NEMATOCYST REPLACEMENT IN THE SEA ANEMONE AIPTASIA PALLIDA
FOLLOWING PREDATION BY LYSMATA WURDEMANNI: AN INDUCIBLE
DEFENSE?
by
Lucas Jennings
A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Science
in Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, Florida
August 2014
ACKNOWLEDGEMENTS
The author would like to thank his wife, Rachel Jennings and daughter Kathryn
Jennings for their understanding, support and encouragement. Committee members Dr.
Susan Laramore, Dr. Clayton Cook and Dr. Joshua Voss provided help and support
throughout this process. Dr. Shirley Pomponi and Dr. John Scarpa contributed use of
equipment and facilities. Support from the Broward Shell Club and Proaqutix was also
greatly appreciated.
iii
ABSTRACT
Author:
Lucas Jennings
Title:
Nematocyst Replacement in the Sea Anemone Aiptasia pallida
Following Predation by Lysmata wurdemanni: An Inducible
Defense?
Institution:
Florida Atlantic University
Thesis Advisor:
Dr. Susan Laramore
Degree:
Masters of Science
Year:
2014
The sea anemone Aiptasia pallida is a biological model for anthozoan research.
Like all cnidarians, A. pallida possesses nematocysts for food capture and defense.
Studies have shown that anthozoans, such as corals, can rapidly increase nematocyst
concentration when faced with competition or predation, suggesting that nematocyst
production may be an induced trait. The potential effects of two types of tissue damage,
predator induced (Lysmata wurdemanni) and artificial (forceps), on nematocyst
concentration was assessed. Nematocysts were identified by type and size to examine the
potential plasticity associated with nematocyst production. While no significant
differences were found in defensive nematocyst concentration between shrimp predation
treatments versus controls, there was a significant difference in small-sized nematocyst in
anemones damaged with forceps. The proportions of the different types of nematocysts
iv
between treatment types were also found to be different suggesting that nematocyst
production in A. pallida is a plastic trait.
v
NEMATOCYST REPLACEMENT IN THE SEA ANEMONE AIPTASIA PALLIDA
FOLLOWING PREDATION BY LYSMATA WURDEMANNI: AN INDUCIBLE
DEFENSE?
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
Introduction ......................................................................................................................... 1
Inducible defenses ........................................................................................................... 2
Production of new defensive structures .......................................................................... 3
Increase in existing defensive structures ......................................................................... 4
Anthozoan defeNse ......................................................................................................... 5
Aiptasia pallida ............................................................................................................. 11
Lysmata wurdemanni .................................................................................................... 12
Hypotheses ........................................................................................................................ 14
Methods............................................................................................................................. 15
Animal Collection ......................................................................................................... 15
CharacterizaTion of the Aiptasia pallida Cnidom ........................................................ 16
Animal Acclimation ...................................................................................................... 17
Artificial predation ........................................................................................................ 19
Shrimp predation ........................................................................................................... 19
Nematocyst counts ........................................................................................................ 20
Results ............................................................................................................................... 23
Determination of Aiptasia pallida Cnidom ................................................................... 23
Overall MANOVAs and anovas ....................................Error! Bookmark not defined.
vi
Artificial Predation ........................................................................................................ 24
Shrimp Predation ........................................................................................................... 26
Artifical vs. shrimp predation........................................................................................ 26
Discussion ......................................................................................................................... 29
Conclusions ....................................................................................................................... 36
References ......................................................................................................................... 49
vii
TABLES
Table 1. Nematocyst size class designations…………………….……………………...37
Table 2. Summary of MANOVA and ANOVA statistics for nematocyst counts………38
FIGURES
Figure 1. Induced spines in Membranipora membrancea from Harvell 1990………….39
Figure 2. Inducible shape of Chthamalus anisopoma from Lively 1986a.....……..…….40
Figure 3. Anatomy of a sea anemone from Shick 1991…………………………………41
Figure 4. Damage treatments used by Chornesky 1983………………………………...42
Figure 5. Means of different nematocyst types for tentacle data………….………...….43
Figure 6. Means of different nematocyst types for column data………………………..44
Figure 7. Means of different nematocyst types for whole anemone data…………...…..45
Figure 8. Total nematocyst means…….…………………………………………..…….46
Figure 8. Nematocyst proportions for each nematocyst type for tentacle data….……...47
Figure 9. Nematocyst proportions for each nematocyst type for column data….………48
INTRODUCTION
Effective defenses against predation and competition are important for sessile or
semi-sessile organisms, including anthozoans. The diversity of defensive strategies and
morphologies suggest strong evolution pressure on these defensive traits among
organisms who cannot simply avoid a predator or competitor by relocating. While it is
difficult to show experimentally, it is generally agreed that the development of defenses
is energetically costly (Tollrian and Harvell 1999). Due to these costs, an organism that
possesses the ability to rapidly allocate energy to the development of defense only when
needed is thought to have a selective advantage compared to the same organism with
fixed defensive phenotypes (Schlichting 1986; Sultan 1987; Adler and Harvell 1990;
Slattery et al. 2001). Species that invest in constitutive (fixed) defense must allocate
energy to the production and maintenance of defensive structures or compounds at all
times while those investing in plastic (induced) defenses forgo these costs until defense is
needed (Karban and Baldwin 1997; Agrawal and Karban 1999). The development and/or
increase of defensive structures only when needed are collectively characterized as
inducible defenses. Inducible defense is a form of phenotypic plasticity where an
interaction with a predator or competitor triggers a defensive phenotypic response within
a short time frame (Trussell 1996; Tollrian and Harvell 1999; Trussell and Nicklin 2002).
