Forum
Laboratory Containment
of Transgenic Arthropods
MARJORIE
R
Gaskalla
Keierleber
Capinera
A. Hoy, RICHARD D. GASKALLA, JOHN L. CAPlNERA, AND CAROLYN N. KEIERLEBER
ELEASES OF EXOTIC AND TRANSGENIC
OR-
ganisms have created serious controversies and concerns among scientists, environmental groups, government regulatory
agencies, and the general public. It is likely
that these controversies increasingly will involve entomologists as transgenic arthropods are developed for use in pest management programs.
Only one transgenic arthropod is known
to have been released into the environment
(M. A. Hoy, unpublished). This planned release occurred after extensive review by
regulatory authorities and involved a beneficial phytoseiid predator, Metaseiulus oecidentalis (Nesbitt), containing a nonfunctional molecular marker gene. The release
involved a short-term experiment in an isolated site, and the predator, for climatic reasons, was not expected to persist throughout the year even if it escaped from the
Florida release plot. Despite the low level of
risk associated with this transgenic predator
release, publicity surrounding the release
resulted in the following newspaper headlines: "Mutant bugs: Genetically altered heroes or spineless menaces?" (Washington
Post, 18 December 1995), "Mite fakes [sic]
fight storm of environmental controversy"
(Tampa Tribune, 16 May 1993), and "UF
entomologist opens a can of mites" (St. Petersburg Times, 20 February 1996).
The following examples, which include
transgenic plants and microorganisms as
well as nontransgenic arthropods, describe
some of the problems and issues that have
emerged in the past few years regarding releases of either transgenic or exotic organisms into the environment.
Accidental or Unauthorized Releases
Purposeful or accidental releases of
transgenic or exotic organisms made without approval of appropriate governmental
authorities can create serious public relations problems. For example, the environmental group Greenpeace charged that biotechnology companies conducted field trials
of genetically engineered plants without in206
Hoy
>
forming authorities in Guatemala and without taking appropriate biosafety measures
(Abbott 1996).
Public and scientific concerns were created when a rabbit calicivirus disease escaped from an island off the coast of Australia onto the mainland. The virus, a
potential biological control agent of the introduced rabbits, was feared because it
could cause" ...major ecological damage to
Australia ...through a mass wave of extinctions due to prey switching" (Lawson
1995). The concern over the accidental release was surprising to some because wild
rabbits are considered Australia's number
one pest. These rabbits have caused eco-
in Montana to control Dutch elm disease
(Roberts 1987). A public debate erupted
immediately as to whether Strobel violated
state and federal regulations despite precautions he took to prevent the organism from
spreading from the release site. According to
the U.S. Department of Agriculture, " ...if
Strobel used fungus that originated in the
state, then he did not need a federal
permit ... .It sounds as if he took extra measures, he followed good field procedures.
But he failed to notify EPA or USDA. And
that, by all accounts, is the crucial step. Even
if an experiment seems safe...we want to
have someone who is more sure than you are
look at it, someone with nothing to gain or
lose. That's what we have regulatory agencies for" (Roberts 1987).
If we agree that all releases
should be planned releases,
then transgenic arthropods
should be maintained in the
laboratory in containment
facilities using procedures
that are thorough and
adhered to consistently.
Releases of Arthropod Vectors of
Disease
nomic and ecological disaster throughout
Australia by decimating dozens of native
plant and animal species to the point of extinction and causing the erosion of millions
of acres of topsoil through feeding damage
(Drollette 1996). The accidental release on
the mainland precluded an orderly process
of evaluation, approval, and public education prior to the planned release in March
1997. This educational effort particularly
was important as one scientist had charged
that the calicivirus could infect humans,
and his critique led to fears of a "biological
time bomb" that threatened human health
(Drollette 1996).
