain (IASP; Merskey and Bogduk 1994),
increased pain sensitivity occurs in the form of hyperalgesia
(greater pain in response to a normally painful stimulus) and
allodynia (pain evoked by a stimulus that is not normally
painful).
185
In a few molluscs and other invertebrates, mechanisms of
nociceptive sensitization have been investigated intensively
in hopes of discovering fundamental processes that contribute to hyperalgesia and allodynia in humans and other animals. Indeed, behavioral and neurophysiological alterations
in nociceptors during nociceptive sensitization appear
remarkably similar in snails and rats (Walters 1994, 2008;
Woolf and Walters 1991), and these alterations involve many
intra- and extracellular biological signaling pathways that
are highly conserved (Walters and Moroz 2009).
Interpreting Nociceptive Differences
across Phyla
There are numerous differences between the neuronal systems that mediate nociceptive sensitization in molluscs and
in mammals (as well as presumably enhanced pain in the
latter). At the axonal level, molluscs and other invertebrates
lack myelination, so conduction of information from one
part of the nervous system to another is usually much slower
than in vertebrates. At the synaptic level, an evolutionarily
recent expansion of the synaptic proteome in vertebrates
may underlie unique cognitive capabilities of this group (Ryan
and Grant 2009) and in principle also contribute to a potentially unique capacity of vertebrates to experience pain.
Furthermore, at the neuroanatomical and, presumably,
neural network levels, little homology exists across any of
the phyla, which diverged before most of the evolution of
neuroanatomical structures in contemporary animals (Farris
2008). Thus noxious information in vertebrates is relayed
from primary nociceptors via neurons in the dorsal horn of
the spinal cord to brain structures including the thalamus and
the somatosensory, insular, and anterior cingulate cortices
(Peyron et al. 2000), but homologues to these brain structures do not exist in invertebrates. The fact that these areas
are not present in the invertebrate central nervous system
(CNS1) does not prove that invertebrates cannot feel pain;
independently derived neural structures might, in principle,
have evolved the capacity to mediate the same functions. For
example, some invertebrates (many cephalopods and some
insects) can process highly complex visual information even
though they lack a structure homologous to the mammalian
visual cortex. While it is plausible that the more elaborate
neural structures of mammals confer a capacity for the experience of pain, analogous processing in other phyla might
mediate painlike experiences using neural structures unrelated
to and quite different from those in the mammalian brain.
Similarity across phyla of nociceptive behavior, mechanisms of nociception, and nociceptive sensitization implies
either deep homology of underlying mechanisms or convergence arising from similar selection pressures among diverse
and distantly related taxa. Either of these possibilities may
permit insights into mechanisms important for pain and its
alleviation in vertebrates from studies of animals with far
1Abbreviation that appears ≥3x throughout this article: CNS, central nervous
system
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simpler nervous systems. Invertebrates thus present two considerable advantages: (1) research on a complex process in a simpler system often permits a clearer picture of what is really
important, and (2) the study of states bearing similarities to pain
or unpleasantness in an animal that appears to have less capacity to interpret and “feel” such sensations is more palatable to
scientists, to the public, and to animal welfare committees.
The presence in invertebrates of some nociceptive mechanisms that are homologous and/or convergent with those
important for pain in humans presents a question of ethics: If
scientists are willing to appeal to evolutionary conservatism
to support the use of “lower” animals to study physiological
building blocks that in humans contribute to pain and suffering (assuming that the information revealed will translate
to humans and other “higher” vertebrates), is it acceptable to
ignore possible implications of evolutionary conservation
that similar processing of nociceptive information by higher
and lower animals might in each case produce suffering? Are
there reasons to think that experimental unpleasantness is
less keenly felt by a snail or fly than by a mouse, monkey, or
human, and if so where should one draw the line when conducting experiments that might cause suffering?
Nociception Versus Pain
Humans tend to experience nociception and pain as a single
phenomenon, but for the study of animals it is important to
draw a distinction between sensory activation and emotional
perception (see Braithwaite 2010 for an excellent discussion
of the separable nature of the two processes).
Defining Nociception and Pain
Nociception is a capacity to react to tissue damage or impending damage with activation of sensory pathways, with
or without conscious sensation. Activity in nociceptive sensory pathways usually results in reflexive behavioral responses
and may or may not result in other responses. Reflexive withdrawal responses tend to be mediated by very simple sensorimotor circuits optimized for speed and reliability and can
occur without input from higher processing centers, although
more complex escape and avoidance behaviors involve more
complex neural circuits (Chase 2002; Walters 1994). Even in
humans the initial reflexive response to a noxious stimulus is
sometimes faster than can be consciously perceived, and
nociceptors can sometimes be activated without conscious
sensation (Adriaensen et al. 1980). Invertebrates that lack
appropriate processing centers may be capable of only this
rapid, unconscious processing.
The definition of pain widely accepted by scientific investigators is “an unpleasant sensory and emotional experience
associated with actual or potential tissue damage, or described in terms of such damage” (Merskey and Bogduk 1994).
The emotional component required by this definition of pain
makes its identification in other species, and especially in
species quite different from humans, extremely difficult, if
ILAR Journal
not impossible. This is because emotion is usually defined in
terms of conscious experience (e.g., Izard 2009), and while
evidence of consciousness in some animals is available,
proof of consciousness is not (e.g., Allen 2004).
It is therefore important to distinguish between nociception as detection of a noxious stimulus (which can be recognized scientifically by unambiguous behavioral and neural
responses), and pain as the unpleasant feeling associated
with that stimulus (and inferred by behavioral and neural responses of uncertain relation to consciousness).
Moreover, the emotional response during pain may be
linked to cognition, “knowing” in some sense that the sensation is negative and involves a threat to the body. Whereas
nociception leading to a nociceptive response can be mediated by the simplest of neural circuits (in principle just a
single nociceptor connected to an effector system—e.g., a
muscle), pain requires neural circuitry that incorporates
additional functions, some of which might entail highly
complex processing by very large numbers of neurons.
Identifying Nociception and Pain in Animals
In invertebrates, like mammals, responses to noxious stimulation can be complex. Indeed, immediate defensive responses
to injury or noxious stimulation are followed by a longerterm phase (sometimes lasting weeks or months) where damaged regions are hypersensitive (e.g., Walters 1987b) and some
invertebrates remember associations of the noxious event with
its context (e.g., Colwill et al. 1988; Walters et al. 1981).
To date, long-term nociceptive sensitization in invertebrates has been explained by long-term alterations of primary
sensory neurons (especially nociceptors) (e.g., Montarolo et al.
1986; Scholz and Byrne 1987; Walters 1987a) and motor
neurons (e.g., Cleary et al. 1998; Glanzman 2008; Weragoda
et al. 2004); there is little evidence for electrical activity or
alterations in other types of neurons that outlast a noxious
stimulus for more than tens of minutes (see Cleary et al.
1998; Marinesco et al. 2004). Interneurons and modulatory
neurons have received far less experimental attention because they are much more difficult to identify and sample
with the intracellular recording methods used for neurophysiological experiments on invertebrates. Truly systematic
investigations of the distribution and duration of enhanced
activity (possible neural correlates of pain) across an entire
nervous system will benefit from the development of functional imaging methods for invertebrates (for a start, see Frost
et al. 2007; Zecevic et al. 2003) equivalent to those used to
examine patterns of activity in the mammalian brain after
noxious stimulation (e.g., Peyron et al. 2000; Tracey and
Bushnell 2009).
Several considerations suggest that pain may be absent
in at least some invertebrates. Capacities for processing all
types of information, including that involved in pain or painlike phenomena, increase with the size and complexity of a
nervous system, and mammals with the most complex brains
have, thus far, shown the most evidence for a capacity for
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2011
humanlike pain, although this might reflect a far greater experimental effort directed at vertebrates rather than genuine
differences in the invertebrate pain experience.
Nociceptive reflexes and nociceptive plasticity can occur
without conscious, emotional experience because these responses are expressed not only in the simplest animals but
also in reduced preparations, such as spinalized animals
(Clarke and Harris 2001; Egger 1978) and snail ganglia
(Walters et al. 1983b). Similarly, in human patients nociceptive reflexes can occur without conscious awareness below a
level of complete spinal transection (Finnerup and Jensen
2004).
But conservation or convergence of physiological and
molecular mechanisms of nociception and nociceptive sensitization across distant phyla does not necessarily imply that
higher-order phenomena (such as pain) that can be supported
by these mechanisms are equivalent. Even if the critical
mechanisms turn out not to be equivalent, it is not possible to
be certain that an animal does not feel pain, and thus the ethical questions remain (Allen 2004).
How can researchers balance the opportunities some
invertebrates offer for discovering mechanisms that, for
example, may alleviate chronic pain or slow dementia in
humans, with the possibility that the invertebrate subjects
might suffer? This is a fundamental problem for all of biomedical research, but has received very little consideration
in invertebrate studies. We refer readers to other articles in
this issue (Elwood 2011; Mather 2011) dealing with ethical
and philosophical considerations of invertebrate use.
Nociception and Painlike Phenomena
in Mollusca
Molluscs have provided invaluable models for neuroscience,
yielding a wealth of basic information applicable to humans
and other vertebrates. A consideration of their nociceptive
behavior and physiology will inform choices about their optimal use for biomedical research and improve the welfare of
molluscs used in the laboratory.
Mollusca is a highly diverse and successful metazoan
phylum with over 100,000 species (Ponder and Lindberg 2008)
distributed among terrestrial, aquatic, and marine environments
and divided into seven classes: Aplacophora, Polyplacophora, Monoplacophora, Scaphopoda, Bivalvia, Gastropoda,
and Cephalopoda. Along with great diversity in body plan,
lifestyle, and ecology, Mollusca encompasses enormous
intraphylum variation in sensory organs and neuroanatomy
(Bullock and Horridge 1965). Because the capacity for sensing and integrating information about the environment and
an individual’s own body is probably important for the ability to feel pain or experience distress, this large variation in
neural and sensory complexity suggests that welfare concerns may differ among groups.
The primary model species considered here are from the
gastropod and cephalopod clades; members of the other
classes are discussed briefly.
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Aplacophora, Polyplacophora,
Monoplacophora, and Scaphopoda
The classes less commonly used in neurobiological research
tend to be the primitive, sedentary, or deepwater-dwelling
groups.
Aplacophora contains small wormlike molluscs that burrow into the substrate (Salvini-Plawen 1981) and have paired
ventral and dorsal nerve cords running along the body as
well as paired cerebral and buccal ganglia in the head. Visual
and vestibular organs are absent but putative mechanosensory
neurons innervate the oral surface (Shigeno et al. 2007).
Little is known about their behavior and nothing about the
physiology of sensory neurons.
Polyplacophora contains the chitons, generally intertidal
marine animals with multiple platelike shells covering the
dorsal surface. They have a simple nervous system without
pronounced cephalization. There are rows of primitive photoreceptors along the dorsal surface and mechanosensory
organs around the mouth.
Monoplacophora is also an exclusively marine taxon of
limpetlike, conically shelled molluscs. Scaphopoda have a
single, tusklike shell through which water is pumped while
the animal remains mostly buried in the substrate. In both of
these groups the neural networks are simple and apparently
unspecialized and the ganglia are small. The simplicity of
their nervous systems and their behavior suggest that the
possibility of these animals experiencing painlike responses
to tissue insult is remote.
Bivalvia
The Bivalvia (e.g., oysters, clams, mussels, and scallops) are
abundant in both marine and freshwater environments. Their
nervous system includes two pairs of nerve cords and three
pairs of ganglia (Brusca and Brusca 2003). There is no obvious cephalization and the nervous system appears quite simple. A population of mechanosensory neurons is activated
during the foot withdrawal reflex in a razor clam, but it is not
known if these are nociceptors (Olivo 1970).
Clams and scallops have simple eyes and chemosensory
organs located along the periphery of the mantle and they
initiate escape swimming if a threat is detected, thus some
integration of information and basic decision making occurs.
Escape swimming in scallops is driven by a motor pattern
generator in the cerebral ganglion and usually occurs after
chemosensory detection or contact with a starfish predator
that would normally precede tissue destruction (Wilkins
1981), suggesting that nociception may be involved. However, to our knowledge there are no published descriptions of
behavioral or neurophysiological responses to tissue injury
in bivalves.
Although a number of studies have claimed that endogenous opioids (e.g., Stefano and Salzet 1999) and opioid
receptors (e.g., Cadet and Stefano 1999) are expressed in
the mussel Mytilus edilus, particularly in immunocytes,
188
neither genes for proopiomelanocortin (POMC) nor opioid receptors are found in Drosophila melanogaster or
Caenorhabditus elegans, and their reported existence in
other invertebrates, including molluscs, is controversial (Dores
et al. 2002; Li et al. 1996).
Gastropoda
This class includes terrestrial, freshwater, and marine species (e.g., snails, slugs, limpets, whelks, and many others).
Typically the shell and body are coiled, although in some
taxa (e.g., terrestrial slugs, sea hares, nudibranchs) the shell
is absent.
Gastropods have more diverse and specialized sensory
organs than the groups above and are typically motile and
active foragers. Along with increased range of habitats and
associated morphologies, their behavioral range is greater
and this is reflected in increased neural complexity. The basic
molluscan nervous system is present (Bullock and Horridge
1965) but is expanded in terms of both the number of cells
and their specialization. Many gastropods have giant neuronal somata, which have been used to advantage for neuronal analyses of behavioral mechanisms (Chase 2002;
Kandel 1976) and perhaps most prominently for investigations of learning and memory mechanisms (Kandel 2001).
Gastropods have also provided the largest number of studies
of nociceptive behavior and sensitization in invertebrates, in
part because noxious mechanical and electrical stimuli have
been used in many learning and memory studies. Some of
these studies have used pulmonate (air-breathing) snails but
most have used opisthobranch (rear-positioned gills) snails.
We know of no studies of nociceptive behavior or physiology
in the remaining group of gastropods—the prosobranchs.
Pulmonates
Pulmonates descended from gastropod molluscs that moved
from the sea to terrestrial and freshwater habitats. The most
extensively studied pulmonate is Helix (several different
species on different continents), which is prey to many generalist predators such as birds and frogs. Thus the welldeveloped defensive and aversive behaviors elicited by noxious
sensory input during failed predation attempts in this genus
should be subject to continuing selection.
When presented with a potentially threatening tactile
stimulus to soft tissue, Helix, like other pulmonates, withdraws reflexively, a behavior mediated by simple neural circuitry including sensory neurons that appear to be nociceptors
(Balaban 2002; Ierusalimsky and Balaban 2007). Noxious
stimulation can sensitize this behavior. Repeated electric
shocks to the foot (which probably activate nociceptors) result in a reduced response threshold to an innocuous mechanical stimulus that persists several days after the shocks,
and this long-term behavioral sensitization is associated with
several alterations in the circuit that mediates the withdrawal
response (Balaban 1983, 1993; Prescott and Chase 1999).
ILAR Journal
Other pulmonates used to investigate responses to noxious stimuli include Lymnaea stagnalis (Sakharov and Rozsa
1989) and Megalobulimus abbreviatus (Kalil-Gaspar et al.
2007). The latter study is of interest because it provided
pharmacological evidence for nociceptive actions of an ion
channel highly expressed in mammalian nociceptors (the transient receptor potential cation channel subfamily V member
1, or TRPV1, the capsaicin receptor).
As explained above, the ability to perceive noxious information as emotionally unpleasant is a delineating point
between simple nociception and pain. Helix, with a brain
containing only about 20,000 neurons in 11 ganglia, might
be presumed to be incapable of higher-order processing necessary for emotional responses. Interestingly, however, some
evidence suggests an emotionlike reaction in Helix.
Experiments using electrodes implanted into two different ganglia allowed animals to “self-stimulate” either of the
two neural regions by pressing a bar (Balaban 1993; Balaban
and Chase 1991). Results from this experiment suggested
that different CNS regions of snails have some rudimentary
“emotional coloration”: snails self-stimulated more frequently when the electrodes were placed in the mesocerebral
region of the cerebral ganglion (containing some neurons involved in sexual behavior) and less frequently when they
were placed in the rostral portion of the parietal ganglion, in
an area where electrical stimulation of putative nociceptive
sensory neurons underlying reflexive withdrawal behavior
was likely (Balaban 2002). But relatively little is known
about the anatomical organization and actual functions of
most neurons in either of these ganglionic regions. A lack of
follow-up reports of snail self-stimulation raises questions
about the robustness of this finding.
An apparent ability to remember and choose to avoid a
negative stimulus would suggest that a snail can selectively
modify its behavior on the basis of aversive experience in
ways similar to mammals. This would not show that Helix
can experience pain but it would suggest that fundamental
components of painlike information processing in vertebrates
might be present in a rudimentary fashion in some molluscs.
Opisthobranchs
The marine opisthobranch Aplysia californica is the leading
invertebrate model system for analyzing cellular bases of
behavioral and neural plasticity in many contexts (Kandel
1976, 2001). Aplysia has nine central ganglia containing
only about 10,000 neurons (Cash and Carew 1989), of which
several hundred have been identified by soma size and location, electrophysiological properties, synaptic connections,
and behavioral effects. Aplysia and many other molluscs
also have numerous neuronal cell bodies in peripheral nerve
nets (Bullock and Horridge 1965; Moroz 2006).
Most studies of learning and memory mechanisms in
Aplysia have used known mechanosensory neurons. Initial
studies used these neurons in the abdominal ganglion that
innervate the siphon (Byrne et al. 1974), but later studies
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2011
used homologous sensory neurons in each pleural ganglion
that innervate most of the rest of the body surface (Walters et al.
1983a, 2004). Moreover, both sets of neurons function as
nociceptors (Illich and Walters 1997; Walters et al. 1983a)
and thus their rich plasticity is highly relevant to nociceptive
functions. Both sets of nociceptors display prominent sensitizing effects, associated with short- and long-term sensitization of defensive behavior (gill and siphon withdrawal, tail
withdrawal, head withdrawal) after noxious stimulation.
These effects include enhancement of synaptic transmission
(reviewed by Kandel 2001; Walters 1994) and hyperexcitability of the nociceptor expressed in its peripheral terminals
(especially near a site of injury), neuronal cell body, axons,
and near its presynaptic terminals (Billy and Walters 1989;
Gasull et al. 2005; Reyes and Walters 2010; Weragoda et al.
2004). Such alterations are similar to those described in
studies of nociceptive sensitization in rodents and humans,
suggesting that some mechanisms that promote pain in vertebrates are also present in molluscs and, most likely, in
other invertebrate taxa as well (Walters and Moroz 2009).
Interesting similarities also exist in the behavioral responses of Aplysia and mammals to noxious stimulation.
Aplysia displays the nearly ubiquitous pattern of immediate
withdrawal reflexes, rapid escape, and prolonged recuperative behaviors exhibited across all major phyla (reviewed by
Kavaliers 1988; Walters 1994; see also Babcock et al. 2009;
Walters et al. 2001).