Researchers agree that an inducible defense is favored over a constitutive defense by
1
natural selection if (1) the risk of predation is variable in both time and place, and is
occasionally intense, but does not result in the mortality of the prey species, (2) the
production of the defense requires costs and, or, there are tradeoffs associated with the
defensive phenotype, (3) the advantage of the defense outweighs any and all costs
associated with the production of the plastic defense, and (4) cues for the plastic trait are
reliable with the defensive phenotype only occurring in the presence of the appropriate
predator (Harvell 1986; Lively 1986a; Sterns 1989; DeWitt et al. 1998; Tollrian and
Harvell 1999).
INDUCIBLE DEFENSES
Inducible defenses are found in a wide variety of marine organisms both sessile
and mobile. When faced with a competitor or predator, organisms that exhibit an
inducible defense either (1) produce new structures or compounds that were not
previously present such as spines (Harvell 1986) or (2) increase defensive structures that
were previously present, for example shell thickening (Trussell 1996) or defensive
nematocysts (Gotchfeld 2004). Examples of the production of new defensive structures
include the production of defensive spines in a bryozoan (Harvell 1984), shelldimorphism in an acorn barnacle (Lively 1986b) and the production of sweeper tentacles
in anthozoans (Chornesky 1983). Examples of increases in defensive structures already
present include increased shell thickness in a marine snail (Trussell 1996), the increase in
secondary metabolites in soft coral (Slattery et al. 2001), and the increase in defensive
nematocysts in hard coral (Gotchfeld 2004).
PRODUCTION OF NEW DEFENSIVE STRUCTURES
The nudibranch Doridella steinbergae is a specialist predator which only feeds on
marine bryozoans of the genus Membranipora (Harvell 1986). Experiments have shown
that both partial predation (Harvell 1984) and water born cues (Harvell 1986) of D.
steinbergae lead to the development of protective spines in Membranipora membranacea
along the periphery of the colony within 36 hours (Figure 1). While the presence of
spines is advantageous, they are produced at a cost. Although the spines reduce predation
by 60%, individuals who develop them have a reduced growth rate which leads to a
reduced reproductive output (Harvell 1986).
Acorn barnacles (Balanomorpha) are sessile, colonial animals commonly found in
intertidal zones. Chthamalus anisopoma is a small species of barnacle found throughout
the Gulf of California. It is often preyed upon by the gastropod Acanthina angelica
during low tide. When C. anisopoma recruits are grown in the presence of A. angelica,
they grow in a bent form rather than the normal, conical form (Lively 1986b). The
conical form has the aperture (opening) of the shell perpendicular to the rock face while
the bent form has the aperture parallel (Figure 2). When feeding, A. angelica attacks by
standing its shell up vertically and pushing its proboscis through the valves of the
barnacle in a downward motion (Lively 1986b). The bent form prevents A. angelica
from successfully performing this attack (Lively 1986a). Both forms are commonly seen,
and although the conicals outnumber the bents, only the bents are seen near crevices in
the rocks where A. angelica seeks refuge during high tides (Lively 1986b).