Plant pathologist Gary Strobel carried
out an "unauthorized" release of a modified
microorganism in a controversial field test
The mass release of mosquitoes or other
arthropods that can vector disease agents
already has created serious concerns among
the public and the media. For example, the
proposed release of 20,000 mosquitoes
(Culex tarsalis Coquillett) in a scientific experiment designed to monitor their dispersal
in southern California in 1988 was blocked
by fears of the community that the mosquitoes would act as vectors of St. Louis encephalitis (Palca 1988). In another controversial project, males from a tropical
population of the vector mosquito Aedes albopietus (Skuse) were released into a temperate region (Illinois) to determine whether
the overwintering ability of a field population would be reduced. This release apparently was conducted without any review by
regulatory agencies. Afterwards, the director of the Division of Vector-Borne Infectious Diseases for the Centers for Disease
Control and Prevention in Fort Collins, CO,
recommended that a " ...committee be
formed, perhaps by the AMCA [American
Mosquito Control Association], to review
and make recommendations on such releases" (Gubler 1993). The director expressed
concern and strongly urged " ...that such
AMERICAN ENTOMOLOGIST
•
Winter 1997
experiments be conducted cautiously and
with consideration of the public health implications of releasing exotic mosquitoes or
exotic genomes into the field" (Gubler
1993). These examples illustrate a particularly high level of concern about regulatory
and scientific reviews of releases of arthropod vectors of diseases. If the release of only
20,000 mosquitoes was blocked in California, what degree of concern would be raised
about the release of millions of transgenic
mosquitoes, even if they are part of a pest
management program designed to reduce
disease transmission?
Risks and Biological Control
Concern over potential risks associated
with releases of arthropod natural enemies
also has been raised (Howarth 1991). Until
recently, biological control has been perceived as an appropriate and safe technology with significant benefits to the environment because pesticide applications to
control pest arthropods could be reduced or
eliminated (van den Bosch 1978). The era of
regarding biological control as a completely
benign pest management tactic appears to
have ended, and concerns about unintended
effects have surfaced that will be germane
when transgenic biological control agents
are released.
For example, releases of predators of
mosquitoes have been criticized as harming
environmental diversity by causing the local
extinction of the California newt, Taricha
torosa (Rathke), in coastal regions around
Los Angeles. Two species, a crayfish (Procambarus clarkii Girard) and a mosquitofish (Gambusia affinis Baird and Giraud),
may have fed on newts as well as mosquitoes
(Diamond 1996). Both species were introduced into California and have become established permanently. The mosquitofish
also is being mass reared and released by a
local government agency to control mosquitoes. The article describing the negative
environmental effects of these introduced
species is entitled "A-bombs against amphibians," which transmits an extremely
negative image of the augmentative biological control program (Diamond 1996). Despite the fact that the mosquitofish releases
were designed to reduce the use of toxic pesticides and their negative effects on humans
and the environment, this article suggested
that the ecological harm of some biological
control programs might outweigh the benefits. Another example involved efforts to release both a parasitic wasp and a fungus to
control grasshoppers that periodically produce outbreak populations in western U.S.
range lands (Goodman 1993). Releases
were stopped after concerns were raised that
AMERICAN ENTOMOLOGIST
•
Winter 1997
the imported natural enemies might damage
nontarget grasshopper species, some of
which are beneficial, rare, or endangered.
Concern About Risks and Public
Perceptions
As responsible scientists, we must be concerned about risks and negative public perceptions of our efforts to manage arthropod
pests. A substantial component of the public
does not understand what we do or why we
do it. Many people recall how pesticides
have been misused (Carson 1962, van den
Bosch 1978) and distrust bland assurances
that scientists know best. We need to
counter negative perceptions about scientists and recombinant DNA technology
raised by some components of the media, as
exemplified by the movie Jurassic Park. Jurassic Park provided the public with images
of scientists cloning the DNA of dinosaurs
from the stomach contents of blood sucking
insects that had been trapped in amber. The
dinosaurs managed to escape containment
to cause destruction and mayhem. As pointed out by Bains (1993), the movie effectively
transmitted the message that scientists are
" ...fools ...meddling with powers beyond
their reach." The movie portrayed scientists
as "...unfettered by laws, regulations, common sense, or brains of any sort."