In addition, Aplysia responds with a motivational state
resembling conditioned fear to previously neutral chemosensory stimuli associated with noxious electric shock (Walters
et al. 1981). For example, after pairing with shock, the smell
of shrimp evoked a state that was not obvious unless combined with other stimuli—indeed, when only the shrimp
extract was presented, the animal exhibited a response reminiscent of the freezing exhibited by rats to a conditioned fear
stimulus (Walters et al. 1981). When tested in combination
with weak tactile stimulation, the shrimp extract greatly facilitated head and siphon withdrawal responses, defensive inking, and escape locomotion. Moreover, when delivered to a
feeding animal, the conditioned smell inhibited the feeding.
These extensive and motivationally consistent associative alterations (see also Colwill et al. 1988) suggest that
memory of a noxious event in snails can be linked to a fearlike motivational state that can dramatically alter the animal’s
response to other biologically significant stimuli. Unfortunately,
almost nothing is known about the neuroanatomical loci and
specific neurons involved in this “higher-order” processing
of nociceptive information in opisthobranch ganglia.
Nociceptive sensitization has also been reported in the
marine nudibranch Tritonia diomedea (Frost et al. 1998),
and associatively conditioned avoidance behavior after noxious conditioning stimulation has been investigated in the
notaspid Pleurobranchaea californica (Jing and Gillette 2003;
Mpitsos and Davis 1973). Both of these species offer many
advantages for cellular analyses, but they are difficult to obtain commercially and have been investigated much less
extensively than has Aplysia.
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In most animals, including Aplysia, nociceptive pathways show a period of inhibition after strong noxious stimulation as the animal engages in escape and active defense
(Mackey et al. 1987; Walters 1994). Opioids contribute substantially to nociceptive inhibition in vertebrates but, despite
indirect evidence for opioid signaling—such as pharmacological actions of opioids (e.g., met-enkephalin) and opioid
antagonists (e.g., naloxone) in molluscs (Kavaliers 1988;
Leung et al. 1986)—molecular evidence does not yet provide firm support for true opioid signaling in invertebrates
(Dores et al. 2002). Moreover, application of enkephalins
fails to produce inhibitory effects on the gill withdrawal reflex (Cooper et al. 1989) or known nociceptors (Brezina et al.
1987) in Aplysia. Instead another peptide, FMRFamide, may
be a major transmitter that produces immediate and longterm suppressive effects on nociceptor excitability, synaptic
transmission, and defensive reflexes in Aplysia (Belardetti
et al. 1987; Mackey et al. 1987; Montarolo et al. 1988).
Cephalopoda
Cephalopoda is an exclusively marine class that comprises
squid, cuttlefish, octopus, and nautilus. The cephalopod shell
is chambered and external in nautilus, internal in cuttlefish,
and reduced or absent in squid and octopuses. Cephalopods
are the most neurologically complex invertebrates, with centralized brains divided into specialized lobes capable of processing and integrating complex visual, chemical, and tactile
sensory inputs. The octopus CNS has around 500 million
cells (Young 1963), vastly more than that of gastropods. The
octopus also has an enormous peripheral nervous system,
separate from the CNS (Rowell 1966; Young 1963). Indeed,
the number of neuronal cell bodies in the arms of the octopus
is close to that of the central brain.
Cephalopods show complex behavior and are excellent
learners, with reports of sensitization, habituation, associative
learning, spatial learning, and even (although controversial)
observational learning (see reviews by Hanlon and Messenger
1998; Hochner et al. 2006). Despite the extensive literature
on cephalopod behavior, there have been no systematic behavioral or physiological investigations into nociception and
nociceptive plasticity.
Cephalopods are also less commonly used for studies of
neurophysiology due to the complex neuroanatomy of the
cephalopod CNS, the small size of the neuronal cell bodies,
and the lack of overshooting action potentials or large synaptic
potentials that can be recorded from the cell bodies (Hochner
et al. 2006). Nothing is known about where nociceptive information is processed in the cephalopod brain. Evidence for
nociception in cephalopods is therefore exclusively behavioral.
In various learning paradigms electric shock to the arms
of an octopus has been used effectively as a negative reinforcement (Boycott and Young 1955; Darmaillacq et al. 2004;
Shomrat et al. 2008; Young 1961), indicating that the animal
finds this stimulation aversive, but nociceptors have not yet
been described in any cephalopod. Octopuses are capable of
190
regenerating damaged or amputated arms, but there are no
published studies of behavioral adaptations to damaged or
healing tissue.
Although electric shock elicits defensive responses, an
intriguing anecdote from Jacques Cousteau (1973, 23-24) describing an octopus’ behavior during exposure to damaging heat suggests the absence of a response to intense thermal
stimulation:
One day at Octopus City, in the Bay of Alicaster, Dumas
dived with an underwater rocket and began waving it in
front of an octopus’ house. Nothing happened. The animal reacted not at all. He did not try to hide, or to escape.
Dumas then turned the beam directly onto the octopus,
which did not even draw [sic] its arms. The game was
called off, however, when Dumas saw that it was becoming cruel. The octopus showed signs of having been
burned. But even then it had not tried to escape from it.…
This surprising insensitivity to fire has been confirmed by
Guy Hilpatric, one of the pioneers of diving, who told us
that he has seen an octopus, which had been brought onto
shore, cross through a fire to get back into the water.
Although it is potentially valuable for an animal (particularly
an intertidal species) to sense when environmental temperatures are approaching dangerous levels, selection pressure
for nociceptors tuned to extreme heat is unlikely in most
aquatic animals. It would certainly be interesting to examine
this question in marine invertebrates adapted for life around
hydrothermal vents where extreme heat exposure may be a
hazard. An interesting question following from Cousteau’s
anecdote is whether the burned octopus would eventually
sense and respond to signals released by the damaged flesh
or to inflammatory mediators released during repair of the
tissue, as occurs in mammals.
Apart from the nautilus, which retains its external shell,
cephalopods have traded the protection of the molluscan
shell for an increase in locomotive efficiency and speed.
Their soft bodies, almost completely unprotected, should be
subject to occasional survivable injuries during failed predation attempts, food contests, and mating competitions. It thus
seems probable that nociceptors exist in cephalopods. We
have begun our own investigations into this possibility by
examining behavioral responses of the squid (Loligo pealei)
to peripheral, sublethal injury to an arm (R. Crook, T. Lewis,
R. Hanlon, and E.T. Walters, unpublished observations).
Sensitization of defensive responses to tactile and visual
stimuli occurs and persists for at least 48 hours after injury,
suggesting that basic behavioral responses to injury in cephalopods may be similar to those in gastropods and vertebrates, but further study is needed.
Notwithstanding the lack of evidence for pain perception
in cephalopods, in the United Kingdom octopus are covered
by the same welfare act that governs procedures on vertebrates (UK Animals [Scientific Procedures] Act 1986)2 and
2Available
online (www.legislation.gov.uk/ukpga/1986/14/contents), accessed
on April 4, 2011.
ILAR Journal
in Canada and Europe an approved welfare protocol is required for the use of cephalopod species in research (CCAC
1993; EU directive 86/609/EEC, updated September 20103).
Cephalopods have far more complex brains and behaviors than any other invertebrate and are capable of impressive cognitive tasks (see Hochner et al. 2006, for review).
For example, both the brainy octopus (Boal et al. 2000) and
“primitive” nautilus (Crook et al. 2009) are capable of vertebratelike spatial learning. Given these sophisticated cognitive abilities, an important question is whether nociceptive
responses in cephalopods are accompanied by affective and
cognitive processing functionally similar to some of the processing that is important for pain states in mammals.
Anesthesia in Molluscs
Until the early 1970s physiological and biochemical studies
of molluscs dispensed with any anesthetics. This practice reflected the widespread assumption that invertebrates cannot
feel pain, a view that is still common (and used to condone,
for example, the preparation of living cephalopods, snails,
and lobsters for human meals by methods that would cause
intense, prolonged stimulation of nociceptors if applied to
mammals).
Several studies of the mechanisms of action of widely
used mammalian anesthetics exploited the experimental
advantages of the giant axon of the squid and the giant cell
bodies in gastropod molluscs such as Aplysia (e.g., Frazier
et al. 1975; Winlow et al. 1992). But these anesthetics were
not used by researchers during their dissections, in part because many of the mammalian anesthetics and analgesics,
including opioids (Cooper et al. 1989), were not very effective in molluscs (although there were interesting exceptions,
including volatile general anesthetics that potently open potassium channels that hyperpolarize and reduce excitability
in Aplysia nociceptors; Winegar et al. 1996; Winegar and
Yost 1998). The widespread use of anesthesia during dissection of Aplysia began when investigators interested in learning and memory realized that dissection without anesthesia
could cause sensitizing effects that would interfere with the
neuronal plasticity they were studying.
The anesthetic of choice for both gastropods (Pinsker
et al. 1973) and cephalopods (Messenger et al. 1985; Mooney
et al. 2010) is isotonic magnesium chloride solution, typically applied by injection for gastropods and immersion for
cephalopods. Magnesium ions provide an ideal anesthetic
(and muscle relaxant) because they are normally present at
relatively high concentrations (especially in marine molluscs) and thus are relatively nontoxic, their effects are rapidly reversible, and the agent is both inexpensive and highly
effective (Walters 1987b). The effectiveness of magnesium
chloride in gastropods is a result of its inhibition of neurotransmitter release at chemical synapses, its depressive effect on voltage-gated sodium channels, reducing membrane
3Available
online (http://ec.europa.eu/environment/chemicals/lab_animals/
home_en.htm), accessed on April 4, 2011.
Volume 52, Number 2
2011
excitability (e.g., Liao and Walters 2002), and the fact that it
is used at high concentrations and injection volumes (>25%
of the animal volume) in combination with a balanced reduction of sodium ions (a reduction that by itself reduces excitability). Effectiveness probably also depends on leakiness of
the primitive blood-ganglion or blood-nerve barriers of most
molluscs (Abbott 1987). Applied magnesium chloride does
not penetrate these barriers in vertebrates, precluding its use
as an anesthetic in mammals. Given that cephalopods also
have a highly effective blood-brain barrier (ibid.) and relatively impermeable skin, the mechanisms underlying the
anesthesia that occurs during the animal’s immersion in isotonic magnesium chloride are an interesting mystery.
We refer readers to Cooper (2011, in this issue) for further discussion of analgesia and anesthesia in invertebrates.
Evolutionary Selection Pressures and
Painlike Phenomena in Molluscs
Insight into the extent to which nociception may lead to pain
in molluscs can come from considering possible selective
advantages of pain during evolution.
Nociception, like any prominent trait in animals, has
been selected and refined over millions of years by evolutionary processes that act to enhance survival and reproductive success. The adaptive value of nociception is obvious
and probably universal: it permits rapid avoidance of a damaging stimulus and escape from the context in which such
damage occurs. Escape may be from predators, aggressive
conspecifics, or threatening environmental features (e.g.,
rough surf).
The adaptive value of experiencing pain is more difficult
to identify, although clues are available from likely consequences of nociceptive responses for survival under natural
conditions. A strong negative emotion motivates rapid avoidance learning, decreasing the chances of reexposure to a noxious stimulus. This interpretation raises the question of how
much neural processing power is required for emotional responses. Behaviorally expressed motivational states (defined
without reference to consciousness) mediated by the actions
of neuromodulators on relatively simple circuits in Aplysia
have been suggested to have functional similarities to emotional states in mammals (Kupfermann 1979), and this is
consistent with properties of the conditioned fearlike state in
Aplysia described above. However, from a functional and
evolutionary point of view, there is no need for these states
to involve conscious experience by the animal.
In social animals, awareness and communication of injury can be advantageous, allowing not only the behavioral
alterations that guard or rest an injured body part (at the expense of other behaviors such as foraging) but also the recruitment of caregivers to help protect and provide for an
incapacitated individual during recovery. In nonsocial animals
(which may include all molluscs, as even those that aggregate do so opportunistically for exploitation of a local
resource, not for social interactions), obvious behavioral
191
changes resulting from injury may be neutral or maladaptive
(attracting the attention of predators or aggressive conspecifics). Thus it is unlikely that painlike states would have
evolved in molluscs to promote communication with potential caregivers.
Animals with high metabolic rates, such as squid, must
forage frequently to survive and thus cannot suppress active
behavior for very long during recuperation (when pain is often worst in mammals), so this common behavioral correlate
of pain would be maladaptive in such animals, although ongoing pain that promotes minor behavioral changes to protect
an injured appendage as a tradeoff against small reductions
in foraging success may be highly adaptive. Animals under
predation pressure (which includes almost all molluscs, and
particularly those lacking primary defenses such as a hard
shell or aposematic signals) probably derive minimal value
from sustained painful “feelings” if their expression renders
them more vulnerable to predators. Thus, while an acute response to injury promoting escape and survival should be
strongly selected, and long-term increases in sensitivity to
potentially threatening stimuli are likely to be adaptive (Walters
1994), persistent painlike states that profoundly alter ongoing behavior (e.g., decreasing foraging or mating activity)
may not be adaptive in molluscs.
Conclusions
All molluscs examined have shown a capacity for nociception as demonstrated by behavioral responses and/or by
direct recording from nociceptors and other neurons. Nociception and nociceptive sensitization at the level of primary
nociceptors make use of neuronal mechanisms that appear to
be highly conserved and widespread throughout the animal
kingdom.
But not all mechanisms related to nociceptive biology
are widely shared. For example, analgesiclike effects mediated
by true opioids and opioid receptors may be absent in invertebrates (Dores et al. 2002), and vertebrates may possess
some synaptic mechanisms that are absent in invertebrates
(Ryan and Grant 2009). Moreover, the sharing of many basic
molecular building blocks does not imply sharing of higherorder processes that depend on those building blocks. For
example, nearly all known brain functions in most phyla
depend on action potentials generated by the operation of
highly conserved sodium channels, but only a few species
have brains with the capacity to learn a spoken language or
do arithmetic—thus voltage-gated sodium channels are essential for learning German or solving equations, but their
presence does not imply the capacity for proficiency in German or algebra. At some level this must also be true for the
capacity to experience pain.
Immediate and longer-term neuronal and behavioral responses (including nociceptive memory and simple associative learning) can be mediated entirely by a single small
ganglion, such as the abdominal ganglion in Aplysia, proving that these effects do not need complex neural structures
192
(e.g., Antonov et al. 2003; Illich and Walters 1997). Some
gastropods, which have simple nervous systems compared to
cephalopods, exhibit changes in state with apparent functional similarities to emotional states, as illustrated by “conditioned fear” and “self-stimulation” in Aplysia and Helix,
respectively (Balaban and Chase 1991; Walters et al. 1981).
Given the capabilities of relatively simple molluscan
nervous systems, and if a key to the experience of pain is the
size and complexity of the nervous system, one must seriously consider the possibility that cephalopods can experience some form of pain. The most complex and behaviorally
sophisticated of molluscs, cephalopods have vastly more
complex central nervous systems, with up to 500 million
neurons, distinctive internal divisions of the brain, specialized
integrative regions where diverse sensory inputs are processed (Boycott and Young 1955), and dense, specialized innervation of the periphery (Hochner et al. 2006). If subjective
pain experience requires a minimal level of network complexity and processing power, these animals’ brains might
approach that level.
Scientifically accepted definitions of pain and nociception neatly distinguish these concepts (e.g., Merskey and
Bogduk 1994), but drawing a line between the two can be
difficult in practice. Furthermore, no experimental observation
of nonverbal animals (nonhumans) can demonstrate conclusively whether a subject experiences conscious pain (Allen
2004). Suggestive evidence for painlike experiences in some
animals is available, and nociceptive responses measured at
the neural and behavioral levels in molluscs have provided
evidence that is both consistent and inconsistent with painlike states and functions. Unfortunately, inferences drawn
from the relatively small body of relevant data in molluscs
are limited and prone to anthropocentrism. Identifying signs
of pain becomes increasingly difficult as the behavior and
associated neural structures and physiology diverge from
familiar mammalian patterns of behavior, physiology, and
anatomy, making interpretation of responses in molluscs
particularly difficult.
In the laboratory, molluscs are often subjected to manipulations that produce nociceptive responses, either as the aim
of an experiment or as a byproduct. Profound differences
between molluscs and mammals in the size, complexity, and
structure of their nervous systems, as well as their lifestyles
and evolutionary history, suggest that painlike phenomena, if
they exist in some molluscs, are likely to be quite different
from pain in mammals, although it does not follow that molluscs are incapable of experiencing pain. Gastropod and
cephalopod molluscs have shown long-lasting behavioral
alterations induced by noxious experience, which probably
involve motivational states that can be used flexibly to alter
defensive and appetitive responses. This suggests that some
molluscs may be capable not only of nociception and nociceptive sensitization but also of neural states that have some
functional similarities to emotional states associated with
pain in humans.
While it seems improbable that any mollusc has a capacity to feel pain equivalent to that evident in social mammals,
ILAR Journal
the existence of some similarities in nociceptive physiology
between molluscs and mammals, the paucity of systemic investigations into painlike behavior in molluscs, and the logical impossibility of disproving the occurrence of conscious
experience in other animals all suggest that it is appropriate
to treat molluscs as if they are susceptible to some form of
pain during experimental procedures.
In conclusion, we recommend that the design of experiments using molluscs, particularly those with larger and more
complex ganglia or brains (especially cephalopods but also
gastropods), take into account the possibility of a capacity
for painlike experience in these animals. Effective anesthetics (e.g., magnesium chloride) should be used during dissections and, to the extent possible, during any procedure that
produces tissue damage or possible stress. Investigators whose
experiments unavoidably produce noxious stimulation should
employ efforts similar to those required for vertebrate subjects to reduce both the number of animals and the potential
for suffering to the minimum needed to test their hypotheses.
Balancing the benefits from knowledge gained in molluscan experiments with the potential for inflicting pain and
distress in the experimental subjects should be an explicit
consideration in molluscan studies. (For related guidance to
IACUC members, we refer readers to Harvey-Clark 2011, in
this issue.)
Acknowledgments
Supported by NIH grant NS35979 (ETW) and a Marine Biological Laboratory Fellowship (RJC).