3
As with induced spine development in M. membranacea, the induced bent form of
C. anisopoma reduces predation but comes at a cost. Barnacles growing in the bent form
have slower growth rates and lower fecundity when compared to those growing in the
typical, conical fashion (Lively 1986a). It is thought that the bent form has a reduced
food uptake due to the position of the aperture and less volume within the shell to devote
to brooding (Lively 1986a).
INCREASE IN EXISTING DEFENSIVE STRUCTURES
Inducible defenses are thought to be common among clonal and colonial animals
(Harvell 1986, 1990; Lively 1986b; Tollrian and Harvell 1999b) and rare among solitary
animals (Trussell 1996). One example of an inducible defense in a solitary animal is
shell thickening of Littorina obtusata in response to the green crab, Carcinus maenas, a
common snail predator (Trussell 1996; Trussell and Nicklin 2002). In the past 50 years,
C. maenas, an invasive species to North America, has undergone a northward range
expansion in the Gulf of Maine, but is more common in the warmer, southern portion of
the Gulf (Trussell and Nicklin 2002).
C. maenas crushes snail shells and then feeds on
their tissue. Trussell (1996) found that the southern populations of L. obtusata have
thicker shells compared to northern populations. Mesocosm experiments showed that
when snails from northern populations were raised in the presence of C. maenas, they
grew thicker shells compared to control snails (Trussell 1996). Later studies showed that
both northern and southern populations of L. obtusata raised with C. maenas fed a diet of
live L. obtusata grew thicker shells compared to snails raised with C. maenas fed a fish
diet (Trussell and Nicklin 2002). C. maenas exerted greater force to crush the thicker
4
shells (Trussell and Nicklin 2002) which suggests that production of a thicker shell
reduces the risk of predation. The costs associated with the production of thicker shells
includes reduced body mass, reduced body growth and a reduction in shell length
(Trussell and Nicklin 2002). The reduction in body mass and growth are thought to be
caused by a reduction in interior shell space (Trussell and Nicklin 2002) similar to that
reported following predation of barnacles by gastropods (Lively 1986a).
ANTHOZOAN DEFENSE
Anthozoans exhibit a wide array of defensive strategies that are both constitutive
and plastic. These include the synthesis of chemical compounds (allelopathic defense)
(Stachowicz and Lindquist 1997; Lages et al. 2006 and 2012; Clavico et al. 2013),
defensive behaviors (Edmunds et al. 1976; Harris and Howe 1979) and the development
of morphological structures (Edmunds et al. 1976; Kass-Simon and Scappaticci 2002;
Anderson and Bouchard 2009; Ӧstman et al. 2010). The development of allelopathic
defenses appears to be constitutive in most anthozoans with few studies examining the
plastic nature of secondary metabolites within this group. Plastic morphological defenses
include the development of sweeper tentacles by corals (Chornesky 1983) and gorgonians
(Sebens and Miles 1988), and the increase in defensive nematocysts in response to partial
predation (Gochfeld 2004).
Scleractinian corals and gorgonians use sweeper tentacles as an inducible defense.
Sweeper tentacles are feeding tentacles that have undergone a phenotypic defensive
switch (Wellington 1980). They are not always present and when present, are not found
on every polyp (Chornesky 1983; Williams 1991). Chornesky (1983) performed both
5
transplants and chemical cue experiments using the coral, Agaricia agaricites and found
that when faced with interspecific competition, A. agaricites will develop sweeper
tentacles, usually within 30 days. Sebens and Miles (1988) found similar results with
Caribbean gorgonians; a higher number of sweeper tentacles were formed that were
longer when compared to sweeper tentacles found in species of coral common to the
same area.
In order to determine what cues are responsible for sweeper development in
Agaricia agaricites, Chornesky (1983) conducted experimental trials with various types
of damage to small colonies (Figure 3). Three separate experiments tested the possibility
that artificial cues could elicit the formation of sweeper tentacles in A. agaricites. In the
first experiment artificial tentacles were made of monofilament and placed in continual
contact with colonies of A. agaricites to simulate contact with another individual. In the
second experiment, small amounts of concentrated hydrochloric acid (HCl) were injected
into colonial tissues to simulate digestion by sweeper tentacles. In the third experiment
colonies were subjected to both types of artificial damage. In addition, two transplant
experiments were performed to simulate natural damage caused by competing corals; in
the first, A. agaricites was placed in contact with another species of coral and in the
second A. agaricites was both placed in contact with another species of coral and
injected with HCl. Formation of sweeper tentacles occurred only in treatments where a
competing species was present and was not induced by any artificial damage treatments
(Chornesky 1983).