To allay public concerns, we need to take
the initiative and deal proactively with issues surrounding development of transgenic
arthropods for pest management programs.
The public and some scientists are concerned about the potential for negative ecological effects or negative health effects from
the release of foreign or genetically engineered organisms into the environment
(Astwood and Fuchs 1996, Comai 1993).
There is a growing concern that releases of
any organism, even those intended to be
beneficial, could have unexpected negative
attributes (e.g., Ehler 1991, Howarth 1991,
Hoy 1992, Office of Technology Assessment 1993, Ruesink et al. 1995, Simberloff
1992, Tiedje et al. 1989). This commentary
focuses on the procedures and practices that
should be adopted to ensure containment of
transgenic arthropods in the laboratory until they have been evaluated and approved
for release. This topic largely has been ignored by both entomologists and Drosophila geneticists.
Containment of Transgenic
Arthropods
Thousands of transgenic strains of the
fruit fly Drosophila melanogaster Meigen
have been developed in hundreds of laboratories around the world since 1982, when
the P transposable element was demonstrated to be an effective means for inserting
genes into its genome (Rubin and Spradling
1982, Spradling and Rubin 1982). Transgenic Drosophila strains have been produced that contain genes from a variety of
species as well as Drosophila genes. Little
discussion has occurred as to the optimal
methods by which these transgenic colonies
can be contained within the laboratory. Although there are no published reports indicating that transgenic Drosophila have escaped from these laboratories or have
harmed the environment after escape, the issues surrounding the containment of transgenic Drosophila should be discussed from a
variety of viewpoints, and standard guidelines for containment should be adopted.
Furthermore, because rapid advances
have been made recently in developing the
genetic tools necessary to develop other
transgenic insects (and mites) for pest management programs (e.g., Beard et al. 1992,
Besansky and Collins 1992, Crampton and
Eggleston 1992, Crampton et al. 1990, Hoy
1994, International Atomic Energy Agency
1993, Loukeris et al. 1995, Mori et al. 1995,
Oakeshott and Whitten 1993), methods and
procedures for containing other transgenic
arthropod species should be developed after
broad discussion. Several arthropod species-including mosquitoes, the Mediterranean fruit fly Ceratitis capitata (Wiedemann), the silkworm Bombyx mori (L.), and
beneficial predatory mites, among othershave been genetically modified using recombinant DNA methods. Research to develop
more effective methods for genetically transforming other pest and beneficial insects is
accelerating (Carlson et al. 1995, Handler
and O'Brochta 1991, Hoy 1994, International Atomic Energy Agency 1993). Potentially useful genes and their regulatory sequences are being identified. Short-term,
medium-term, and long-term plans are being made to develop effective pest management programs using transgenic vectors of
disease (e.g., mosquitoes that cannot transmit disease) and transgenic plant pests (e.g.,
sterile males developed through recombinant DNA methods).
The first field release of a transgenic arthropod, a beneficial predatory mite, Metaseiulus occidentalis (Nesbitt), was authorized in March 1996 by the United States
Department of Agriculture, Animal and
Plant Health Inspection Service (USDAAPHIS). This release was approved using
guidelines for assessing the potential risks
associated with the release of transgenic arthropods into the environment for shortterm field trials in the United States. An application was submitted to USDA-APHIS.
(Such applications can be submitted via the
207
internet to: http://www.aphis.usda.gov:80/
bbep/bp/arthropod/#tgenadoc,
and the applications and the subsequent assessments
of risk are available for review and comment
on the internet.) The public had the opportunity to comment on the application, and,
before issuing a permit, USDA-APHIS consulted with the Florida Department of Agriculture and Consumer Services, Division of
Plant Industry, about pest risk and with the
U.S. Fish and Wildlife Service about threatened and endangered species. Issues surrounding the assessment of potential risks of
transgenic arthropods are evaluated on a
case-by-case basis. The authority by which
USDA-APHIS evaluates transgenic arthropods is based on the arthropods' potential
role as plant pests in U.S. agriculture (Federal Plant Pest Act and the Plant Quarantine
Act). It is not clear which agency should be
consulted for approval to release transgenic
insects that are vectors of human or animal
diseases. Furthermore, no agency has published any guidelines for containing transgenic arthropods in the laboratory prior to
their purposeful release.