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Anesthesia, Analgesia, and Euthanasia of Invertebrates
John E. Cooper
Abstract
Introduction
Invertebrate animals have long played an important role in
biomedical research in such fields as genetics, physiology,
and development. However, with few exceptions, scientists, veterinarians, and technicians have paid little attention to the anesthesia, analgesia, and euthanasia of these
diverse creatures. Indeed, some standard research procedures are routinely performed without anesthesia. Yet various chemical agents are available for the immobilization or
anesthesia of invertebrates, ranging from gases or volatile
liquids that can be pumped into either an anesthetic chamber (for terrestrial species) or a container of water (aquatic
species), to benzocaine and other substances for fish. Many
invertebrates are not difficult to immobilize or anesthetize
and the procedures recommended in this article appear
to be safe; however, none should be considered totally
risk-free. Analgesia of invertebrates is as yet a largely unexplored field; until scientific data are available, other measures can promote the well-being of these animals in the
laboratory. For euthanasia, various methods (physical or
chemical or a combination of both) have been recommended for different taxa of invertebrates, but most have
not been properly studied under laboratory conditions and
some can be problematic in the context of research procedures and tissue harvesting. Furthermore, relevant data are
scattered, sometimes available only in languages other than
English, and there is no international approach for seeking
and collating such information. In this article I review various methods of anesthesia, analgesia, and euthanasia for
terrestrial and aquatic invertebrates, as well as areas requiring further research.
Invertebrates: An Overview
Key Words: analgesia; anesthesia; euthanasia; humane care;
invertebrate; laboratory animal; welfare
John E. Cooper, DTVM, FRCPath, FSB, CBiol, FRCVS, is a Diplomate of
the European College of Veterinary Pathologists and holds various academic
positions including those of visiting professor at the University of Nairobi
and associate lecturer in the Department of Veterinary Medicine at the
University of Cambridge in the United Kingdom.
Address correspondence and reprint requests to Dr. John E. Cooper,
Department of Veterinary Medicine, University of Cambridge, Madingley
Road, Cambridge CB3 0ES, UK or email [email protected].
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M
ost living animals belong to the large group commonly termed “invertebrates.” There are at least
1 million species of invertebrate compared with
4,000 mammals, nearly 10,000 birds, 6,000 reptiles, 6,000
amphibians, and over 25,000 fish. The vertebrates all belong
to one phylum (Chordata), whereas there are over 30 phyla
of invertebrate animals. They range from the single-celled
protozoan to the more complex and larger metazoan (multicellular animals) such as arthropods and molluscs. The
emphasis in this article is on the metazoan species, with
particular reference to those commonly used in research.
Most invertebrates are ectothermic (“cold-blooded”), although a few species (mainly moths [Lepidoptera]) that live
in cold areas or are active in the winter can generate metabolic heat to raise their body temperature sufficiently to
counter freezing. A similar adaptation is evident in bumblebees (Hymenoptera; Bombidae), which can raise their body
temperature by contraction of flight muscles.
Emerging Interest in Invertebrate Welfare
Although some species of invertebrates have been domesticated for hundreds or even thousands of years (Beavis 1988),
relatively little is known about the physiology and responses
of most (with the notable exception of the work by Young
and others on cephalopods; e.g., Young 1963), and therefore
only limited scientific evidence is available to guide research
workers.
Belief in Europe that some insects could be sentient goes
back at least to the Middle Ages. In Measure for Measure
William Shakespeare wrote that “the poor beetle that we
tread upon, in corp’ral sufferance finds a pang as great as
when a giant dies.” In the early 19th century, enlightened
naturalists began to express concern about the treatment
of animals. Some entomologists and collectors introduced
methods of killing insects that were thought to be more humane and in keeping with the rapidly developing philosophy
of “kindness to animals” (see, e.g., Stephens 1834). This
concept of giving invertebrates the benefit of the doubt was
remarkably far-sighted.
In the past 50 years, researchers recognized the need to
manage and work with invertebrates in the laboratory in a
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humane way. Early editions of the UFAW Handbook on the
Care and Management of Laboratory Animals (e.g., UFAW
1967) included chapters on the fruit fly, house fly, and blow
fly, the American cockroach, locusts, crickets, beetles, ticks,
and molluscs.
Until relatively recently, however, little attention was
paid to the health and welfare of invertebrates in captivity
(Cooper 1980, 1987a,b; Cooper and Zwart 2005, 225-245;
Frye 1986, 1990; Williams 2002). The situation is changing
largely because of the importance and value of these animals
when they are kept or managed in zoos, in private collections, and in conservation projects and because invertebrates
play an increasingly significant part in research (see below).
Wilson-Sanders (2011, in this issue) illustrates the range of
research, testing, and educational procedures in which invertebrates are used.
Nonetheless there is much debate as to whether invertebrates can feel pain, although most species show responses
to adverse stimuli (Cooper 2006). Many (e.g., the cephalopods) have a well-developed nervous system, and in some
species researchers have identified opioid systems similar to
those associated with pain sensation in mammals (Kavaliers
et al. 1983).1 Elwood (2011, in this issue) explores in detail
the hypothesis that invertebrates might experience pain,
carefully distinguishing such experience from pain reception
(nociception).
The focus of this article is the use of anesthesia, analgesia, and euthanasia in invertebrates that are kept for research.
Much of the text is also pertinent to the care of these animals
when they are kept in captivity for other purposes such as
display, exhibition, or pleasure (Cooper 1987a, 1998; Cooper
and Zwart 2005).
Anesthesia
Anesthesia of invertebrates is necessary either (1) to immobilize an animal to facilitate examination or sampling or
(2) to permit the performance of procedures (e.g., magnetic
resonance imaging; Figure 1) that may be stressful or painful (in such instances it may be used in conjunction with
analgesia).
In the past, research scientists and others working with
invertebrates tended to perform procedures without anesthesia or use only hypothermia, sometimes with little regard for
the animals’ well-being. This approach is probably not conducive to good experimental technique or accurate scientific
results. It is increasingly unacceptable to conduct invasive
procedures on invertebrates without some form of chemical
restraint, preferably through the use of an agent with known
anesthetic properties (Cooper 2001). There may also be legal
arguments for anesthetizing invertebrates when they are used
1It
is interesting, and perhaps pertinent, to note that electric fences are
among methods used to discourage molluscs from escaping from study
enclosures and to confine animals to trays in helicultural enterprises
(Symondson 1993).
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2011
Figure 1 Magnetic resonance image of an anesthetized Achatina
snail.
in research; animal welfare legislation in some countries
covers some or all invertebrates (ME Cooper 1987).
It is important for researchers and technicians who deal
with invertebrates to be familiar with and able to use appropriate anesthetic techniques for these species. The “higher”
(i.e., more complex) invertebrates are generally not difficult
to anesthetize, and most of the procedures recommended in
the sections below seem to be relatively safe; indeed, some
taxa, such as cockroaches, are remarkably resistant to hypoxia. However, as with other species, no method is totally
risk-free and the old British maxim that “there are no safe
anesthetics, only safe anesthetists” remains true, even when
one is dealing with invertebrates. Particularly in this context,
the scientist, technologist, or veterinarian who is familiar
with biology and natural history and who understands the
needs of the species has an advantage over the person with
no such knowledge.
The excellent book on invertebrate medicine edited by
Lewbart (2006; the second edition is in press, Lewbart 2011)
presents methods for the anesthesia of over a dozen different
taxa of invertebrates. For some that have been widely used in
research, such as the Nematoda (Bodri 2006), a whole range
of techniques is available and there is extensive supporting
literature. However, there is still, it appears, a dearth of information about the use of anesthetic agents in many groups.
For example, Foster (2006, 242) stated that “there are currently no known reports of anesthesia use in chaetognaths”
but, as with other such claims, it is not clear whether the
statement follows a full literature search in languages other
than English. Similarly, Smolowitz (2006, 74) claimed that
“little work has been done to identify appropriate anesthetic
agents” in gastropods and Lewbart (2006, 129) observed that
“there is little in the literature on anesthesia of leeches.”
Clearly, a more international approach is necessary for seeking and collating relevant information on anesthesia and
other topics relating to invertebrates.
The following methods of chemical immobilization for
terrestrial and aquatic invertebrates are recommended based
largely on my experience working with various species in
197
the laboratory, in zoological collections, and in veterinary
practice. I also draw on the observations of Zwart (both cited
in Fehr et al. 2005 and from personal communications).
Although it is not clear whether some of the agents documented produce a true state of anesthesia or merely immobilize the animal,2 they usually prove to be safe (in terms of
patient recovery), are generally well tolerated, and greatly
facilitate the handling and manipulation of individual animals and groups.
Terrestrial Species: Inhalation Anesthesia
Isoflurane (5–10%), sevoflurane, halothane (5–10%), or carbon dioxide (CO2; 10–20%) are now the most commonly
used agents for the anesthesia of terrestrial invertebrates.
They are administered by either a jar or, more appropriately,
an anesthetic chamber (Figure 2). A chamber for use with
insects, arachnids, and crustaceans is available (Applebee
and Cooper 1989), but such a sophisticated and relatively
expensive item is not essential. Suitable chambers can be
improvised from a variety of items, notably from “recycled”
plastic bottles that can be disinfected or discarded after use;
Pizzi (2006) describes the use of this method with spiders. It
is important to place gauze or something similar over the end
of the tubing in such systems because certain invertebrates
may attempt to escape (Chitty 2006).
Induction with an inhalation agent usually takes several
minutes but can vary according to the species and the ambient temperature. Recovery can be prolonged (2–5 hours) but
is usually uneventful.
Many arthropod species can be anesthetized safely with
inhalation agents (Figure 3) and some are remarkably tolerant
to hypoxia. In one unpublished trial I exposed American cockroaches (Periplaneta americana) to 100% CO2 for 2 hours;
recovery took several hours but there were no fatalities. Cockroaches may be unusual in this respect, but I have also found
the routine use of 20% CO2 or 10% isoflurane, halothane, or
sevoflurane to be successful and safe in several other species
of arthropod. If a procedure is considered to be potentially
painful, there may be merit in using isoflurane, halothane, or
sevoflurane rather than CO2 because the extent to which the
latter induces analgesia in invertebrates is not known, and its
use in vertebrate animals is controversial because of concerns
about its effects on the animals’ health and welfare.
Isoflurane is probably the current agent of choice for
anesthetizing terrestrial invertebrates. Chitty (2006) described its use in myriapods and Pizzi (2006) in spiders, and
Bodri (2006) included it in his extensive formulary of anesthetic agents for nematodes.
Terrestrial or amphibious molluscs are often best anesthetized using water-soluble agents (as discussed below).
Snails and slugs can be placed foot down in a glass recepta-
Figure 2 An anesthetic chamber (18 cm deep × 22 cm long × 15 cm
wide) suitable for terrestrial invertebrates.
cle with 0.5 to 5 cm of water (depending on the animal’s
size) containing the chemical.
Aquatic Species
Noga and colleagues (2006) provided an excellent table of
agents that can be effective for immobilizing/anesthetizing
crustaceans and many of the agents and techniques they advocated are likely applicable to other aquatic taxa. Based on my
experience, the following information applies to freshwater
and marine species as well as temperate and tropical aquatic
species (Figure 4). At high (tropical) temperatures, induction
of and recovery from anesthesia usually occur more rapidly,
in keeping with the ectothermic nature of invertebrates.
Anesthetic agents are best administered to invertebrates
in a specific container. I use a clean glass container that, depending on the size of the animal, holds between 0.5 and 5.0
liters of fluid (Figure 5). The water in the container can
either be from the animal’s own tank or, preferably, made up
fresh. The latter must be carefully prepared, especially for
2In
this article the term “anesthesia” implies that an animal is rendered
insensitive to noxious stimuli; “immobilization” means that the animal
cannot move but may or may not be aware of such stimuli.
198
Figure 3 Inhalation methods are effective with large metazoan
species, such as this anesthetized mygalomorph spider.
ILAR Journal
remain wet; in addition to being placed on a damp surface
(Figure 6), it can be sprayed periodically either with the water
that contains the anesthetic agent (to maintain anesthesia) or
with freshly prepared oxygenated water (to hasten recovery).
Oxygenated water is produced by pumping pure oxygen from a
cylinder for 5 minutes through chlorine-free water. I have never,
knowingly, encountered “gas bubble disease” in my invertebrate patients but the possibility must be considered.
Recovery from absorption anesthesia generally takes
place in 30 minutes or more, after which the animal can be
returned to its own tank.
Carbon Dioxide
Figure 4 Aquatic species, such as this cuttlefish (a cephalopod),
need careful handling prior to and during anesthesia to avoid damage to delicate structures such as the mantle.
marine species, for which salt concentration can be crucial.
The water in the container should be at the same temperature
as that from which the animal is transferred. If necessary,
anesthesia can be induced in the animal’s home tank, although complications can arise with this approach if several
invertebrates are present, not all of which require anesthesia,
or if there is a filtration and water circulation system that can
dilute or remove the anesthetic agent.
Absorption Anesthesia Using Tricaine
Methanesulfonate (MS-222) or Benzocaine
Tricaine is soluble in water, but benzocaine must first be dissolved in acetone; in each case 100 mg of the anesthetic
agent is generally used per liter of water. Various species
(e.g., coelenterates) can be anesthetized safely using tricaine
(Stoskopf 2006).
Once immobile, the animal is removed and can usually be
kept out of the water for 10 to 15 minutes, during which it must
Figure 5 An anesthetic container and other equipment for the anesthesia, examination, and microbiological screening of aquatic
species.
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2011
Carbon dioxide offers a reliable and relatively inexpensive technique for the “anesthesia” of aquatic invertebrates. Although
doubt exists about the degree of humaneness and analgesia
induced by CO2, in practice the technique is well tolerated in
invertebrates and appears not to compromise their welfare; it
has, for example, been used on many occasions to immobilize,
examine, and swab medicinal leeches (Hirudo medicinalis)
kept for medical research (Cooper et al. 1986; Cooper 2001).
However, if CO2 is used in water, the resulting changes in pH
can lead to an increase in acidity that may be injurious to a sensitive skin (or gill) and may influence research results.
Carbon dioxide can be bubbled from a cylinder through
water in a suitable container (usually a glass or plastic jar)
and this has the advantage of some control over concentration. Alternatively, the water can be diluted 50:50 with commercial soda water. In a real emergency, a product such as
Alka-Seltzer that liberates CO2 gas can be used but this
should not be considered good practice.
Other Agents
Various other agents have been tried in different invertebrates (see Lewbart 2006). For example, chloretone, ethanol,
Figure 6 This flaccid leech is kept moist while it is out of the water
after removal from its anesthetic jar.
199
and chloroform can be used in water to immobilize various
taxa of invertebrates (Fehr et al. 2005). Scimeca (2006) provided valuable information on the anesthesia of cephalopods and recommended magnesium chloride and ethanol,
either separate or combined, for this purpose. Magnesium
chloride is also effective for marine polychaetes (Lewbart
2006), isobutanol and lidocaine can be given by injection to
crustaceans (Noga et al. 2006), and saturated mephenesin
has been used to anesthetize leeches (Tettamanti et al.
2003).
Limited or Discontinued Methods
Hypothermia can be used in many species to slow the animal’s metabolic rate and to facilitate handling and noninvasive techniques, such as surface sampling. Thirty minutes in
a refrigerator (+4ºC) is usually sufficient for this purpose but
other techniques can be effective; for example, nematodes
can be chilled using a stream of carbon dioxide (Bodri 2006).
However, hypothermia should not be used for surgical or
other invasive procedures. Some invertebrates (e.g., certain
Solifugae, or camel spiders) do not appear to tolerate chilling very well and can die as a result (Pizzi 2006).
The concept of using chemical agents, especially inhalable substances, to immobilize invertebrates is not new—for
hundreds of years apiarists have used smoke to calm honeybees (Apis mellifera) (Figure 7). As mentioned earlier, scientists studying fruit flies (Drosophila melanogaster) have
long used anesthetic ether to immobilize their subjects, to
the extent that strains of Drosophila were categorized on the
basis of whether or not they were “ether sensitive.” But despite its long history and many good features, the use of
ether is no longer advisable for any living organisms.
Traditionally in work with Drosophila, “etherizers”
were developed to anesthetize the flies for experimental
purposes (Demerec and Kaufmann 1962). These sometimes allowed precise administration of ether through the
use of a rubber bulb attached to a chamber containing a wad
of cotton or gauze soaked in the agent; a squeeze of the bulb
forced relatively uniform volumes of ether-air mixture
through rubber tubing into the chamber holding the flies.
For larvae, nitrogen gas was often used. But Zwart (cited in
Fehr et al. 2005) warned that ether, on account of its irritancy, should not come into contact with the head of the
animal and cited chloroform as another possible agent.
Snails (Helix aspersa) exposed to ether vapor demonstrate
an adverse response in the form of marked production of
mucus (Peer Zwart, University of Utrecht, personal communication, 2005). Another alternative, methoxyflurane,
proved useful in various species in the past (Cooper 1985)
but is no longer readily available.
Before and During Anesthesia
Vertebrates and invertebrates should be carefully observed and
if necessary examined and sampled before the administration
200
Figure 7 Use of smoke to calm honey bees.
of an anesthetic agent (Figure 8). An unhealthy animal, or
one that is in poor body condition or dehydrated, is less
likely to tolerate the procedure.
Assessment of depth of anesthesia in invertebrates is problematic and few guidelines exist. Immobility and the loss of a
righting reflex are usually indicators of full anesthesia. Some
species, such as certain Lepidoptera, can “sham death” or go
into a state of immobility that does not necessarily indicate
that they are anesthetized. Twitching of limbs, antennae, and
palps and the presence of muscle tone are features of both induction and recovery in many terrestrial species but are unreliable signs (or perhaps have not been properly studied). Aquatic
invertebrates show similar changes but with marked variation.
In my work on the anesthesia of leeches, I assessed responses
on the basis of (1) whether the leeches remained attached to
the side or bottom of the holding jar, (2) the speed and extent
of the animal’s swimming movements, (3) muscle tone (i.e.,
the extent to which the leeches, when removed, were turgid or
flaccid), (4) whether the caudal sucker was functioning, and
(5) response to stimulation (touching and handling). In gastropod molluscs, a response to pricking of the foot can be used as
an indicator (Cooper 2001; Cooper and Knowler 1991).
ILAR Journal
Figure 8 Preanesthesia examination of a Helix snail to look
for external damage or other lesions and to assess muscle tone and
behavior.
Anesthetic Emergencies
Occasionally problems (e.g., accidental overdose by inexperienced personnel; Cooper 1998) arise during anesthetic procedures and prompt action is necessary to revive the animal
and save the experiment (thus preventing the need to repeat
it and to use additional animals).
If a terrestrial invertebrate takes a long time to recover, or
appears to be dead, it should be returned to the anesthetic chamber and exposed to pure oxygen for 10 to 30 minutes. Failure to
show any signs of recovery 12 hours later—and even then, only
with signs suggestive of death such as rigor mortis or a fetid
odor—is a clear indication that the organism is dead.
As indicated earlier, some species of invertebrate can be
very refractory to hypoxia. Zwart (personal communication,
2005) has questioned whether artificial respiration, by exerting pressure on the body, might help in eliminating inhaled
anesthetic agents, especially in insects where respiration is
maintained by altering the shape of the thorax at each action
of the flight muscles.
Similar emergency measures may be effective with
aquatic species, but the O2 should be bubbled through the
water, either continuously for 10 to 20 minutes or intermittently for 1 to 2 hours.