6
While sweeper tentacles rely on venomous nematocysts for their defensive role,
they also contain another type of cnidae; spirocysts. Similar to nematocysts, spirocysts
consist of a capsule and thread which rapidly ejects when receptors on the capsule are
stimulated. Unlike most nematocysts, spirocysts adhere to objects with adhesive
compounds making them an important component for food capture and prey
manipulation (Shick 1991). Wellington (1980) found that the transition from feeding to
defensive tentacle (sweeper tentacle) resulted in a shift in nematocyst abundance and a
change in the nematocyst to spirocyst ratio. Prior to the defensive shift, feeding tentacles
had a nematocyst to spirocyst ratio of 1:4. After the defensive shift, there was both an
increase in the abundance of nematocysts as well as a shift in the nematocyst to spirocyst
ratio (1:0.2) (Wellington 1980). These results suggest a shift from feeding to defensive
mode when the sweeper tentacle is formed, however only total nematocyst abundance
was recorded and the types of nematocysts associated with the sweeper tentacles were not
identified.
Defensive behaviors are more commonly seen in sea anemones than in other
anthozoans due to their mobility. These defenses include column inflation, tentacle
retraction, pedal locomotion (crawling) and detachment from the substrate (Edmunds et
al. 1976; Turner et al. 2003). Each of these defenses is thought to have a different
purpose. For example, column bulging due to inflation and tentacle retraction help
prevent predatory nudibranchs from consuming sea anemone tentacles, which appears to
be the preferred target for some species of aeolids (Howe and Harris 1978). Harris and
Howe (1979) showed that under laboratory conditions escape responses such as pedal
7
locomotion and detachment did not prevent predation on the anemone Anthopleura
elegantissima by the nudibranch predator Aeolidia papillosa. Instead they suggest that
these behavioral defenses function either to dislodge the anemone allowing it to drift
away from the predator or to buy time in hopes that the predator is dislodged and carried
away by wave action (Harris and Howe 1979).
Constitutive morphological defenses are found in all groups of Anthozoans,
however only those found in anemones are outlined here. These defenses consist of
acrorhagi and acontial threads (Figure 4) both of which contain venomous nematocysts.
Acrorhagi are bulbous sacs found under the tentacular crown of some species of anemone
that contain a large concentration of penetrant nematocysts (Daly 2003). When a
competitor is near, these sacs can be inflated and adhered to the opposing individual.
The adhering tissue, known as a peel, fires a large amount of nematocysts into the
opposing individual eventually resulting in tissue necrosis and possible death. Other
anemones species do not possess acrorhagi but instead have specialized structures called
acontia that can be ejected out of the body wall. These are extensions of the gastric septa
(Blanquet 1968) and due to their structure appear to serve an important role in defense
(Shick 1991; Marino et al. 2008). When provoked by a competitor or predator, they
rapidly evert out of special openings in the column called cinclides (Turner et al. 2003) or
through the mouth (Marino et al. 2008).
Sweeper tentacles, acontia and acrorhagi all contain nematocysts that deliver
venom to predators, competitors and prey (Marino et al. 2008; Thorington et al. 2010)
and in some cases can aid in movement (Kass-Simon and Scappaticci 2002). They are
8
found on feeding tentacles, defense tentacles, along the body column and sometimes in
the basal disc. They consist of an outer capsule wall with an inverted thread inside.
When the appropriate stimulus is applied, the capsule rapidly opens ejecting the thread
out of the cell. Nematocysts can be classified by capsule shape and size and characters
associated with the thread (Mariscal 1974). Nematocysts are morphologically distinct
and can be used for species identification (Fautin 1988). A single species of anthozoan
can have several nematocyst types within its cnidom (Shick 1991).
Gochfield (2004) found that the Hawaiian coral Porites compressa can rapidly
increase the production of microbasic p-mastigophores, a type of nematocyst used for
defense, following partial predation. The butterfly fish Chaetodon multicinctus, a natural
predator of P. compressa was allowed to graze on coral fragments for 24 hours. The
grazed fragments were then cut into halves for feeding trials. The first feeding trial
occurred after 24 hours and the second, after 11 days. During feeding trials, C.
multicinctus was offered both a control (ungrazed) coral and a previously grazed coral.