Every Release a Planned Release
If it is assumed that every field release of
a transgenic arthropod should be conducted
only after thorough peer review by appropriate scientists and regulatory agencies,
then efforts to contain transgenic arthropods in the laboratory prior to their purposeful release into the environment should
be effective and exemplary. With this in
mind, the community of scientists working
to develop transgenic arthropods to improve pest management practices should be
proactive in addressing these containment
concerns. As a starting point in the discussion on appropriate containment facilities
and procedures, we recommend that entomologists adopt the following principles:
• No releases of transgenic arthropods
into the environment should be made
without prior evaluation by regulatory
authorities and peer reviews by knowledgeable scientists.
• To reduce the likelihood that accidental
releases will occur, transgenic arthropods and putatively transgenic arthropods should be contained in the laboratory by appropriate facilities and
procedures.
Lack of Uniform Containment
Guidelines
Local university biosafety committees
apparently are developing containment procedures for transgenic arthropods and recommendations on appropriate containment
208
facilities and procedures on a case-by-case
basis. This is time consuming and difficult,
especially when institutional biosafety committees lack personnel who are familiar with
insect biology and behavior. We suggest,
therefore, that the containment facilities and
procedures for pest and beneficial arthropods that are imported into new geographic
regions in the United States for classical biological control programs be considered the
minimal standards appropriate for transgenic arthropods.
Uniform containment guidelines also are
desirable because, in some cases, no containment efforts currently are employed to prevent the accidental release of transgenic arthropod strains. This oversight probably is
due to a lack of knowledge about appropriate containment procedures and facilities
and a belief that specific transgenic arthropod
species (especially Drosophila) are harmless.
Suggested Containment Procedures
and Facilities
The containment facilities for arthropods used primarily for classical biological
control projects are certified by the USDAAPHIS-PPQ (Plant Pest Quarantine) and
some state departments of agriculture.
These facilities, designed to contain the beneficial arthropod species for study or until
permission has been granted to release them
into the environment, also prevent the escape of undesired organisms including pest
arthropods, plant pathogens, arthropod
pathogens, and hyperparasitoids (Coulson
et al. 1991, Ertle 1993, Fisher 1973, USDA
1978). Personnel working in these facilities
adhere to specific handling and disposal
protocols designed to prevent accidental releases. We suggest that such facilities and
protocols also would provide adequate containment for most transgenic arthropods.
Greenhouses or other general laboratory
facilities usually are inadequate to prevent
the accidental release of arthropods. If
guidelines for transgenic arthropods are
adopted that are equivalent to the guidelines
for containing beneficial (nontransgenic)
natural enemies and their pest hosts or prey,
the containment facilities would include the
following features:
• The laboratory for handling transgenic
arthropods would be insect-tight and
self-contained. For classical biological
control facilities, the facility must have
an anteroom and a containment room,
with the anteroom serving as a buffer
between the containment room and the
other laboratories. The anteroom in
quarantine facilities contains traps that
can capture escaped flying insects. Ac-
cess to the containment laboratory
should be limited to workers trained in
containment procedures.
• The containment room would be constructed to completely seal cracks and
crevices where escaped insects could
hide. The walls would be solid with triple-paned windows to allow natural
lighting, if necessary. The window
panes would be made of wire-reinforced glass (outside) plus thermopane
and frosted pane for light diffusion (inside). Fluorescent lights and other fittings would be flush with walls and ceilings to prevent arthropods
from
escaping through cracks.
• To reduce the accidental transport of
transgenic arthropods out of the containment facility, there would be minimal movement of equipment into and
out of the containment room. Equipment for the room would not be removed without thorough cleaning or
sterilization.