Recovery
Animals should receive supportive care during and after anesthesia, but as yet little is known about the needs of different invertebrates. As in all such work, an understanding of
the physiology of the species is helpful.
Maintenance of fluid balance is particularly important, as
it is in all animal species. Most invertebrates are small, with a
large surface area in proportion to body mass, and are therefore prone to desiccation. Spiders and some other species will
imbibe water or saline given orally with a pipette or syringe
(Figure 9). In describing the use of isoflurane in myriapods,
Volume 52, Number 2
2011
Figure 9 Fluid (saline) is administered per os to a spider to aid
rehydration.
Chitty (2006) advocated humidification of the vapor because
centipedes are very susceptible to dehydration; similar precautions could be adopted with other sensitive species.
Terrestrial gastropods require special consideration as they
are especially susceptible to desiccation. Indeed, they have developed various strategies to conserve water. One such strategy
is “feces-sitting” (Bleakney 1991; the snail rests with its muscular foot on a pile of feces); fresh feces are 80% water and the
snail may limit fluid loss by remaining in contact with them.
Fluid balance is obviously less of a consideration in
aquatic species. However, it can be an important factor if, for
example, surgery or a wound in the animal’s integument permits the ingress or egress of fluids and electrolytes.
Analgesia and Other Methods to Promote
Invertebrate Welfare
For decades scientists and welfarists have debated whether
invertebrates can experience pain or discomfort (Alumets
et al. 1979; Cobby 1988; Cooper 2006; Fiorito 1986;
Wigglesworth 1980), and the debate is likely to continue
(see Elwood 2011). Experiencing pain and “suffering” from
it may not be the same. Until more is known, there would
appear to be merit, on both scientific and humanitarian
grounds, for minimizing the extent to which laboratory invertebrates are exposed to adverse stimuli that may prove
“stressful” or painful. As stated earlier, whenever possible
the animals should be afforded the benefit of the doubt.
Judging from the available literature in English, French,
and German (e.g., Gabrisch and Zwart 2005), the use of analgesics in invertebrates does not yet appear to be feasible.
One alternative is to use anesthesia for any procedures that
might possibly be painful or that may necessitate either
prolonged restraint or disruption of the animal’s normal
behavior.
Whenever invertebrates are kept for research or study,
it is helpful to draw up codes of practice—quite apart
201
from any legal requirements—to help promote high standards of care and welfare (CCAC 1982; Collins 1990; Invertebrate Working Group 1990). There are also practical ways
to maintain the well-being of invertebrates in the laboratory:
•
•
•
Provision of an environment and social groupings that
match, as closely as possible, those that the species favors in the wild. If in doubt, a choice of environmental
parameters and habitat should be offered.
Avoidance of unnecessary or insensitive handling or
restraint.
Maintenance of high standards of management, preferably by personnel with a genuine interest in and knowledge of invertebrates and their care.
Data on behavioral needs and the effect of stressors on
survival are available for some taxa and should be used in
efforts to enhance the well-being of invertebrates in the laboratory. Such information is available for millipedes (Bailey
and Kovaliski 1993; Dangerfield and Chipfunde 1995) and
molluscs (Cowie 1985), and Smith and colleagues (2011, in
this issue) provide detailed information about laboratoryreared cephalopods. For the latter, the provision of a correct
environment is crucial and, no less than the type of research
procedures, may determine whether the welfare needs of
such species are met in the laboratory.
Indeed, an emerging approach to humane care as a means
to not only enhance welfare but also reduce pain—in both
human and veterinary medicine—incorporates physical
therapy and environmental changes as well as the administration of anesthetics, analgesics, and other drugs.
Mather (2011, in this issue) discusses moral and ethical
approaches to invertebrate use in detail. She emphasizes the
importance of knowing about the biology of the invertebrates
with which one works in order to recognize “suffering.” She
further stresses the need for personnel to be properly educated in this respect so that they can participate knowingly in
decisions about the management and use of invertebrates in
laboratories.
Euthanasia
Official texts on the euthanasia of experimental animals
have generally made little or no mention of invertebrates
(e.g., Commission of the European Communities 1993;
Working Party 1996, 1997). In contrast, for over 60 years
the UFAW Handbook series, to its credit, has not only
covered laboratory invertebrates but also incorporated information on anesthesia and euthanasia for many species;
for example, the section on Birds, Poikilotherms, and
Invertebrates (UFAW 1967) included methods for “tranquillizing and killing insects and ticks” as well as more
specific notes on the “narcotisation and anaesthesia” of
molluscs.
Animal welfare publications, on the other hand, increasingly include discussion of the euthanasia of invertebrates.
202
For example, some years ago the World Society for the Protection of Animals (WSPA 1994) cited various methods for
killing crustaceans, ranging from pithing (of crabs) and electronarcosis (crabs and lobsters) to chemical euthanasia (all
species), and criticized the use of hot or boiling water to
prepare crustaceans for cooking. The killing of surplus or
diseased invertebrates in zoological collections has also attracted attention; among others, the Invertebrate Working
Group (1990) provided guidance on humane methods to kill
terrestrial species (e.g., in butterfly houses) and more recently Hackendahl and Mashima (2002) considered the euthanasia of aquatic invertebrates.
In the research literature, various methods of euthanasia
have been recommended for different taxa of invertebrates
(Lewbart 2006), but most have not been properly studied.
However, information is available about some of the idiosyncrasies of ectothermic animals, specifically reptiles and
amphibians; for example, methods that involve severing the
nervous tissue (i.e., the spinal cord in vertebrates) may or
may not be relevant to “higher” invertebrate taxa (Cooper et
al. 1989). Zwart (cited in Fehr et al. 2005) provided a table
of both physical methods (e.g., decapitation of species with
a well-defined head) and chemical methods (e.g., the injection of snails with pentobarbitone). Zwart (personal communication, 2005) has also found that the pH of commercially
available pentobarbitone can vary between 9.5 and 11.0 and
that, when injected into the hemocele of snails, these unbuffered substances produce a rubbery ball of coagulated protein that, inter alia, can make the tissue unsuitable for
histological examination. It seems likely that such an effect
is injurious, and possibly painful, in the live snail.
For the euthanasia of spiders, Pizzi (2006) recommended immersion in 70% ethanol. He warned against
rapid freezing of these (and other) invertebrates because
the resulting tissue damage compromises histological examination, an important consideration in invertebrates kept
for research purposes.
In summary, methods of euthanasia for invertebrates are
inadequately researched and warrant more attention (Cooper
2006; Murray 2006).
Conclusions
As the Council of Europe’s Charter on Invertebrates (Pavan
1986) pointed out, invertebrates are the most important component of wild fauna—providing food and contributing to
agriculture and forestry (e.g., providing useful models for
mathematical and other studies on genetics and biodiversity),
medicine, and industry and crafts (e.g., as the basis of designs
for carvings, bee hives, furniture, carpets, curtains). Moreover,
they are increasingly attracting interest among researchers. As
I have expressed in the context of the care of invertebrates in
zoos and in private hands (Cooper 2006), I believe that the
care of these animals in the laboratory should be of as high a
quality as that for vertebrates and that this care should be reflected in the research in which these animals are used.
ILAR Journal
It is worth noting that over 100 years ago (January 5,
1901), an editorial in the Veterinary Record declared that, for
anesthesia, “The use of chloroform by practitioners is now
common, and the amount of pain and suffering prevented is
enormous. By its aid we are enabled to perform operations
with success which without it were seldom satisfactory.” A
century later, sophisticated methods of anesthesia are used
routinely in both veterinary medicine and laboratory animal
science and those words seem quaint. Yet the ability to use
such methods on invertebrates remains very much at a comparable embryonic stage, as is widespread knowledge of appropriate methods of euthanasia. Research in these areas is needed
to rectify this situation and to ensure both invertebrate welfare
and quality research. Furthermore, with the increasing globalization of research, methods should be established for the
collation and translation of information available only in languages other than English.
Acknowledgments and Dedication
I am grateful to Sally Dowsett for typing this article and to
my wife, Margaret Cooper, for providing most of the photographs and for commenting on the legal section. My friend
and former colleague Ken Applebee produced images of the
anesthesia chamber and agreed to their use. Peer Zwart, a
pioneer of invertebrate medicine and pathology, has been an
ongoing source of inspiration and encouragement. Cameron
H. Fletcher, Managing Editor of the ILAR Journal, has been
most helpful and considerate during the metamorphosis of
this contribution: merci beaucoup!
This paper is dedicated to all veterinarians, research workers, and animal technologists whose interest in invertebrates
has led them to promote a better understanding of the health,
welfare, and conservation of these oft-neglected creatures.
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ILAR Journal
Philosophical Background of Attitudes toward and Treatment of Invertebrates
Jennifer A. Mather
Abstract
People who interact with or make decisions about invertebrate animals have an attitude toward them, although they
may not have consciously worked it out. Three philosophical
approaches underlie this attitude. The first is the contractarian, which basically contends that animals are only automata
and that we humans need not concern ourselves with their
welfare except for our own good, because cruelty and neglect demean us. A second approach is the utilitarian, which
focuses on gains versus losses in interactions between animals, including humans. Given the sheer numbers of invertebrates—they constitute 99% of the animals on the planet—this
attitude implicitly requires concern for them and consideration in particular of whether they can feel pain. Third is the
rights-based approach, which focuses on humans’ treatment
of animals by calling for an assessment of their quality of
life in each human-animal interaction. Here scholars debate
to what extent different animals have self-awareness or even
consciousness, which may dictate our treatment of them.
Regardless of the philosophical approach to invertebrates,
information and education about their lives are critical to an
understanding of how humans ought to treat them.
Key Words: cephalopod; consciousness; contractarian; ethics;
invertebrate; nociception; pain; rights; utilitarian
I
t is probably not surprising that people’s attitudes toward
invertebrates, and indeed all animals, are human-centered.
After all, as infants we are first aware only of ourselves,
truly egocentric (Berk 2000). Gradually we learn that there is
a world separate from us, and by age 3 or 4 a child is busy
exploring and cataloguing it. Later, in adolescence we go
through another stage of egocentrism where we focus tightly
on our place in this burgeoning universe. So anthropocentrism,
the attitude that we are the measure of everything and the universe revolves around us, is predominant in human thinking.
This attitude is fostered by Western science (see Balcombe
2010). Western society is individual rather than communally
Jennifer A. Mather, PhD, is a professor in the Department of Psychology at
the University of Lethbridge in Alberta, Canada.
Address correspondence and reprint requests to Dr. Jennifer A. Mather,
Department of Psychology, University of Lethbridge, C888 (University
Hall), 4401 University Drive, Lethbridge, Alberta T1K 3M4, Canada or
email [email protected].
Volume 52, Number 2
2011
focused, emphasizing competition instead of cooperation and
thus reinforcing an individual focus and increasing the egocentrism. Furthermore, there is a strong belief in the objectivity of this view, so that it is not critically evaluated but
assumed to be correct. This is supported by the Judeo-Christian
view of humans as having dominion over the earth and all
things in it. Some have pointed out that this dominion should
mean protection and care, but in practice the industrial complex has used it as an excuse for exploitation.
This attitude goes hand in hand with the arrangement of
animals on a scala naturae, a sort of tree of life with “lower”
organisms at the base, rising through simpler vertebrates to
the peak—primates and, of course, humans. This hierarchical approach originated with Aristotle as a way of logically
organizing all life, and was taken up by the Christian church.
God was at the top of the chain of life, indicating perfection,
and humans were the next step down, working toward it
(Balcombe 2010).
Surprisingly, there are modern versions, now with three
peaks, showing insects, molluscs, and vertebrates increasing
in neural complexity toward the apex of intelligence. Even
now, people talk of “highly evolved animals” at these peaks.
Of course this is untrue—simpler animals like nematode
parasites and blind cave fishes are very highly evolved for
their demanding environment. And horseshoe crabs, whose
fossils can be found in rocks from the late Ordovician, are an
enduring model that may survive when recent “generalists”
like octopuses and humans have passed on.
No animal is better than any other, yet studies (Bekoff
1994; Eddy et al. 1993) show that humans value intelligent
animals and those similar to us over all others. For example,
although invertebrates comprise 99% of the animals on the
planet, Eddy and colleagues (1993) proved the vertebratecentered view by choosing only 3 invertebrates for their
sample of 30 animals.
Whatever the particular bias, attitudes toward animals
have a philosophical and ethical basis, even though those
who hold them may not realize or explore the meaning of
their attitudes toward animals in general and invertebrates in
particular. There is a lot of variation, but the attitudes can be
roughly categorized as contractarian, utilitarian, and rights
based (Nussbaum 2001).
The Contractarian Approach
This philosophy presumes the complete separation of humans and nonhuman animals. Writing long ago, Descartes
205
proclaimed that humans merited consideration in terms of
pain and suffering because we have souls; all other animals
did not have souls and thus were no better than automata
(Balcombe 2010). He was supported in this by the Christian
church, which sanctioned much cruelty to animals.
Disconcerting as it is to those who care for animals, this
approach has one advantage: it asks us to evaluate the effects
of our interactions with animals for our own good. Rollin
(1985), talking about treatment of animals for research, places
the responsibility for and benefit of ethical behavior on our
shoulders and argues that we must act fairly toward animals.
As people who control animals’ lives, we must treat them well
not because they “deserve” it but because it demeans us not to.
For example, he asks undergraduate students using shock as a
deterrent in their animal studies to first try the shocks on themselves, so that they are aware of what they are doing. He calls
on all humans to be moral actors.
Even industries that inflict suffering on animals can minimize it in the name of moral action. A vertebrate-centered
example of this approach is that of Temple Grandin (1995),
who has described her understanding of the worldview of
cattle and has developed a career in designing holding facilities and slaughterhouses for them. She has designed places
that minimize their daily stress and the trauma of death and
notes her satisfaction in knowing that they are as well cared
for as possible.
Grandin also discusses the effect of working in such
facilities on people. The Jewish man who conducts ritual
slaughter of kosher beef considers this a calling and an act of
religious piety; and Grandin (1995) sees his attitude as reflecting a need for ritual and respect at the death of animals,
a need that is present in many cultures. Even so, routine killing can’t help but affect those who do it, and may engender
internalized pain or repudiation as a means of emotional or
psychological protection from the reality of large-scale animal slaughter. Grandin advises slaughterhouse managers to
rotate the actual job of killing to enable workers to retain a
moral stance—so that the act of killing never becomes commonplace and no one gets callous about life because of it.
A further challenge is that the modern, urbanized, technical world removes humans farther and farther from organisms
of all kinds. In addition, Balcombe (2010) suggests that media
coverage deliberately distances us from animals and distorts
the understanding of their lives by focusing on the “excitement” of the chase and the drama of death. But octopuses, for
example, are predators that actually spend three-quarters of
their daytime lives resting or sleeping, which doesn’t make
“good press” (Mather 1988). Balcombe (2010) also points out
that, in order to make ourselves seem more important, we use
demeaning and uncomplimentary words not only to describe
animals but also to insult humans—think of “beastly,” “brutish,” “savage,” or even simply calling someone “an animal.”
How much more true this uncaring attitude is toward invertebrates! It is not just that their importance is seen as much less
than their numbers indicate, but also either that they are simply
not considered (Bekoff 1995) or, in the case of insects, that we
are uneasy and even fearful around them (Hardy 1988).
206
The most positive of human attitudes toward animals is
Wilson’s (1984) “biophilia.” He writes that humans should
gain an “inherent human affinity for life and lifelike processes” and that we will act ethically if we appreciate the
living inhabitants of the planet in all their diversity. Again,
the point is not the type of animal but that all animals benefit
when we care. Wilson (1984) has a long way to go to persuade us to love all animals.
The Utilitarian Approach
A Gain-Loss Approach to Animal Ecology
The utilitarian approach to the importance of animals is objective: humans should assess gains and losses when making
any decisions and judgments about animals. According to
this practical view, the dominance of invertebrates in terms
of number of animals means that they are critical to the survival of life on the planet and should therefore be respected
and protected (New 1993).
Kellert (1993) makes the point by emphasizing the value
of invertebrates in waste decomposition, as food for humans
and for the organisms that humans eat, as sources of chemicals and drugs of immense benefit to humans, and as indicators of the health of ecosystems. Recent prominent
developments illustrate this close-knit relationship between
invertebrate and human welfare. Observing the decimation
of bee colonies by disease, experts pointed out that bees’
pollination enables the production of many human food
crops. And coral reef animals, which form the backbone of
one of the most productive marine ecosystems (Ponder et al.
2002), are threatened by a number of human actions.
Assessing Pain as a Cost
The gain-loss equation applies to evaluation of the impact of
human actions in particular situations and on specific animals or populations. For example, what are the impacts of
fishing, keeping animals in aquariums, and using them as
experimental subjects? Unfortunately, it is often the human
gains that are the major consideration and the losses to animals secondary.
Debate has focused especially on the possibility that humans inflict pain and suffering on animals and on how these
conditions can be assessed in nonhuman animals. Evaluation
of emotions in animals is fraught with subjectivity; indeed,
many Western scientists refused until quite recently to even
speculate that animals had emotions at all because it was not
possible to properly prove their existence. Griffin’s (2001)
efforts over many years have helped but the problem of evaluating animal emotions is a huge one still.
The Significance of Pain versus Nociception
Pain is valuable from an ecological perspective as it allows
an organism to be aware of danger and to avoid situations in
ILAR Journal
which damage might occur or recur. Yet it is not a simple
sensory modality. Even in humans, who can describe their
physical sensations, it is not easy to understand (see Matlin
and Foley 1997 for a textbook description). Merskey’s
(1986) definition of pain as “information about actual or potential tissue damage, or interpreted in terms of such damage” conveys the variability of the sensation.
The experience of humans shows that receptor signals of
damage are not automatically processed and passed undistorted to the central nervous system and brain. There are
many examples of rituals, sports, and wartime experiences
in which humans are not aware of painful major tissue damage for minutes or even hours (Matlin and Foley 1997);
clinically diagnosed pain disorder (American Psychiatric
Association 2000) is not tightly associated with actual damage, only triggered or even unaccompanied by actual damage; and phantom pain, the sensation from a lost limb, is the
result of a central representation that endures after the periphery is no longer sending signals. Because of all these
variations in pain, it is obvious that it has sensory, emotional, and cognitive aspects in humans, and these make it
difficult to identify in nonhuman animals, whose ways of
communicating discomfort are often less well understood
by humans.
One way to separate these different aspects of pain experiences is to define and evaluate nociception, the purely sensory experience of the damage signal (Kavaliers 1988; also
see Elwood 2011, in this issue). It is too simple to suggest
that nonhuman animals have only nociception, especially if
they exhibit learning and expectation of future stimuli—and
many invertebrates do demonstrate at least simple learning.