C. multicinctus showed a preference for ungrazed corals in both trials, however, the
reasons for this preference varied between trials. Following grazing, P. compressa
withdrawals polyps into the skeleton for several days. Nematocyst counts taken after 24
hours showed no increase in nematocysts and the author concluded that the preference for
ungrazed corals was due to the fact that butterfly fishes are visual hunters (Gotchfeld
2004). After 11 days, C. multicinctus still showed a preference for ungrazed corals
however, the conclusion reached was that preference was due to an increase in defensive
nematocysts. Both nematocyst counts and observations of feeding behavior of C.
9
multicinctus were used to reach this conclusion. Anthozoan feeding fish shake their
heads while feeding which is thought to be caused by nematocyst stings to the mouth
(Gotchfeld 2004). There were significantly more head shakes observed when C.
multicinctus took bites of grazed corals compared to when bites were taken of ungrazed
corals. Nematocyst counts confirmed that grazed corals contained significantly more
microbasic-p mastigophores than ungrazed corals.
With the exception of Gochfeld (2004), few studies have examined how
anthozoan nematocyst diversity changes with damage. This study was designed to test
the potential phenotypic plasticity of nematocyst production in response to different types
of tissue damage.
The sea anemone Aiptasia pallida has become a model organism for
molecular studies especially those related to climate change (Lenhert et al. 2012) but is
most known as a model for symbiosis research (Cook et al. 1988; Goulet et al. 2005) and
for nematocysts venom studies (Blanquet 1968; Hessinger and Lenhoff 1973). Using an
experimental design similar to Chornesky (1983) (Figure 3), both artificial damage and
damage inflicted by a predator will be assessed to examine changes in the A. pallida
cnidom. The shrimp Lysmata wurdemanni will serve as the predator for this experiment
because it is known to be a predator of A. pallida (Rhyne et al.2004). To perform this
experiment, the cnidom of A. pallida will be investigated and the cnidae types will be
characterized.
The aim of this study is to determine if Aiptasia pallida exhibits an inducible
defense in response to partial predation by Lysmata wurdemanni through an increase in
defensive nematocysts associated with acontial threads. Increased nematocyst content
10
has been shown in other species of anthozoans in response to both partial predation
(Gochfeld 2004) and competition (Bigger 1982; Turner et al. 2003). However, only the
study by Gochfeld (2004) focused on defensive nematocysts. The study presented here is
novel in that nematocysts will be identified and quantified based on size and suggested
function in an effort to analyze entire cnidom changes in response to different tissue
damage.
AIPTASIA PALLIDA
Aiptasia pallida, also known as the glass or pale anemone, is a small species of
anemone found in the southern Atlantic and Gulf Coasts, from North Carolina, to Texas
and throughout the Caribbean (Stephenson and Stephenson 1950). A.pallida reproduces
both asexually through pedal laceration (Clayton and Lasker 1985; Schlesinger et al.
2010) and sexually through broadcast spawning (Hambleton et al. 2014). It is often
found in clonal aggregations (Ruppert and Fox 1988) and is a prey source for many
organisms including the peppermint shrimp, Lysmata wurdemanni (Rhyne et al. 2004)
and several species of aeolid nudibranch (Carroll and Kempf 1990; Schlesinger et al.
2009). A.pallida can acquire energy through both autotrophic and heterotrophic means. It
can feed on zooplankton (Clayton 1986) or obtain energy from endosymbiotic algae
known as zooxanthellae (Goulet et al. 2005). Taxonomically, it is a member of
Metridioidea and uses acontial threads for defense. These threads contain a large amount
of microbasic p-mastigophores (Hessinger and Lenhoff 1973; Phelan and Blanquet 1985)
which, given their size and location, have the suggested function of defense (Shick 1991;
Marino et al. 2008).
11
When compared to other anemones, Aiptasia pallida has a simple cnidom. It
consists of spirocysts and three different types of nematocysts: microbasic pmastigophores, microbasic amastigophores, and basitrichs (Carlgren and Hedgpeth
1952). The size of both microbasic p-mastigophores and basitrichs differ by location
within the anemone. Defensive nematocysts are herein defined as large (>40 µm)
microbasic p-mastigophores. These nematocysts are found within acontial threads that
are used for anemone defense (Blanquet 1968). Other size classes include medium
nematocysts (39-20 µm) which function in prey capture and small nematocysts (<19 µm)
for which function has not yet been determined. Microbasic amastigophores are found
only in the actinopharynx (Carlgren and Hedgpeth 1952) and differ from microbasic pmastigophores by having a curve at one end instead of a cigar shape (personal
observation).