Authorized
workers
would wear white laboratory coats so
that visible transgenic arthropods
could be dislodged prior to the workers' leaving the room.
• Air conditioning and heating systems
would be made insect-proof with
screening or filters (such as HEPA filters) to avoid accidental escape. Negative air pressure would be maintained
in the containment facility to reduce the
likelihood of accidental escape of transgenic arthropods.
• Waste produced within the containment facility, including waste water in
the sink, would be sterilized before
leaving the facility. Sterilization could
be achieved either by autoclaving,
chemical treatment, freezing, or microwaving. Excess transgenic arthropods would be killed by autoclaving,
microwaving, or freezing prior to their
removal from the containment facility,
unless permission had been obtained to
release them into the environment.
• Sufficient space would be available in
the containment facility so that rearing
and testing could be conducted on multiple transgenic strains without compromising security.
• A facilities officer should be appointed
to develop a procedures manual and
oversee operations to ensure compliance with the procedures.
Some may argue that these proposed facilities and procedures are too expensive or
time-consuming. In fact, they would be less
stringent than those required for insect
pathogens or parasitic nematodes (Ertle
1993). The proposed facilities and procedures are not always considered adequate
AMERICAN ENTOMOLOGIST
•
Winter 1997
safeguards against accidental release of
pests. Some state departments of agriculture, including those in Florida and California, refuse to allow the importation of pests
that currently are not established in the state
or county where the quarantine facility is
located. Presumably this is due to concerns
that accidental releases of the host or prey
arthropod from the containment facility
could result in the establishment of a new
agricultural pest. For example, it is not possible to rear the Mediterranean fruit fly in
quarantine in Florida because this pest is not
established permanently in the state. Nor
can we rear parasitoids of the mealybug
Maconellicoccus hirsutus (Green) on this
host in quarantine until after the pest has
become established in Florida-despite the
fact that it is expected to invade Florida in
the near future.
Risk Assessment
The level of risk associated with accidental releases of transgenic arthropods will depend upon the arthropod species involved,
the transgenes and the regulatory sequences
concerned, the geographic location of the
release site, the presence or absence of con-
specific populations, the method by which
the transgene was inserted into the arthropod, and the weather conditions at the time
ofthe release (Hoy 1995, Tiedje et al. 1989).
If we agree that all releases should be
planned releases, then transgenic arthropods should be maintained in the laboratory
in containment facilities using procedures
that are thorough and adhered to consistently. Although not all accidentally released
transgenic arthropods would become established in the environment, many arthropod
species exhibit an extraordinary ability to
colonize new environments, and we have to
assume that some transgenic arthropods
would be able to establish after an accidental release.
How flexible can the containment and
handling procedures guidelines be? We recognize that accidental releases of transgenic
subtropical or tropical insects would not be
risky during the winter in most temperate
climates because the insects could not establish. Accidental releases of a beneficial arthropod containing a molecular marker
should be less hazardous than the accidental
release of a pest arthropod containing a
functioning transgene. Releases of transgenic arthropods with active transposable ele-
ment vectors or active viral vectors could be
more hazardous than releases of transgenic
arthropods with genetically inactivated vectors because the possibility of accidental
horizontal gene transfer (the movement of
genetic information from the genome of one
species to the genome of another) would be
reduced. Although horizontal gene transfer
is a rare event that occurs on an evolutionary time scale, we do not understand the
mechanisms by which this process occurs,
making assessments of risk difficult (Kidwell 1993, Robertson and Lampe 1995).
It has been argued that accidental releases of most laboratory strains of arthropods
are harmless because these populations are
unlikely to be sufficiently fit to establish and
survive in the wild due to deleterious effects
of the genetic load caused by the transgene
as well as the deleterious effects of laboratory adaptation, genetic drift, inbreeding, inadequate diets, and diseases (King and Leppla 1984, Lenski and Nguyen 1988, Roberts
1989). However, risks could be increased if
the transgenic laboratory strain could interbreed with wild individuals in the environment and transfer the transgene to the wild
population or if natural selection operated
continued on page 255
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209
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