Evidence of Pain in Vertebrates (Fish)
Braithwaite’s (2010) study of whether fish feel pain shows
how such an investigation could be carried out for invertebrates. She notes that fish, as vertebrates, should have structural and brain similarities with mammals that experience
pain, and her assessment of the anatomy of receptor systems
similar to those of other vertebrates does show a clear parallel. She looks at data about brain regions and finds that, although the specific brain arrangement is different among the
vertebrate classes, similarly functioning regions are present
(this is harder to show in invertebrates). She then measures
responses to stimulus situations that would be considered
painful for mammals and demonstrates clearly comparable
results, though of course not in brain location.
In light of her observations, Braithwaite asks whether
pain-inducing stimuli trigger differences in responses that
matter to the animals’ daily lives, such as whether a fish is
willing to tolerate the possibility of electric shock so that it
can school with conspecifics (the answer is different for two
fish species). The evidence indicates that it is important to
look at interference with nonreflex (i.e., “planned”) behaviors and evaluate investigation of novelty as an example of
such behavior.
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2011
Given the positive answers for these studies, Braithwaite
concludes that yes, fish do feel something like pain.
Assessing Pain versus Nociception in Invertebrates
This structural parallel between fish and other vertebrates
makes it easier to prove pain in those species than in invertebrates, but an examination of nociception across different
invertebrate phyla is a good starting point.
Cnidarian sea anemones, for example, have stinging cells
that they use on each other and that are also painful to humans and repellent to animals of many species (Braithwaite
2010; Mather 2001). The anemones live in clones (groups
of identical individuals), holding fast to the rocks of the
seashore and catching drifting small animals and detritus.
Clones that encounter each other have “wars,” stinging each
other and inflicting considerable damage. They flinch from
these attacks and eventually one clone retreats from the other
and is the loser in the encounter. These animals have no centralized brain and only a nerve net, yet they clearly exhibit
nociception in this situation.
The anemones’ weapons are also used by other animals,
in situations that seem to demonstrate learning. Hermit crabs
pull anemones off the substrate and place them on their
borrowed gastropod shells where they repel both crabs and
octopuses, which learn to avoid the crabs as prey (Maclean
1983). One study showed that chemical stimuli in water that
contained an octopus stimulate hermit crabs to put anemones
on their shells (Ross and von Boletzky 1979), presumably to
repel predators. However, there is no evidence that the hermit crab learned this behavior.
The physiology of stress responses is clearly similar
across many phyla. Stefano and colleagues (2002) point out
that the immediate rise in immunocytes and a later increase
in opiates in mussels and leeches subject to cold water shock
are not only normal stress responses but the same as found in
humans after coronary artery bypass surgery. These animals
also show adrenocorticotropic hormone (ACTH) downregulation of immunocyte activation, again similar to that of
mammals. Even more interesting is the heart rate increase of
juvenile queen scallops (Aequipecten opercularis) under
predation threat when on a substrate that offered no refuge
(Kamenos et al. 2006). And the heart rate of mussels increases in response to a chemical cue in the effluent of their
predator, the dog whelk (Nucella lapillus; Rovero et al.
1999). While these observations are scattered, they make it
clear that the physiological systems are very similar across
widely diverse vertebrate and invertebrate animals.
Elwood (2011) and his associates are the only researchers that have explicitly studied whether the nociception that
an invertebrate shows to noxious stimuli could be extended
to pain. The first study (Barr et al. 2007) was on prawns’
antennal grooming. Prawn antennae are crowded with tactile
and chemical receptors and are a major area for evaluation
of waterborne sensory stimuli. Application of chemicals or
gentle pinching caused grooming of the specific antenna and
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rubbing against the substrate, which was reduced by application of the local anesthetic benzocaine (which did not
reduce general arousal, only antennal grooming). Because
the grooming is a targeted and not a generalized response,
the authors suggested that it parallels the responses of fish
and other vertebrates in similar situations and can be considered evidence of pain.
Elwood (2011) argues that separating immediate nociceptive responses from longer-term cognitively guided pain
responses will always be difficult because pain is such an
imperative system that response is usually immediate. According to the utilitarian approach, the solution is simply to
accept the importance of minimizing the effect (i.e., “costs”)
of these stimuli on animals, whatever their perception of the
stimulus might be.
The Rights-Based Approach
According to the rights-based approach both the agent and
the receiver in any interaction have value and thus deserve
consideration and respectful treatment (Regan 2003). This is
the only one of the three viewpoints that focuses on the individual and accords animals the right to bodily integrity and
physical liberty. The spotlight is thus on the experience of
the animals themselves.
Nussbaum (2001) suggests taking into account an animal’s life, health, physical integrity, and emotional well-being. Such a perspective raises some difficult questions, such
as whether it is defensible to exhibit animals (including invertebrates) in aquariums and zoos, thus depriving them of
liberty. And one of my colleagues asked whether this approach requires consideration of the potentially conflicting
welfare of a parasite or its host.
Furthermore, it is necessary to understand the basic
physiology of the animal itself, whereas such knowledge is
missing for most invertebrates. How can humans protect the
right of the clam, the luna moth (Actias luna), or the nereid
worm without understanding how the animal lives? Davis
and colleagues (1999) make this point about the ascidians, a
primitive chordate that is common but whose ecology is very
poorly known: we might like or need to protect them if we
knew how.
The rights-based approach demands close study of the
experiences and awareness of animals and evaluation of the
situations to which humans subject them. Does the animal
have the learning capacity to recognize and respond to a
stimulus that signals an event? Does it have the self-awareness to know how trouble will affect it and the mobility to
avoid trouble? The animal’s capacity to learn is important;
Bekoff (1994) points out that suffering might be less bearable without cognition, which remembers the past and plans
for the future, only dealing more effectively with an unpleasant present. Many people say that invertebrates do not have
consciousness or self-awareness, so it is important to examine their capacity in this area to evaluate what humans’ treatment of them might mean.
208
According to Broom (2007, 99), a sentient animal is
“one that has some ability to evaluate the actions of others in
relation to itself and third parties, to remember some of its
own actions and their consequences, to assess risk, to have
some feelings, and to have some degree of awareness.” This
account of cognition and awareness certainly applies to cephalopod molluscs, which studies have shown are heavily
dependent on learning (Alves et al. 2008; Wells 1978) and
may have consciousness (Mather 2008). The following sections therefore focus largely, but not exclusively, on these
invertebrate species.
Self-Referencing, Self-Awareness, and
Self-Consciousness
Bekoff and Sherman (2004) propose three levels of understanding of self: self-referencing, self-awareness, and selfconsciousness. They believe that an animal’s fit in these
categories is dictated more by its behavioral ecology (e.g.,
whether it is social) than its brain size (within limits) or phylogenetic derivation. And presumably the animal’s place in
these categories should dictate the treatment it receives from
humans. For instance, a Korean-style restaurant in New York
City cooks mixed shellfish and octopuses alive in a frying
pan at the table. Do some of these animals or none of them
deserve this treatment?
Self-Referencing
Self-referencing is the matching of a target individual to
oneself and does not usually involve learning or cognition.
Courtship of hetero- or homosexual individuals entails the
identification of species, sex, and readiness to reproduce before mating actually takes place.1 Dual-sexed hermaphrodites may compete with one another to see which will be
which sex in the reproductive act (Anthes 2010). Simple
awareness of self and of the identity of the target animal is
necessary.
Self-Awareness
Self-awareness involves recognition that one is a self and
that conspecifics are others, as well as a sense of one’s place
in the world or possessions (e.g., shelter, territory). The animal may demonstrate learning and cognition, and the factors
that influence its “decisions” may be simple or complex.
Hermit crabs, for example, fight one another over the possession of shells in which to hide their vulnerable abdomen;
they evaluate new shells for different durations and fight
others with different intensities in different circumstances,
including whether the contended shell is of a more desirable
species (Elwood 1995). There may be evidence that they
1Although
some marine animals do not even do this—sessile species such
as coral and bivalves use broadcast fertilization, simply releasing gametes
into the water.
ILAR Journal
assess both their ability in relation to a rival and the “value”
of the shell possession to calculate whether a fight should
proceed (Elwood 2011).
Further demonstrating self-awareness, cephalopods are
excellent navigators for short distances (Alves et al. 2008),
and some of their ability to locate themselves in the environment is learned. Field studies of octopuses (Mather 1991a)
showed they forage freely across the ocean bottom, returning to a sheltering “home” from a distance and over a time
that necessitates spatial memory. They can return home by
detours even after they have been displaced from their foraging path (Mather 1991b). They also remember which areas
they have been foraging in over the last few days and do not
repeat searches in these locations where no prey is likely to
be found.
Interestingly, cephalopods’ “decision” of whether to
consume a prey species in hiding near the capture location or
whether to take it home to consume it is based on the distance to the home (Mather 1991a), clearly an indication of
the awareness of oneself and one’s relative location. This
ability has also been proven in the laboratory for octopuses
and cuttlefish, and is an interesting parallel with the spatial
ability of bees and mammals (Shettleworth 2010), suggesting parallel competence across several phyla.
Self-Consciousness
What of self-consciousness, which likely involves cognition? The mirror test has become the critical evaluation of
this capacity in primates (Gallup et al. 2002) and lately has
been used on other vertebrates, although not without controversy (Moses 1994). A simple version of the test is to expose
the animal to its image in a mirror and evaluate reaction. A
more stringent test is to make an innocuous mark on a nonvisible area of the animal’s body (e.g., its head), then to expose the animal to a mirror image of the area and see if it
touches the mark.
Cephalopods have the best potential of self-consciousness of all the invertebrates, as they exhibit exploration and
play, personalities and problem solving (Mather 2008).
Octopuses exposed to mirrors show alerting and approach
behavior, no different from their reactions to a view of another octopus (Mather and Anderson, submitted).
Two problems have been cited concerning the exposure of
animals such as octopuses and cuttlefish to mirrors, assuming
their failure at the task (Bekoff and Sherman 2004; Mather
and Anderson, in press). First, although octopuses have excellent visual acuity, they may not depend on that sense in the
same way that mammals do. Second, they are generally asocial
and so may not have complex behavioral responses to either
the image or the actual presence of another octopus.
Deception
Another way to evaluate invertebrate animals for self-awareness is to look at whether they show deception. Again the
Volume 52, Number 2
2011
ability comes in three levels, with the simplest and most
automatic being permanent deception (e.g., visual or other
sensory camouflage); many invertebrates have a deceptive
appearance—for example, the lemon yellow dorid nudibranch gastropod (Tochuina tetraqueta) is a perfect match
for the yellow sponges on which it lives and feeds. Similarly,
kelp crabs (Pugettia producta) are excellent matches for the
algae on which they live, and several species of shrimp
mimic their gorgonian resting places exactly. Some insects
have exquisite matching to aspects of the environment such
as leaves and sticks, and moths mimic the bark of trees on
which they hide. But these deceptive appearances are permanent and selection is the machinery that determines them; no
learning or cognition is present.
A second level is deception based on time and place.
Cephalopods with their changeable skin show such ability
with ease and often with a wide repertoire of displays for use
in different situations. One example of such deception is the
deimatic dots on the dorsal surface of cuttlefish (Langridge
et al. 2007) and squid (Mather 2010). These dots appear on
the skin surface in the presence of a low-level threat, as when
a potential but not imperatively dangerous predator approaches. The cuttlefish shows two dots and the squid two of
four on their large dorsal surfaces, presumably mimicking
the eyes of a larger animal, as the dorsal surface is often
turned toward the approaching fish. Indeed, there is a significant correlation of appearance of lateral dots with the direction of the approaching fish, as the animals exhibit dots on
the part of their body that can be most easily seen by the
potential predator. Squid do not direct such warning displays
toward conspecifics but toward a chosen target (Mather
2010), thus they seem not to be automatic but chosen.
True deception occurs when animals give misinformation about resource-holding power or their ability to win
contests. Caribbean reef squid (Sepioteuthis sepioidea) have
“honest” formalized zebra display contests, in which the intensity of the display is greater on the squid taking a position
above, and that individual is the one that claims resources in
terms of consortships with a nearby females (Mather 2004).
There is no deception, as the animals trade places if the display of the lower animal is more intense. Juvenile squid,
however, respond to the mating displays of an adult pair by
engaging the male with a high-intensity zebra that does not
accurately represent its ability to win a fight. Such a deceptive display intensity might mean that the juvenile later was
able to mate with the female.
Similarly, male cuttlefish have alternate morphs (skin
displays) when they are courting a female (Hall and Hanlon
2002). One of these displays signals dominance, and the
male sets up a consortship and later mates with the female.
An alternate deceptive strategy is for a male to adopt the
same display as female cuttlefish and become a “sneaker,”
changing its body pattern to then court and mate with a female when her consort is temporarily away. Unfortunately,
Hall and Hanlon (2002) did not continue to watch sneakers
over time, so they did not observe whether tactics differed
from one individual to the next or changed with maturity (for
209
a discussion of alternate reproductive strategies, see Taborsky
and Brockmann 2010). In reef squid, males adopt peripheral
positions and sneaking strategies as subadults, then join visual
display contests and hold consortships as they grow to maturity
(Mather, unpublished observations).
A striking example of deception over time is the mantis
shrimp fighting technique (Caldwell 1986). Individuals occupy
and often successfully defend their burrows in the substrate.
The meral claw strike is dangerous, so shrimp seldom actually fight but favor visual displays to indicate their size and
ability to hold the burrow. When females lay eggs or when
any shrimp molts, their resource holding potential is considerably reduced and fights often result in displacement from
a burrow. Weakened individuals instead use a meral claw
spread to indicate size and resource holding potential. They
also initiate fights and are more aggressive in other ways just
before they molt, enhancing their “reputation” just before it
could most easily be challenged.
Scientific Evidence in Support of the
Rights-Based Approach
Given this evidence that many invertebrates may have simple
sentience sensu Broom (2007), and that the rights theorists
believe that these animals have the right to a full, rich life,
how should humans behave morally when interacting with
them? It is helpful to look beyond simple evaluations and tap
into the life history of the animal to see to what extent its
natural behavior and needs are fulfilled in captivity.
Moltschaniwskyj and colleagues (2007) offer wide-ranging guidance for appropriate care of a variety of cephalopod
species. They also urge use of the 3Rs (reduce numbers of
animals, refine procedures, and replace animals with alternatives) for invertebrate subjects in experimentation. This advice is important because there is a trend to replace vertebrate
animals in experimental work with invertebrates; but this reduces animal welfare concerns only if people believe invertebrates are not aware of the consequences of many human
actions.
For example, crabs caught in commercial pot traps are
declawed and then returned to the ocean (Patterson et al.
2007, 2009), a practice that seems justified by the fact that
crabs sometimes autotomize a claw when threatened by a
predator; declawing is thus seen as “natural” and not detrimental to the crab. The opposite turns out to be true: the effects are extensive and change several important aspects of
the crabs’ lives. Studies show that claw removal is a significant stressor (Patterson et al. 2007), raising the level of glucose, lactate, and glycogen in the hemolymph much higher
than baseline both in handled crabs and in crabs induced to
autotomize. Furthermore, glucose and lactate were higher
and glycogen lower when crabs were placed with conspecifics after claw removal. Crabs are aggressive with one another and the crab with one or more claws missing, whether
by autotomy or removal, is at a significant disadvantage to
compete for and hold high-quality territories.
210
Not only do crabs without a chela move down in the hierarchy, they are at a disadvantage in feeding. Crabs with
only one claw had significant difficulty consuming mussels,
their common prey (normally the crab holds the mussel with
one chela and crushes it with the other). In captivity they
consumed a lot of alternative food (pieces of fish), but this
option is likely not available in the wild. Some of the declawed animals even died from hemolymph loss and others
would have starved or lost significant amounts of weight.
Thus an intervention that looks like a harmless mimic of
a natural situation is instead devastating to all aspects of the
crab’s life. Patterson and colleagues (2009) therefore strongly
recommend that this method be stopped.
The Importance of Supporting
Species-Specific Normal Behavior
For rights theorists, even benign captivity can deprive an
animal of its rights, and acting morally means ensuring all
animals a full and complete life, including the rights to appropriate housing, stimulation, reproduction, and feeding
opportunities (see Broom 2001 for an extensive discussion
of these topics mostly in mammals).
The need to carefully choose appropriate species for captivity was recently highlighted by Mason (2010; although
she ignored the invertebrates, as pointed out by Carere et al.,
in press). Again a good example is the cephalopods, for
which Moltschaniwskyj and colleagues (2007) note clear
limits in knowledge of their biology. They point out that ommastrephid squid suffer 100% mortality in rearing and are
therefore not suitable for captivity.
Octopuses, for example, explore and learn well, play,
have personalities, and solve problems (Mather 2008). Is it
right to keep these intelligent animals in a barren, restrictive
aquarium tank? Studies clearly show that enrichment makes
a difference to their biology: captive young cuttlefish in an
enriched environment grew faster and ended up larger than
those in a barren one (Dickel et al. 2000).2 Enrichment for
these (and many other) animals helps to maintain healthy
activity levels, alleviate the effects of confinement, and enable animals to pursue species-specific normal activities.
A particular risk for intelligent and social animals is boredom (Wemelsfelder 1993). Common symptoms of boredom
are repetitive route retracing, constant attempts to break out of
confinement, and abnormal sleep and resting patterns. Octopuses in captivity are well known for their ability to escape
from the confinement of their tank, taking advantage of the
manipulative ability of their arms. Aquarium owners use a
wide variety of techniques to confine them, not always successfully. The first recorded event was in the Brighton Aquarium
over 100 years ago, when the captive octopus visited a
neighboring tank and dined on a lumpfish each night for several nights before returning to its home. But bored octopuses
2Wells
(1978), however, has argued that octopuses are used to hiding in a
protective home and moving out for food, and that their solitary lifestyle
makes them preadapted to thrive in captivity.
ILAR Journal
can also be destructive; Anderson (2005) describes an octopus
that attacked her tank by repeatedly moving rocks around and
scratching the glass, blowing gravel up from the bottom, and
finally biting through the ties holding the undergravel filter in
place, pulling it up, and tearing it into pieces, which were
found floating at the water surface the next morning.
One way to provide enrichment for octopuses in an
aquarium is to provide substrate that is comparable to the
animal’s natural environment, with rocks to make a sheltering home and gravel (cuttlefish bury in gravel and should
never be deprived of it; Mather 1986). Enrichment for octopuses also includes the provision of novel objects for manipulation and play (Anderson and Wood 2001). Octopuses
will take apart any complex many-pieces object, and the
children’s Mr. Potato Head toy is a favorite at several
aquaria.
If appropriate and at all possible, enrichment should also
prepare the animal for release into its natural environment;
for instance, hatchery-reared salmon are notoriously unaware of predators and are easily picked off (Brown and Day
2002). Cephalopods should receive live prey so that they can
exercise their natural predatory response.