LYSMATA WURDEMANNI
The peppermint shrimp Lysmata wurdemanni is species of cleaner shrimp found
along the Atlantic coast from New Jersey to Brazil and throughout the Caribbean
(Williams 1984). It is often found in rock rubble, near jetties, piers and buoys and is
sometimes found living in tube sponges (Sefton and Webster 1986). These shrimp are
commonly sold in the aquarium trade due to their unique coloration and ease of care
(Zhang et al. 1998; Rhyne et al. 2004). They are known to eat Aiptasia pallida and are
often sold to control A. pallida outbreaks in home aquaria (Rhyne et al. 2004) and yet, L.
wurdemanni appears to be sensitive to nematocyst discharge. L. wurdemanni tends to
12
attack the anemone’s tentacle first. While feeding on A. pallida, L. wurdemanni can be
seen quickly swimming away after contact with the expelled acontial threads of A.
pallida but returning to feed on the anemone even after repeated exposures (personal
observation).
13
HYPOTHESES
Null hypothesis 1: Shrimp predation will have no effect on the concentration of large
(>39 µm) microbasic p-mastigophore concentration of Aiptasia pallida.
Alternative hypothesis 1: Shrimp predation will result in an increase in large defensive
nematocyst in Aiptasia pallida.
Null hypothesis 2: Tissue damage (removal of tentacles) will not result in a change in the
Aiptasia pallida cnidom.
Alternative hypothesis 2: Removal of tentacle s will cause a shift in the cnidom of
Aiptasia pallida.
Null hypothesis 3: There will be no difference in the Aiptasia pallida cnidom between
individuals damaged artificially and individuals damaged by shrimp predation.
Alternative hypothesis 3: Different types of tissue damage will result in different
cnidoms of Aiptasia pallida.
Null hypothesis 4: Nematocyst composition and concentration will be similar between
the tentacular crown and the column of Aiptasia pallida.
Alternative hypothesis 4: Nematocyst composition will differ between the body regions
of Aiptasia pallida.
14
METHODS
ANIMAL COLLECTION
Aiptasia pallida was collected from the algae mariculture facility at Harbor
Branch Oceanographic Institute at Florida Atlantic University (hereafter HBOI-FAU)
located in Fort Pierce, Florida. The facility uses a flow through design where water is
pumped into large holding tanks from the nearby Indian River Lagoon (IRL). Anemones
were carefully removed from a single holding tank and placed into a bucket containing
water from the IRL. Care was taken to choose anemones attached to pieces of algae or
the sandy bottom of the tank rather than those attached to the side walls to reduce any
possible damage to the anemones during collection. After collection, anemones were
transported to the lab at HBOI-FAU in buckets and acclimated to a salinity of 35 psu over
the course of 24 hours with two 50% water changes, using 1 µm filtered, UV sterilized
salt well water. After acclimation, anemones were placed in a 10 gallon aquarium with
aeration, carbon filtration and full spectrum, fluorescent lighting with a 12/12
photoperiod. Tanks were maintained at 24 oC using aquarium heaters and anemones were
fed Instant Baby Brine Shrimp (Ocean Nutrition, San Diego, CA). Salinity was
monitored using a refractometer and maintained using reverse osmosis deionized (RODI)
water.
15
Eighteen Lysmata wurdemanni were obtained from Proaquatix (Vero Beach,
Florida) and held in a 10 gallon aquarium filled with 1 µm filtered, UV sterilized salt
wellwater and provided with aeration and carbon filtration. Temperature was maintained
at 24 oC using aquarium heaters. Salinity was monitored using a refractometer and
maintained at 35 PSU using RODI. Shrimp were fed a diet consisting of thawed Artemia
adults and live Aiptasia pallida every other day.
CHARACTERIZATION OF THE AIPTASIA PALLIDA CNIDOM
Carlgren and Hedgpeth (1952) report that the cnidom of Aiptasia pallida consists
of spirocysts, microbasic amastigophores, and varying sized microbasic p-mastigophores
and basitrichs. The capsule length of microbasic p-mastigophores and basitrichs vary by
the location they are found (Carlgren and Hedgpeth 1952). Prior to the tissue damage
experiments, these nematocyst size classes were defined and verified. Five anemones
were removed from the same holding tank as mentioned previously, brought back to the
lab and separated into individual Petri dishes. The anemones were agitated with forceps
to cause them to expel their acontia. The expelled acontia were collected using forceps
and a pipette. Acontial threads were kept whole and were not homogenized for analysis.