Anderson (2000), for example, describes the release of
the giant Pacific octopus (Enteroctopus dofleini) Ursula, who
had come to the Seattle Aquarium by donation when she was
very small. She was growing too large for her aquarium tank,
so her keepers fed her live crabs for several days before her
release to give her the opportunity to catch appropriate prey.
When she was released (to a huge blast of publicity), divers
followed her progress down to the seafloor below the waterfront aquarium. The divers checked on her for the next 40
days and noted the remains of three species of local crabs
piling up in front of her den, so she was hunting successfully.
When she was last seen, three males were also observed near
her, so it is likely that she also fulfilled the rights criterion of
being able to reproduce.
Thus it is clearly possible with captive octopuses to accommodate the special needs of an intelligent invertebrate.
Conclusion
Although theorists have not necessarily thought specifically
of invertebrates in postulating humans’ attitudes toward animals, human attitudes are important to the welfare of these
(and all) animals. Contractarian theorists value our humanness in caring about animals, utilitarians consider the importance of the 99% of animals that invertebrates represent, and
rights theorists’ concentration on animals’ essential needs is
useful for enriching the everyday lives of invertebrates.
Education is key (Meehan 1995): as invertebrates are
better understood, people—whatever their value system—
will come to appreciate and take better care of them.
Acknowledgments
Thanks to Roland Anderson for years of research partnership.
Volume 52, Number 2
2011
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ILAR Journal
IACUC Challenges in Invertebrate Research
Chris Harvey-Clark
Abstract
A Passion for Invertebrates?
With billions of individuals and possibly hundreds of thousands of genera, invertebrates represent the largest number
and greatest diversity of all animals used in research. Although the capacity for nociception is recognized in many
invertebrate taxa, researchers and IACUC members are challenged by a lack of clear understanding of invertebrate welfare and by differing standards of moral concern for these
taxa. In practice this has led IACUCs to consider invertebrates in two major groups: species worthy of increased
moral concern approximating that shown to vertebrate species (this group includes cephalopods and to some extent
decapod crustaceans) and all others. This dichotomy has led
to differences in how invertebrate research is regulated and
documented. This article presents two case studies illustrating specific concerns in invertebrate research protocols and
then provides relevant information to address practical
IACUC matters related to regulatory and ethical issues,
sourcing and record keeping, risk management, assessment
of pain and nociception in invertebrates, housing and husbandry, invasive procedures, veterinary care, and humane
endpoints.
Introduction
The sheer number of invertebrate species is what impressed
the biologist Haldane. Within one order (Coleoptera) of the
phylum Arthropoda there are over 300,000 species, and the
total numbers of arthropod taxa—in excess of 750,000 species—outnumber all other animals on the planet threefold.
Invertebrate species range from comparatively simple
single-cell life forms (e.g., protozoa) to colonial aggregations of cell types (e.g., sponges and coelenterates) to
complex animals that share morphological and physiological convergence with vertebrates (e.g., cephalopods and
crustaceans).
Some of the longest-lived colonial and noncolonial animals on the planet are invertebrates: the common red sea
urchin (Strongylocentrotus franciscanus) may live 200 years
(Ebert and Southton 2003); it has been estimated that vestimentiferan worms found near deep-sea hydrothermal vents
live up to 250 years, and some corals and sponges may be
thousands of years old (Bergquist et al. 2000).
Invertebrates in Research
A
fundamental concern of institutional animal care and
use committee (IACUC) members is the welfare of
all animals used in research regardless of their phylogenetic position. The increase in knowledge of functional
and comparative genomics has revealed extensive genetic
homology between humans and other species and underscores the fact that although there are great differences, there
are also fundamental similarities in all eukaryotes. Yet with
invertebrates humans seem to be more aware of the differences than the similarities, notwithstanding the vast numbers
of organisms and variety of species that populate every corner of the planet, accounting for over 90% of animal
biodiversity.
Chris Harvey-Clark, DVM, is Director of the Animal Care Centre at the
University of British Columbia in Vancouver, Canada.
Address correspondence and reprint requests to Dr. Chris Harvey-Clark,
Animal Care Centre, University of British Columbia, 6199 South Campus
Road, V6T 1W5 Vancouver BC, Canada or email chclark@interchange.
ubc.ca.
Volume 52, Number 2
The Creator would appear as endowed with a passion for
stars, on the one hand, and for beetles on the other.
– JBS Haldane (1949)
2011
Invertebrates are used both in a wide range of field research
on biodiversity and conservation and in the laboratory as
animal models for a variety of science questions. Uses range
from acute toxicity assays in aquatic invertebrates such as
Hydra and Daphnia to invasive neurophysiology in the sea
hare Aplysia. Wilson-Sanders (2011) describes a large number of well-defined invertebrate models using fruit flies and
the nematode C. elegans in diverse research areas such as
drug screening, cell death, aging, retrovirus biology, memory, muscular dystrophy, Parkinson’s disease, wound healing, aging, amyloidosis, programmed cell death, diabetes,
and immunology.
The diversity of invertebrates requires particular IACUC
care in dealing with research ethics issues. A central challenge
is the lack of scientific consensus on what constitutes pain and
suffering in these species and whether they are applicable to
even “advanced” invertebrate species. Elwood (2011) discusses
efforts and methods to distinguish between pain and nociception in invertebrates, and Crook and Walters (2011) elaborate on
the nociceptive behavior and physiology of molluscs; but
213
scientific evidence of pain and suffering in invertebrates remains poorly researched and controversial. This subject is discussed in greater detail in the section below on the Challenge of
Assessing Pain and Nociception in Invertebrates.
IACUC Invertebrate Protocol Case Studies
The following case studies illustrate the challenges that
IACUC members confront in their review of protocols for
research involving invertebrates. These cases raised questions about proper scientific procedure, animal welfare, containment, handling and human safety, and attitudes toward
invertebrates. Both cases underscore the importance of
thoughtful and informed IACUC review.
Case Study 1
The Protocol
An IACUC received a protocol from a newly recruited principal investigator (PI) working with orb-weaving spiders.
The PI proposed to conduct single-cell electrode recordings
from the giant ganglia in the spider’s leg, which he proposed
to pull off without the use of anesthesia. One or two legs
would be sufficient for each day’s recording sessions, and
each spider would yield a total of eight legs over the course
of 1 to 2 weeks. The PI asserted that this was an accepted and
widespread practice among neurophysiologists working
with this model. He also indicated that stepping on the spiders was the preferred method of physical euthanasia.
IACUC Concerns and Resolution
The IACUC was sufficiently concerned about issues of both
human safety and animal welfare that they requested a meeting with the investigator, a neurophysiologist, to discuss the
protocol.
As a justification for the technique proposed the PI made
the point that this species of spider has a natural detachment
line at the base of each leg for separation of limbs by autotomy and does so to avoid predation in the wild. He also
stated that a number of investigators worldwide use this
technique and that it is accepted practice in his discipline.
When asked how many legs could be harvested, he answered
“all the legs.” When asked if the spider would be able to selffeed with fewer than three or four legs, he was unsure. When
asked if he did indeed step on the spiders to euthanize them
he indicated that he had been facetious and that they were
generally killed with ether in a bell jar.
The committee consulted with the institutional veterinarian who cited evidence in the veterinary literature that arachnids could be anesthetized with veterinary gas. The PI, while
concerned that this could result in the accidental death of
valuable animals before their full use for research purposes,
214
agreed to work with the veterinarian to refine the model, use
gas anesthesia for the removal of legs, and restrict the number of legs removed after recovery to four, after which the
last leg removal would be a terminal procedure. The PI
agreed to use gas anesthetic overdose for euthanasia (the use
of ether was prohibited because of the risk of explosion).
The IACUC included a member from the department of
occupational health and safety who asked the PI about safety
measures for the handling and use of venomous spiders. The
PI indicated that there was no specific antidote for the venom
of the spiders and that their venom was not fatal in humans but
analogous to a wasp sting. The room the animals were held in
had not only an insect escape–proof anteroom and doorway
for secondary containment but also a primary screen-sealed
holding container for each spider. The room was locked and
secure at all times and labeled that it contained venomous animals. The animals were physically handled by the PI wearing
gloves and using a set of long forceps.
Finally, the PI was informed that the protocol form was a
legal record and that facetiousness was inappropriate, and he
agreed to modify the form before approval to reflect best
practices as discussed.
Case Study 2
The Protocol
The IACUC was approached by a PI who had been contracted
by a company involved in the transportation of live lobsters.
The purpose of the protocol was to determine the critical maximum and minimum temperatures that caused mortality in
lobsters during transport. The proposal was to raise or lower
the water temperature by 5°C per hour until the lobsters
stopped moving, which was the proposed endpoint.
IACUC Concerns and Resolution
The committee was concerned about the project, which was industrially driven and lacked any form of academic review (although it was fully funded), and requested a peer review of the
protocol. The IACUC members met with the PI and discussed
the endpoints. It was agreed that a more precise endpoint would
be the point at which the lobster stopped responding to a gentle
pinch to the antennae and/or failed to right itself when inverted.
The temperature change regime would be fixed at 2°C per hour.
The IACUC agreed to a pilot study of five animals until the
behavioral/welfare issues could be assessed through observation by a subcommittee of the IACUC, which would decide on
a full project based on the pilot results.
Attitudes and Their Impact on
Regulations and Review
As Mather (2011) eloquently explains, how animals are used
in research depends not only on the regulatory and ethical
ILAR Journal
environment but also on attitudes about which species are
considered worthy of moral concern. It is clear from a survey
of the literature that in practice invertebrates tend to be
ranked at the lowest levels of moral concern for living animals; indeed, some institutions require no formal review of
their use for research, although funding approval generally
entails at least a scientific merit review and institutional approval (primarily vested in the IACUC) is usually linked to
the release of research funds.
Among IACUCs, many members adopt a binary approach to invertebrate welfare, classifying the animals as
“advanced” invertebrates (i.e., cephalopods and occasionally
decapod crustaceans) and all other species. Boyle (1991, 7)
explained the basis for the special status conferred on some
cephalopod species, in particular octopuses:
In the laboratory, numerous experimental studies have
described remarkable capabilities of sensory discrimination, especially of visual stimuli; they have demonstrated
that true learning occurs and is likely to be an integral
part of the normal life of an octopus. These qualities of
behavioral complexity, sensory discrimination and learning in cephalopods bear comparison with those of many
lower vertebrates and provide ample cause for considering their welfare in the laboratory for humane and scientific reasons.
However, in the absence of evidence to the contrary, such a
distinction is not appropriate and all invertebrates should receive care and treatment that ensure their welfare.
International Regulations and Guidelines
At the regulatory level, fish and invertebrates are excluded
from the US Animal Welfare Act, for example, although
Public Health Service (PHS)–approved research protocols
require ethical justification for all animal use (PHS 2000).
Thus whether invertebrates meet the legal definition of an
animal under this policy is unclear. In Canada, Canadian
Council on Animal Care guidelines for the use of invertebrates are more specific in articulating ethical concern for,
and IACUC oversight of, research using advanced invertebrates such as cephalopods (CCAC 1991):
Protocols must be submitted to an appropriate review
committee for all studies and courses which involve the
use of vertebrates and some invertebrates in Categories B
through E. Cephalopods and some other higher invertebrates have nervous systems as well developed as in some
vertebrates, and may therefore warrant inclusion in Category B, C, D, or E.
The CCAC guidelines, which have been translated and
used as the basis for animal care practices in a number of
countries, do not specify which “other higher invertebrates”
are indicated based on nervous system complexity, but in
practice some IACUCs extend this guideline to species of
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2011
arthropods, notably decapod crustaceans. Otherwise, the
CCAC categorizes experiments using “most invertebrates”
at the lowest invasiveness level (Category A1), similar to
studies that involve the use of tissues collected after slaughter of domestic animals.
In the United Kingdom, the Cruelty to Animals Act 1876
specifically excludes invertebrates. On the other hand, the
Council of Europe issued a “Charter on Invertebrates” that
recognizes the “compelling positive values of invertebrates
including their use in science and medicine” (New 1995,
16).
As a further example of the relatively limited moral concern for invertebrate use at the undergraduate level, Youth
Science Fair Canada (YSF 2010) regulations governing students’ use of animals in science fairs permit the unrestricted
use of bacteria, fungi, protozoa, insects, plants, and invertebrate animals, but are far more restrictive where vertebrate
animals are concerned, prohibiting their use in any fashion
that may be “deleterious to the animals.”
IACUC Review
Many institutions have developed a policy of reviewing all
protocols involving invertebrate use, whereas other IACUCs
may refuse to review invertebrate protocols in jurisdictions
where invertebrates do not meet the legal definition of “animal.” When IACUC review of invertebrate research is not
strictly required legally, some investigators object to it as an
unnecessary regulatory burden. Review is nonetheless appropriate as part of the due diligence of research oversight
and principles of parsimonious, ethical animal use. One institution, San Jose State University, has a voluntary invertebrate protocol review policy that embraces this concept:
For the use of invertebrate species, the IACUC requires
review and approval of projects that entail permission
from a government agency to access, collect, or deploy
the species being studied…. The provision for IACUC
approval of invertebrate studies also extends to work involving animal species considered venomous or a threat
to public health, endangered, threatened or of special
concern [Endangered Species Act, 1972] and for projects
involving invertebrate species in which the pain and distress category is considered a category level V (as defined
by the IACUC for vertebrate species…). All other invertebrate studies do not require IACUC review and approval.
However, it is highly recommended…that investigators
pursue committee approval for graduate or research projects to be kept on file with the University Animal Care
office.2
1Category
A covers “experiments on most invertebrates or on live isolates,”
including, for example, “the use of tissue culture and tissues obtained at
necropsy or from the slaughterhouse; the use of eggs, protozoa or other singlecelled organisms; experiments involving containment, incision or other invasive
procedures on metazoa” (from the CCAC website, http://ccac.ca/en_/standards/
policies/policy-categories_of_invasiveness, accessed on April 4, 2011).
2Available online (www.sjsu.edu/gradstudies/iacuc), accessed on April 4,
2011.
215
The value of a conscientious IACUC in oversight of the
use of invertebrates in research cannot be overstated, reflecting the leadership and overall institutional conscience concerning the use of all animals.
Special Considerations for the IACUC
Permits, Collection, and Transfers
Many species of invertebrates used in research may be exotic
to the region where they are being used, where they may
represent a threat to biodiversity or a direct disease or pest
threat not only to other laboratory animals but also to agriculture and aquaculture, so their housing and containment
may be subject to local, regional, and federal regulation.
In the United States fish and wildlife regulatory authorities in each state typically enforce requirements for collection permits and annual reports of animals collected from the
wild. In coastal regions of Canada, the use of laboratoryreared cuttlefish other than in complete containment systems
is subject to licensing by the federal Department of Fisheries
and Oceans (DFO) and permits are issued under the local
DFO Introductions and Transfers Committee. This committee delegates the responsibility to local DFO aquaculture
authorities and veterinarians to inspect and regulate the proposed holding facility for the nonendemic species; such
inspection may entail visits, effluent water containment,
continuous effluent water decontamination and testing, and
testing of the introduced species for the presence of unwanted or reportable disease entities.
For at-risk, threatened, or endangered invertebrates or
those listed by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) there
may be additional layers of federal or state/provincial permitting required.
Sourcing Invertebrates
The sources, transportation methods, procedures for quarantine, and disease monitoring methods for invertebrates are as
diverse as the animals themselves. Some species are available
from mainstream laboratory or biological supply sources, together with turnkey life support holding systems, special diets, and good information sources for standards maintenance.
For some species, laboratory-reared pathogen-free specimens are available from a well-defined source (e.g., the National Resource Center for Aplysia; http://aplysia.miami.edu/)
analogous to the commercial suppliers of research rodents in
terms of microbial and genetic integrity. Other species may be
available only from the wild, pet stores, or specialized hobbyists, or they may be obtained in ways that are on the margins
of the comfort zone for the average IACUC member. In these
instances communication between the PI, the IACUC members, and the animal care and veterinary personnel is particularly important.
216
Record Keeping
Many institutions require little record keeping for the research use of invertebrates other than cephalopods. IACUC
forms for invertebrate use are often abbreviated and contain
minimal information, limited to the name of the PI, the species used, the end use of the animals in teaching or research,
brief descriptions of the techniques used in the study, disposition of the animals at the end of the study, and safety issues
if isotopes or hazardous chemicals are involved.
In Canada, Invertebrate Protocol Forms for the majority
of invertebrate species (other than cephalopods) may simply
report the species used, numbers of animals used, and the
grant number of the PI. Furthermore, institutions are not obligated to keep records of the numbers of invertebrates used,
although they are not discouraged from developing animal
use practices and culture for the use of invertebrates, such as
record keeping, standard operating procedures (SOPs) for
the care and maintenance of the animals, and IACUC review
of animal care protocols. For cephalopods, the categorization of protocols and their ethical review parallel those for
the use of vertebrates, with requirements for anesthesia, reasonable aseptic conditions for surgery, postoperative monitoring, and endpoint definition.
Numbers of Invertebrates Used
Apart from cephalopods, the numbers of invertebrates used
for research are seldom tracked, at least in part because many
of the species studied (Drosophila, C. elegans, Artemia) may
teem in uncountable millions in a single research laboratory,
let alone in an institution, region, or country, and accurate
accounting for such numbers is not feasible. When the research involves advanced invertebrates such as cephalopods
or rare and endangered invertebrates of greater economic or
ecological value, there may be more proactive efforts to track
the numbers of animals used for purposes of accounting to
regulatory agencies.3
Risk Management: Toxic and Injurious
Species, Field Work
Invertebrates can be a hazard to people and the environment,
capable of escape, harmful environmental impacts if introduced as pest species, and injury to humans through physical
or chemical means (e.g., the venomous bite of the blueringed octopus, the sting of a lion’s mane jellyfish, the venom
of certain spiders used in neurophysiology). The IACUC
needs to be satisfied that the PI and holding facility can
maintain hazardous invertebrates in safe conditions for animals and humans alike (more on this under Housing and
Husbandry below).
3However,
a review of the CCAC figures for animal use in 2008 showed
that, of 2,272,815 animals used, just 7 were cephalopods; it is likely that this
figure represents underreporting.
ILAR Journal
Risks are inherent in some types of field and laboratory
work, for example in scuba diving for specimen collection or
census purposes and in the handling of venomous or injurious species. Such work requires primary and secondary containment, escape mitigation plans, emergency supplies and
training, and special training for first responders and other
individuals not directly involved with the project who may
need to access the holding area in an emergency. In all cases
it is important to enumerate such risks and to mitigate them
by taking precautions at the institutional and local levels to
ensure that (1) SOPs, equipment, and animal holding/housing are in place and approved for the safe handling of the
species in question, (2) antivenin and other emergency treatment supplies are on hand, (3) medical personnel are identified and familiar with the risks and treatment, and (4) records
are maintained through the institutional occupational health
and safety program.
The Challenge of Assessing Pain and
Nociception in Invertebrates
Because IACUCs represent the ethical conscience of their
institutions and on a wider scale reflect the morality of the
culture and community where they exist, it is essential that
their members understand and address issues of pain and
suffering in invertebrates when making decisions about their
research use (see Cooper 2011; Elwood 2011). Such understanding begins with a grasp of the distinction between pain
and nociception in animals.
The International Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage or
described in terms of such damage” (Merskey and Bogduk
1994). The cognitive-emotional component of pain, which
requires higher brain center function through the limbic system in vertebrates, is considered its important aspect, not the
activation of pain sensors (nociceptors). “Activity induced in
the nociceptive pathways by a noxious stimulus is not pain,
which is always a psychological state, even though…pain
most often has a proximate physical cause” (Merskey and
Bogduk 1994). Under this definition, species capable of feeling pain are those that have not only the nociceptors but also
the brain neuroanatomical and neurophysiological features
and behaviors that allow the expression of fear, anxiety, distress, and terror, akin to human and other advanced vertebrate responses to noxious stimuli.
Many species of invertebrates, including annelids, nematodes, molluscs, and insects, have the capacity for nociception and withdraw from damaging stimuli (St. John Smith
and Lewin 2009), and in some species there are putative connections between nociceptors and brain learning centers
(Puri and Faulkes 2010; Sandeman et al. 1992). In molluscs
the central nervous system is not required for pain withdrawal reflexes, which can be mediated by the peripheral
nervous system (Crook and Walters 2011). In leeches, specialized sensory nociception cells induce bending away from
Volume 52, Number 2
2011
damaging stimuli, and the bending reflex can function with
as few as 10 abdominal ganglial cells (Sahley et al. 1994). In
arthropods, recent work suggests that nociceptive fibers, like
all other sensory neurons, can be inhibited by a variety of
neuromodulators (Pfeiffer and French 2009). And studies in
Aplysia provide evidence that nociception and sensitization
can lead to long-term influences on memory and behavior in
invertebrates (Woolf and Walters 1991).
However, the presence or modulation of nociception
does not conclusively demonstrate that invertebrates are capable of experiencing the mental/emotional state of pain that
is acknowledged in many vertebrate species. Imaging studies in mammals expressing painlike behaviors in response to
noxious stimuli have demonstrated activation of brain areas
(e.g., the limbic system) associated with emotion (Malisza
et al. 2003). This type of cause and effect evidence is lacking
in invertebrates, and the poorly understood nature of invertebrate nervous systems makes proof by analogy difficult.
Some insect neurobiologists have argued that the distributed nature of the invertebrate nervous system (except in cephalopods) precludes higher-order information processing,
including the existence of any kind of mental state (Eisemann
et al. 1984). Invertebrates other than cephalopods also have
much lower (by two to three orders of magnitude) numbers
of neurons compared to vertebrates, a difference that could
preclude the ability to experience mental states such as emotion and pain, and there is not much evidence in the scientific
literature of an emotional state akin to “distress” in invertebrates such as insects.
Importantly, none of the evidence cited is conclusive in
either confirming or refuting the possibility of pain and distress homologues in invertebrate species. Clearly this is fertile ground for study. In the absence of conclusive results,
however, there is no evidence that the scientific community
should ignore the welfare of invertebrates used as research
animals.
Standards of Care
Given the exceptional diversity of invertebrates, housing and
husbandry needs can range from accommodation for the
very large (e.g., for Enteroctopus dofleini at 40 kg) to the
very small and even microscopic (e.g., for Drosophila or C.
elegans). The IACUC, which in most institutions likely deals
only occasionally with invertebrate protocols, needs to concern itself with the details of species-specific requirements
and of the life support systems, containment measures for
hazardous species, and experience and training of the personnel who will maintain the animals.
The myriad details of invertebrates’ basic care requirements, food, a healthy and enriched environment, and safe
handling for both operators and animals is beyond the scope
of this paper. Smith and colleagues (2011, in this issue) provide an overview of the maintenance of invertebrates; there
is an abundance of literature on the care, maintenance, and
housing of various invertebrate species, from the laboratory
217
animal, zoo/aquarium, wildlife rehabilitation, and pet and
hobbyist literature; and the Association of Zoos and Aquariums (www.aza.org) has guidelines for the maintenance of
many species of invertebrates in captivity.
In addition, both the IACUC members and the scientists
using invertebrates usually benefit from mutual education,
the former through laboratory visits and other methods to
become familiar with the particular invertebrate models in
use, and the PI through protocol writing workshops and
other opportunities to interact with the IACUC and educate
its members.
or biofiltration) and other treatments such as ozonification.
For all marine invertebrate species there should be an emergency backup for life support systems.
The importance of measures to maintain water quality
cannot be overstated; in one case in the author’s experience
zoonotic Vibrio vulnificus was isolated in pure culture from
the anterior chamber of cuttlefish (Sepia officinalis) exhibiting bilateral hypopyon as a primary symptom. The source
of the Vibrio was intake seawater from a sewage-polluted
source in a marine flow-through system.
The CCAC has extensive guidelines for maintaining
marine species on its website (www.ccac.ca).
Housing and Husbandry
Housing and Containment
Housing design is important for both the provision of life
support and, particularly in the case of insect pest and toxic/
injurious species, containment to prevent escape (together
with clear procedures for recapture in the event of an escape). In general species should be housed separately to prevent aggression or predation but there are polyculture and
experimental exceptions to this rule.
Rooms may require higher than normal temperature and
humidity, and lighting systems should be controllable for
photoperiod. Many invertebrate species benefit from some
form of environmental complexity such as branches or hollow pipes for hiding spaces. All surfaces (including those of
items provided for environmental enrichment) should be
sanitizable.
It may be necessary to ensure the availability of features to contain escaped individuals, such as an “air knife”
at the door level to keep flying insects inside, or a bath or
moat system to keep crawling insects inside containers. In
some cases electrified “bug zappers” or nonlethal electric
barriers are used to enhance containment and prevent escaped specimens from spreading through a facility. There
should also be screens or other means to isolate the area
and the HVAC systems from surrounding areas to prevent
the appearance of unwanted guests in other areas of the
building.
It is highly advisable to hold escape-prone and destructive pest species (e.g., dermestid beetles, which damage biological and book collections) and species that generate
strong odors (e.g., Phanecia carrion flies) in isolated buildings away from other activities, valuable collections, and
personnel.
Water
Water sources for invertebrates, whether for drinking or life
support, require careful attention as the addition of certain
chemicals in city water supplies may be acutely or chronically toxic to some species. Marine species require highquality seawater that has undergone the removal of
particulates, metabolites, and pathogens (e.g., through UV
218
Food
Provision of foodstuffs for invertebrates could be the subject
of an entire paper. General principles are similar to those for
other species: food must be nutritionally balanced and fresh,
and food that is contaminated (e.g., with mold) or that may
have been sprayed with insecticides or other toxic materials
should be strictly avoided. Food should not be permitted to
undergo decomposition, although even here there are exceptions (for instance, autoclaved cow’s liver provided to blowflies for the production of fly larvae may be in the process of
autolysis).
Contamination of invertebrate food may have unforeseen
consequences. In one case contamination of fruit flies used
as a protein source for captive rufous hummingbirds (Selasphorus rufus) proved to be the means of transmission of epiornitic Aspergillus infection in the birds (Harvey-Clark
1993).
Invasive Procedures, Veterinary Care,
and Treatment
Surgery, anesthesia, and veterinary care for invertebrates are
a relatively new subspecialty of zoo and exotic animal veterinary medicine, and may therefore need some translation
both by PIs and by veterinarians serving on IACUCs to be
fully understood and effectively evaluated. Collegial communication and consultation with persons versed in methods used in invertebrate research, and especially in invasive
procedures, are important. Experience with the species in
question often enables recognition of subtle signs of health
status changes and earlier interventions, supporting better
outcomes for both the research and the animal care and
welfare.
Resources are available to IACUC members for information on the health management and veterinary care of invertebrates (Cooper 1980, 2004, 2011; Lewbart 2006; Smith
et al. 2011). However, due to the lack of comprehensive information on many of these species, clinical assessment
and treatment options may be limited and this may mean
that euthanasia is exercised earlier and/or more frequently
than in comparable studies with research animal species that
are well characterized.
ILAR Journal
Humane Endpoints and Euthanasia
Acknowledgments
The American Veterinary Medical Association Guidelines
for Euthanasia (AVMA 2007) fail to include techniques and
ethics for invertebrate species. For invertebrates that show
clear signs of morbidity, standardized guidelines and methods are needed to ensure timely intervention by skilled personnel. In many cases, euthanasia may be the first and most
compassionate option.
In practice, as for mammals, when feasible the use of
physical euthanasia methods should be preceded by a
chemical method that obtunds the animal, unless the PI
can provide compelling scientific arguments to the contrary. Various chemical and physical means are available
to achieve rapid, humane euthanasia for invertebrate species and are reviewed elsewhere (Cooper 2004, 2011;
Lewbart 2006).
After euthanasia the IACUC needs to ensure that animal remains are disposed of in an appropriate fashion (e.g.,
biowaste incineration), as components of some species
may remain venomous after death, some invertebrates are
vectors for infectious or zoonotic diseases, and others may
harbor viable eggs or propagules that can cause invasive
species infestations, damage to agriculture, or other unintended effects.
I thank Shelley Adamo (Department of Psychology, Dalhousie University, Halifax, Nova Scotia, Canada) for her valuable comments on and contributions to the manuscript, and
Cameron Fletcher for countless valuable insights and edits.
Conclusion
The wise man regulates his conduct by the theories both
of religion and science. But he regards these theories not
as statements of ultimate fact but as art-forms.
– JBS Haldane (1927)
The art of working with the immense diversity of invertebrate species—for which investigators and IACUC members alike do not always fully apprehend all aspects
of care and biology—lies in the principles of humane
parsimony.
In the absence of a definitive understanding of welfare
implications in these species, scientists and IACUC members should strive to respect life, follow 3Rs principles
(reduce, refine, replace), and minimize the trauma and
severity of procedures when possible. Investigators can
bring much to the table by educating the members of the
IACUC in the biological and scientific aspects of invertebrate models, and the IACUC needs to work collegially
with the PI to ensure the fulfillment of its role in ethical
oversight and the thoughtful and responsible use of research animals.
Work with species that are not fully characterized as animal models will continue to be iterative and requires a flexible institutional culture that incorporates good scholarship,
best practices, and use of the most current information. In all
cases, invertebrates should not be regarded as “second-class
citizens” by either the IACUC or investigator but should, as
with all research animals, be subject to rigorous professional
standards of animal care oversight.
Volume 52, Number 2
2011
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Pfeiffer K, French AS. 2009. GABAergic excitation of spider mechanoreceptors increases information capacity by increasing entropy rather than
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PHS [US Public Health Service]. 2000. Public Health Service Policy on
Humane Care and Use of Laboratory Animals. Bethesda: Office of Laboratory Animal Welfare, National Institutes of Health.
Puri S, Faulkes Z. 2010. Do decapod crustaceans have nocicepters for extreme pH? PLoS One 5:e10244. Available online (www.plosone.org/
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Sahley CL, Modney BK, Boulis NM, Muller KJ. 1994. The S cell: An interneuron essential for sensitization and full dishabituation of leech shortening. J Neurosci 14:6715-6721.
Sandeman D, Sandeman R, Derby C, Schmidt M. 1992. Morphology of the
brain of crayfish, crabs and spiny lobsters: A common nomenclature for
homologous structures. Biol Bull 183:304-326.
Smith SA, Scimeca JM, Mainous ME. 2011. Culture and maintenance of
selected invertebrates in the laboratory and classroom. ILAR J 52:153164.
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St. John Smith E, Lewin G. 2009 Nociceptors: A phylogenetic view. J Comp
Physiol A Neuroethol Sens Neur Behav Physiol 195:1089-1106.
Wilson-Sanders SE. 2011. Invertebrate models for biomedical research,
testing, and education. ILAR J 52:126-152.
Woolf CJ, Walters ET. 1991. Common patterns of plasticity contributing to nociceptive sensitization in mammals and Aplysia. Trends Neurosci 14:74-78.
YSF [Youth Science Fair Canada]. 2010. Research Ethics and Safety Policy.
Available online (www.ysf.ca/files/PDF/governance/policy/en/4.1.2_
Animals.pdf), accessed on August 20, 2010.
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Recently Released
The Guide for the Care and Use of Laboratory Animals has been updated by a committee of experts
to incorporate new scientific information on commonly used laboratory animals, including aquatic
species, and provides extensive references. It is organized around major components of animal
use: key concepts of animal care and use; animal care and use program (including the roles and
responsibilities of the institutional official, attending veterinarian, and institutional animal care and use
committee; regulatory considerations; program and personnel management; and program oversight);
animal environment, husbandry, and management; veterinary care (including animal procurement
and transportation, preventive medicine, and clinical care and management); and physical plant
design considerations.
To reserve your copy:
Contact the National Academies Press at www.nap.edu or
Call 888-624-8373 or 202-334-3313
For veterinarians, investigators, IACUCs, and animal care staff
This second in a two-part update of ILAR’s 1992 report Recognition and Alleviation of Pain and
Distress in Laboratory Animals aims to educate readers on the current scientific and ethical
issues associated with pain experienced by laboratory animals. It covers basic principles, causes,
and signs of pain, and treatment options and their potential deleterious effects. It also provides
practical guidelines for those involved in laboratory animal care. Finally, the report identifies areas
in which further scientific investigation is needed to improve laboratory animal welfare.
To reserve your copy:
Contact National Academies Press at www.nap.edu
Call 888-624-8373 or 202-334-3313
ILAR Publications
Amphibians: Guidelines for the Breeding, Care and
Management of Laboratory Animals
This report of the Subcommittee on Amphibian Standards
should serve as a useful guide to all users of amphibians,
lead to success in the normal maintenance of amphibian
colonies, and continue to stimulate efforts toward improving
the quality of utilization of these animals. In addition to
guidelines for animal care and quality, certain terminology is
suggested. ISBN 0-309-07767-2; 1974, 162 pages, 6 × 9,
paperbound
Chimpanzees in Research: Strategies for Their Ethical
Care, Management, and Use
Chimpanzees in biomedical and behavioral research constitute a national resource that has been valuable in addressing
national health needs. However, the expected level of use of
the chimpanzee model in biomedical research did not materialize, creating a complex problem that threatens both the
availability of chimpanzees and the infrastructure required
to ensure their well-being. This report examines the issues
and makes recommendations. ISBN 0-309-05891-0; 1997,
108 pages, 6 × 9, paperbound
Definition of Pain and Distress and Reporting
Requirements for Laboratory Animals: Proceedings
of the Workshop Held June 22, 2000
The goal of this ILAR/NIH joint workshop was to provide
feedback from the scientific community to the USDA regarding the lack of a functional definition of “distress” as well as
the efficacy of continuing to use current categories to report
pain and distress. Speakers’ areas of expertise and perspectives
ranged from scientific research to animal welfare policy,
protocol review, and relevant organizations or institutions.
ISBN 0-309-0698-6; 2000, 132 pages, 6 × 9, paperbound
The Development of Science-based Guidelines for
Laboratory Animal Care: Proceedings of the
November 2003 International Workshop
The purpose of this workshop was to bring together experts
from around the world to assess the available scientific
knowledge that can affect the current and pending guidelines for laboratory animal care. Workshop presentations
and discussions focused on identifying gaps in the current
knowledge to encourage future research endeavors; assessing potential financial and outcome costs of nonscientifically
based regulations, facilities, and research; and determining
possible negative impacts of arbitrary regulations on animal
Volume 52, Number 2
2011
welfare. ISBN 0-309-09302-3; 2004, 264 pages, 6 × 9,
paperbound
Education and Training in the Care and Use of
Laboratory Animals
Federal law requires that institutions provide training for
anyone caring for or using laboratory animals. This volume
provides the guidelines and resources needed to coordinate a
quality training program, as well as to meet all legal requirements. ISBN 0-309-08691-4; 1991, 152 pages, 8.5 × 11,
paperbound
Guide for the Care and Use of Laboratory Animals,
8th ed.
The Guide for the Care and Use of Laboratory Animals has
been updated by a committee of experts to incorporate new
scientific information on commonly used laboratory animals,
including aquatic species, and provides extensive references.
It is organized around major components of animal use: key
concepts of animal care and use; animal care and use program
(including the roles and responsibilities of the institutional
official, attending veterinarian, and the institutional animal
care and use committee; regulatory considerations; program
and personnel management; and program oversight); animal
environment, husbandry, and management; veterinary care
(including animal procurement and transportation, preventive medicine, and clinical care and management); and physical plant design considerations. ISBN 0-309-15400-6; 2010,
248 pages, 6 × 9, paperbound
Guidelines for the Care and Use of Mammals in
Neuroscience and Behavioral Research
Expanding on the Guide for the Care and Use of Laboratory
Animals, this report provides current best practices for animal care and use and discusses how the regulations and
guidelines provided by the Guide, the Animal Welfare Act,
the Animal Welfare Act Regulations, and PHS Policy can be
applied to neuroscience and behavioral research. The report
treats the development, evaluation, and implementation of
animal-use protocols as a decision-making process, not just
a decision. It encourages the use of professional judgment
and careful interpretation of regulations and guidelines to
develop performance standards that ensure animal wellbeing and high-quality research. This report is an indispensable resource for researchers, veterinarians, and institutional
animal are and use committees. ISBN 0-309- 08903-4; 2003,
224 pages. 6 × 9, paperbound
225
Guidelines for the Humane Transportation of
Research Animals
Transporting research animals is a necessary part of the biomedical enterprise that can have substantial effects on the
physiological and psychological condition of the animals.
Individuals at research facilities often find arranging transportation of animals a challenge. In order to address a plethora
of sometimes confusing and burdensome regulations pertaining to transportation of research animals, this report recommends that an interagency working group be established
to coordinate federal inspections and permitting activities. It
further recommends that steps be taken to ensure the availability of safe, reliable air and ground transportation for research animals. The report also establishes science-based good
practices for transporting research animals and advises that
research institutions designate a single individual to be responsible for ensuring safe shipment and receipt of animals.
ISBN 0-309-10122-0; 2006, 160 pages, 6 × 9, paperbound
Immunodeficient Rodents: A Guide to Their
Immunobiology, Husbandry, and Use
This volume is an indispensable reference on the nature of
immune defects in rodents and the special techniques necessary to maintain and breed them. The authors describe 64 inbred, hybrid, and mutant strains of rodents, each with some
immune defect; explain mechanisms for ensuring genetic
purity; and provide a standardized nomenclature for different varieties. ISBN 0-309-03796-4; 1989, 260 pages, 6 × 9,
clothbound
Infectious Diseases of Mice and Rats
This edition—a must for all researchers who use these animals—provides practical suggestions for breeding, keeping,
and identifying pathogen-free laboratory rodents. ISBN
0-309-06332-9; 1991, 415 pages, 6 × 9, paperbound
Companion Guide to Infectious Diseases of Mice and Rats
This companion to Infectious Diseases of Mice and Rats
makes practical information on rodent diseases readily accessible to researchers. ISBN 0-309-04283-6; 1991, 108
pages, 6 × 9, paperbound
International Perspectives: The Future of Nonhuman
Primate Resources, Proceedings of the Workshop Held
April 17-19, 2002
Nonhuman primates (NHP) continue to play an important
role in the research of many human diseases such as malaria
and AIDS. Changes in the need for different species of NHP,
the adequacy of the current supply of NHP, and projections
of future needs for NHP are issues that concern scientists,
veterinarians, and funding authorities from countries that are
major users of NHP, as well as countries that produce and
supply these animals. In this volume, workshop participant
discussions relate to current shortfalls and excesses in NHP
breeding and exportation programs, the status of breeding and
conservation programs internationally, the development of
226
specific pathogen-free colonies, difficulties in transporting
NHP, and challenges in the management of NHP colonies.
ISBN 0-309-08945-X; 2003, 262 pages, 6 × 9 paperbound
Microbial and Phenotypic Definition of Rats and Mice:
Proceedings of the 1998 US/Japan Conference
This workshop is part of a long-term program to nurture international collaborative and information-exchange activities.
As genetics and genomics affect the study of biology and
medicine, the role of comparative medicine cannot be understated. Workshop contributors seek to enhance the genetic
and microbiologic integrity of laboratory rat and mice colonies worldwide. The mouse has been a critical model for the
discovery of genes responsible for several cancers and many
other diseases. The rat model “functionally” characterizes
mammalian model systems. This meeting sought to help global
scientific enterprise harmonize the mouse and rat models
and to meet the research challenges of the 21st century. ISBN
0-309-07389-8; 1999, 110 pages, 6 × 9, paperbound
Monoclonal Antibody Production
Monoclonal antibodies are important reagents used in research, diagnosis, and treatment of diseases. They are produced by injection into the abdominal cavity of a suitably
prepared mouse or by tissue culturing cells in plastic flasks.
This report weighs the costs and benefits of each method
and makes recommendations for their uses. ISBN 0-30907511-4; 1999, 74 pages, 6 × 9, paperbound
National Need and Priorities for Veterinarians in
Biomedical Research
This report identifies various factors that contributed to creating an unfulfilled need for veterinarians in the biomedical
research workforce, including an increase in the number
of NIH grants utilizing animals and the burgeoning use of
transgenic rodents, without a comparable change in the supply of appropriately trained veterinarians. The committee
developed strategies for recruiting more veterinarians into
careers in biomedical research. ISBN 0-309-09083-0; 2004,
102 pages, 6 × 9, paperbound
Nutrient Requirements of Laboratory Animals, 4th ed.
In the years since the third edition of this indispensable
reference was published, a great deal has been learned about
the nutritional requirements of common laboratory species:
rat, mouse, guinea pig, hamster, gerbil, and vole. The fourth
edition presents the current expert understanding of the lipid,
carbohydrate, protein, mineral, vitamin, and other nutritional
needs of these animals. The extensive use of tables provides
easy access to a wealth of comprehensive data and resource
information. ISBN 0-309-05126-6; 1995, 192 pages, 8.5 ×
11, paperbound
Occupational Health and Safety in the Care and Use of
Nonhuman Primates
The field of occupational health and safety constantly
changes, especially as it pertains to biomedical research.
ILAR Journal
New infectious hazards are of particular importance at nonhuman-primate facilities. For example, the discovery that
B virus can be transmitted via a splash on a mucous membrane raises new concerns that must be addressed, as does
the discovery of the Reston strain of Ebola virus in import
quarantine facilities in the U.S. The risk of such infectious
hazards is best managed through a flexible and comprehensive occupational health and safety program (OHSP) that
can identify and mitigate potential hazards. This report is
intended as a reference for vivarium managers, veterinarians,
researchers, safety professionals, and any other persons who
are involved in developing or implementing an OHSP dealing with nonhuman primates. This report attempts to list the
important features of an OHSP and provide the tools necessary for informed decision-making in developing an optimal
program that meets all particular institutional needs. ISBN
0-309-08914-X; 2003, 184 pages, 6 × 9, paperbound
Occupational Health and Safety in the Care and Use of
Research Animals
Much has been written about the care of research animals,
yet little guidance has appeared on protecting the health and
safety of the people who care for or use these animals. This
report, an implementation handbook and companion to the
Guide, identifies principles for building a program and discusses the accountability of institutional leaders, managers,
and employees for a program’s success. ISBN 0-30905299-8; 1997, 168 pages, 6 × 9, paperbound
The Psychological Well-Being of Nonhuman Primates
A 1985 amendment to the Animal Welfare Act requires
those who keep nonhuman primates to develop and follow
appropriate plans for promoting the animals’ psychological well-being. The amendment, however, provides few
specifics. The Psychological Well-Being of Nonhuman Primates recommends practical approaches to meeting those
requirements. ISBN 0-309-10359-2; 1998, 184 pages, 6 ×
9, paperbound
Recognition and Alleviation of Distress in Laboratory
Animals
The use of animals in research adheres to scientific and
ethical principles that promote humane care and practice,
and these principles and standards of practice must be updated based on scientific advances in our understanding of
animal physiology and behavior. This report focuses on
the stress and distress experienced by animals used in research. It aims to educate laboratory animal veterinarians;
students, researchers, and investigators; institutional animal care and use committee members; animal care staff;
and animal welfare officers about current scientific and
ethical issues associated with stress and distress in laboratory animals. The report evaluates pertinent scientific literature to generate practical guidelines, focusing on the
following areas: scientific understanding of causes and
functions of stress and distress; the transformation of
Volume 52, Number 2
2011
stress to distress; and the identification of principles for
the recognition and alleviation of distress. The report also
discusses the role of humane endpoints in situations of
distress and principles of minimization of distress in laboratory animals. Finally, the report identifies areas in which
further scientific investigation is needed to improve laboratory animal welfare. ISBN 0-309-10817-9; 2008, 118
pages, 6 × 9, paperbound
Recognition and Alleviation of Pain in Laboratory
Animals
This report, the second of two revising the 1992 publication
Recognition and Alleviation of Pain and Distress in Laboratory Animals, focuses on pain experienced by animals used
in research. This book aims to educate laboratory animal
veterinarians; students, researchers, and investigators; institutional animal care and use committee members; and animal care staff and animal welfare officers about the current
scientific and ethical issues associated with pain in laboratory animals. The committee evaluated pertinent scientific
literature to generate practical and pragmatic guidelines
for recognizing and alleviating pain in laboratory animals,
focusing on the following areas: physiology of pain in
commonly used laboratory species; pharmacologic and nonpharmacologic principles to control pain; identification of
humane endpoints; and principles for minimizing pain associated with experimental procedures. Finally, the report
cites areas in which further scientific investigation is needed
to improve laboratory animal welfare. ISBN 978-0-30912834-6; 2009, 270 pages, 6 × 9, paperbound
Recognition and Alleviation of Pain and Distress in
Laboratory Animals
Clear guidelines on the proper care and use of laboratory
animals are being sought by researchers and members of the
many committees formed to oversee animal care at universities as well as the general public. This report provides comprehensive information about behavior, pain, and distress in
laboratory animals. ISBN 0-309-07525-4; 1992, 160 pages,
6 × 9, paperbound
Science, Medicine, and Animals, 2nd ed.
Science, Medicine, and Animals discusses how animals
have been and continue to be an important component of
biomedical research. It addresses the history of animal research and what it looks like today, and gives an overview of
some of the medical advances that would not have been
possible without animal models. Finally, it looks at the
regulations and oversight governing animal use, as well as
efforts to use animals more humanely and efficiently.
ISBN 0-309-08894-1; 2004, 52 pages, 8.5 × 11, paperbound
Science, Medicine, and Animals Teacher’s Guide
Science, Medicine, and Animals explains the role that animals
play in biomedical research and the ways in which scientists,
227
governments, and citizens have tried to balance the experimental use of animals with a concern for all living creatures.
An accompanying Teacher’s Guide is available to help teachers of middle and high school students use Science, Medicine, and Animals in the classroom. As students examine the
issues in Science, Medicine, and Animals, they will gain a
greater understanding of the goals of biomedical research
and the real-world practice of the scientific method in general.
The Teacher’s Guide was reviewed by members of the
National Academies’ Teacher Associates Network and is recommended by the National Science Teacher’s Association.
ISBN 0-309-10117-4; 2005, 24 pages, 8.5 × 11, paperbound
Scientific and Humane Issues in the Use of Random
Source Dogs and Cats
This report examines the value of random source animals in
biomedical research funded by the National Institutes of
Health (NIH) and the role of Class B dealers who acquire
and resell live dogs and cats to research institutions. The report addresses (1) the important biomedical research questions and common research topics in contemporary NIHfunded research where Class B dogs and cats are desirable/
necessary as well as the frequency of these research
topics (i.e., number of grants where the potential exists or
the source of the animal is identified as a Class B dealer); (2)
the specific characteristics (e.g., physiological, anatomical,
or genetic) of the animals that make them particularly well
suited for certain types of research; and (3) recommendations for the use of Class B dogs and cats. ISBN 978-0-30913807-9; 2009, 136 pages, 6 × 9, paperbound
Strategies That Influence Cost Containment in Animal
Research Facilities
This second report of the National Research Council’s
Committee on Cost of and Payment for Animal Research
presents the committee’s conclusions and recommendations
regarding cost containment methods for animal research facilities. This publication follows the Committee’s initial report which examined interpretation of governmental policy
228
(OMB Circular A-21) concerning institutional reimbursement for overhead costs of animal research facilities. ISBN
0-309-07261-1; 2000, 168 pages, 6 × 9, paperbound
Use of Laboratory Animals in Biomedical and
Behavioral Research
Scientific experiments using animals have contributed significantly to the improvements of human health. Animal experiments were crucial to the conquest of polio, for example,
and they will undoubtedly be one of the keystones in AIDS
research. However, some persons believe that the cost to the
animals is often high. Authored by a committee of experts
from various fields, this report discusses the benefits that
have resulted from animal research, the scope of animal research today, the concerns of advocates of animal welfare,
and the prospects for finding alternatives to animal use. The
authors conclude with specific recommendations for more
consistent government action. ISBN 0-309-07878-4; 1988,
112 pages, 6 × 9, paperbound
Laboratory Animal Management Series
Rodents
Since ILAR (formerly the Institute of Laboratory Animal
Resources) issued its last report on the general management
of rodents, advances in biomedical technology and increased
public awareness of laboratory animal issues have created a
new research environment. This volume brings researchers
up to date on both of these aspects of laboratory investigation and provides a comprehensive resource manual for
management of laboratory rodents. ISBN 0-309-04936-9;
1996, 180 pages, 6 × 9, paperbound
Dogs
This revised edition incorporates the regulatory requirements and improved practices for laboratory animal care.
ISBN 0-309-04744-7; 1994, 152 pages, 6 × 9, paperbound
ILAR Journal
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Amphibians: Guidelines for the Breeding, Care and Management of Laboratory Animals
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Companion Guide to Infectious Diseases of Mice and Rats
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Quantity Price ea Total
2011 52(2) Spineless Wonders: The Welfare and Use of Invertebrates in the Laboratory
and Classroom
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Introduction: Laboratory Invertebrates: Only Spineless, or Spineless and Painless?
Invertebrate Models for Biomedical Research, Testing, and Education
Culture and Maintenance of Selected Invertebrates in the Laboratory and Classroom
Invertebrate Resources on the Internet
Anesthesia, Analgesia, and Euthanasia of Invertebrates
Pain and Suffering in Invertebrates?
Nociceptive Behavior and Physiology of Molluscs: Animal Welfare Implications
Philosophical Background of Attitudes toward and Treatment of Invertebrates
IACUC Challenges in Invertebrate Research
52(1) Animal Models of Aging: Something Old, Something New
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Introduction: Animal Models of Aging: Something Old, Something New
Mice as a Mammalian Model for Research on the Genetics of Aging
Heterogeneous Stocks and Selective Breeding in Aging Research
The Collaborative Cross: A Recombinant Inbred Mouse Population for the Systems Genetic Era
Mindspan: Lessons from Rat Models of Neurocognitive Aging
Successful Aging and Sustained Good Health in the Naked Mole Rat: A Long-Lived Mammalian Model
for Biogerontology and Biomedical Research
The Marmoset as a Model of Aging and Age-Related Diseases
Calorie Restriction and Aging in Nonhuman Primates
The Development of Small Primate Models for Aging Research
Candidate Bird Species for Use in Aging Research
Aging Research 2011: Exploring the Pet Dog Paradigm
IACUC Issues Related to Animal Models of Aging
2010 51(4) Birds as Animal Models in the Behavioral and Neural Sciences
• Introduction: Contributions of Bird Studies to Behavioral and Neurobiological Research
• Japanese Quail as a Model System for Studying the Neuroendocrine Control of Reproductive and
Social Behaviors
• The Role of Hair Cell Regeneration in an Avian Model of Inner Ear Injury and Repair from
Acoustic Trauma
• Auditory Processing, Plasticity, and Learning in the Barn Owl
• Applications of Avian Transgenesis
• The Songbird as a Model for the Generation and Learning of Complex Sequential Behaviors
• Sleep, Learning, and Birdsong
• A Social Ethological Perspective Applied to Care of and Research on Songbirds
• The Use of Passerine Bird Species in Laboratory Research: Implications of Basic Biology for Husbandry
and Welfare
• Guidelines and Ethical Considerations for Housing and Management of Psittacine Birds Used in Research
• An IACUC Perspective on Songbirds and Their Use in Neurobiological Research
51(3) One Health: The Intersection of Humans, Animals, and the Environment
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Introduction: One Health Perspective
Mainstreaming Animal-Assisted Therapy
Cross Talk from Pets to People: Translational Osteosarcoma Treatments
Modeling Opportunities in Comparative Oncology for Drug Development
Zoonotic Enterohemorrhagic Escherichia coli: A One Health Perspective
Methicillin-Resistant Staphylococcus aureus in Animals
Microbe Hunting in Laboratory Animal Research
Understanding Risk Perceptions to Enhance Communication about Human-Wildlife Interactions and the
Impacts of Zoonotic Disease
Animals as Sentinels: Using Comparative Medicine to Move Beyond the Laboratory
Lessons from Pandemic H1N1 2009 to Improve Prevention, Detection, and Response to Influenza
Pandemics from a One Health Perspective
A Global Veterinary Medical Perspective on the Concept of One Health: Focus on Livestock
Veterinary Manpower Needs in One Health Initiative (online only)
Animal Studies and One Health: IACUC Considerations
Volume 52, Number 2
2011
231
51(2) Disaster Planning and Management
• Introduction: Disaster Planning and Management: A Practicum
• Tropical Storm and Hurricane Recovery and Preparedness Strategies
• Disaster Preparedness in Biocontainment Animal Research Facilities: Developing and Implementing an
Incident Response Plan (IRP)
• Management of Rodent Viral Disease Outbreaks: One Institution’s (R)Evolution
• Crisis Planning to Manage Risks Posed by Animal Rights Extremists
• Verification of Poultry Carcass Composting Research through Application during Actual Avian
Influenza Outbreaks
• Wildfire Evacuation: Outrunning the Witch’s Curse—One Animal Center’s Experience
• IACUC Considerations: You Have a Disaster Plan But Are You Really Prepared?
• Also in this issue: Workshop Summary: Detection, Impact, and Control of Specific Pathogens in Animal
Resource Facilities
51(1) Regenerative Medicine: From Mice to Men
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The Use of Animals in Human Stem Cell Research: Past, Present, and Future
Neural Stem Cell Niches and Homing: Recruitment and Integration into Functional Tissues
Preclinical Assessment of Stem Cell Therapies for Neurological Diseases
From Stem Cells to Bone: Phenotype Acquisition, Stabilization, and Tissue Engineering in Animal Models
Chimeric Animal Models in Human Stem Cell Biology
The Use of Animal Models to Study Stem Cell Therapies for Diabetes Mellitus
Practical Considerations in Regenerative Medicine Research: IACUCs, Ethics, and the Use of Animals in
Stem Cell Studies
• A “Season of Light” in Stem Cell Research and Regenerative Medicine: Observations on Recent
Developments
• (ILAR e-Journal) Analysis of Current Laboratory Animal Science Policies and Administration in China
2009 50(4) Pain and Distress in Fish
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Introduction: Pain and Distress in Fish: A Review of the Evidence
Challenges in Assessing Fish Welfare
Pain Perception in Fish: Indicators and Endpoints
Fish Sedation, Analgesia, Anesthesia, and Euthanasia: Considerations, Methods, and Types of Drugs
Effects of Restraint and Immobilization on Electrosensory Behaviors of Weakly Electric Fish
The Zebrafish: A Model to Study the Endogenous Mechanisms of Pain
Morphologic Effects of the Stress Response in Fish
Testing the Waters: IACUC Issues Associated with Fish
(ILAR e-Journal) Validation of a Method for Measuring Sperm Quality and Quantity in Reproductive
Toxicity Tests with Pair-Breeding Male Fathead Minnows (Pimephales promelas)
50(3) Sleep-Disordered Breathing: Exploring a Human Disorder Using Animal Models
• Introduction: Sleep-Disordered Breathing across the Lifespan: Exploring a Human Disorder
Using Animal Models
• Breathing and Sleep: Measurement Methods, Genetic Influences, and Developmental Impacts
• Cardiovascular Consequences of Sleep-Disordered Breathing: Contribution of Animal Models
to Understanding the Human Disease
• Vascular Effects of Intermittent Hypoxia
• Metabolic Consequences of Sleep-Disordered Breathing
• Insight from Animal Models into the Cognitive Consequences of Adult Sleep-Disordered Breathing
• An IACUC Perspective on Animal Models of Sleep-Disordered Breathing
2008
2007
50(2)
50(1)
49(4)
49(3)
49(2)
49(1)
48(4)
48(3)
48(2)
48(1)
Gene Therapy in Large Animal Models of Human Genetic Diseases
The Neurobiology of Social Behavior
Postapproval Monitoring... Balancing Risk Management with Burden
Detection and Management of Microbial Contamination in Laboratory Rodents
Microbial Quality Control for Nonhuman Primates
Noninvasive Bioimaging of Laboratory Animals
Animal Models Used in the Study of Movement Disorders
Use of Amphibians in the Research, Laboratory, or Classroom Setting
Training and Adult Learning Strategies for the Care and Use of Laboratory Animals
Contemporary Topics for Animal Care Committees
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Spineless Wonders: Welfare and Use of Invertebrates in the Laboratory and Classroom
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In This Issue:
Spineless Wonders:
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Volume 52 • Number 2 • 2011 • pp 121-232