The anemones were then relaxed using an 80% solution of magnesium chloride (MgCl2)
in a 1:1 ratio of seawater to MgCl2. After 20 minutes, a scalpel was used to dissect each
anemone into two parts; the tentacular crown and the body column. This was done by
cutting the anemone just under the tentacles. The tentacles were then removed from the
tentacular crown and the oral disc was discarded. The tentacles were homogenized in
300 µl of RODI water at a medium speed using a Potter-Elvehjem tissue grinder with a
16
Polytetrafluoroethylene (PTFE) pestle attached to a drill. The remaining column was
dissected into two parts by cutting it vertically. Acontial threads were removed with
forceps from the column and discarded in order to differentiate between body wall and
acontial nematocysts. The remaining body column tissue was then homogenized as
described above. The homogenates and whole acontia samples were frozen at -80 oC
until analyzed.
Prior to analysis, the samples were thawed. Whole acontia were placed on a
hemocytometer and, photomicrographs were taken of the entire hemocytometer counting
grid at 100x magnification using an EVOS® FL Auto digital microscope. The
homogenates from both the tentacular crown and body column were vortexed and 11 µl
of the suspended sample was placed on a hemocytometer. Eight replicate sub-samples
from each homogenate were photographed as outlined above. Using CPCe point
counting software, images were scaled using the hemocytometer grid and the lengths of
nematocyst capsule were measured. Nematocysts were identified according to Mariscal
(1974). For each anemone, the first ten nematocysts found lying flat for each size were
measured per body section (total N=10 per nematocyst type per anemone). The average,
median and standard deviation were calculated for large, medium and small microbasic
p-mastigophores. Based on a Shapiro-Wilk test for normality, the data was not found to
be normally distributed and could not be transformed. Using SPSS analytics software
(IBM), a Kruskal-Wallis test was performed to compare relative capsule lengths of
nematocysts among size classes.
17
ANIMAL ACCLIMATION
Experimental treatment groups were housed in a Thermo Electron Corp. low
temperature illuminated incubator. Prior to the start of the experiment, the incubator was
set to a temperature of 24 oC and a 12 /12 photoperiod and irradiance was measured with
a Li-Cor LI-193 Spherical Quantum sensor with a LI-1400 datalogger. The incubator
used for the experiment had a mean irradiance of 76.2 µmol m-2 s-1. Forty anemones with
pedal disc diameters of 5-7 mm were isolated from the laboratory holding tank for the
experiment. Anemones were separated into two equal groups and each group was placed
into a 1200 ml beaker filled with 900 ml of 1 µm filtered, UV sterilized salt well water
and incubated for 7 days. Anemones were not fed during this period and 90% water
changes were performed every other day.
Thirteen Lysmata wurdemanni with carapace lengths between 0.8-1 cm were
placed in separate 800 ml glass bowls filled with approximately 600 ml of 1 µm filtered,
UV sterilized saltwater and placed in the incubator for 7 days. Small Styrofoam bowls
were placed on top of the glass bowls to prevent escape. The Styrofoam bowls were
loose fitting which allowed for gas exchange. During acclimation, 90% of the water was
changed every other day and shrimp were fed a diet of one live Aiptasia every other day.
The shrimp were not fed for 3 days prior to the start of the experiment.
18
ARTIFICIAL PREDATION
To simulate artificial predation, 13 anemones were taken from one of the 1200 ml
beakers. Each anemone had ten entire tentacles removed with forceps. The tentacles
were removed at the junction between the tentacle and the oral disc. After the tentacles
were removed the anemones were placed in individual bowls, returned to the incubator
and allowed to recover for 10 days before further processing and analysis.
SHRIMP PREDATION
To test the effects of predator induced tissue damage, a glass bowl containing a
single shrimp was haphazardly selected from the incubator, and a single, haphazardly
selected Aiptasia pallida from one of the two incubated beakers was placed in the bowl
with the shrimp. Each shrimp was allowed a total attack time of 2 minutes; attack time is
defined as the time actively spent attacking and feeding on the anemone. If the shrimp
attacked but left the anemone before this time, the timer was stopped until the shrimp
returned to feed. Only 1 shrimp was fed at a time. After 2 minutes of attack time, the
shrimp was removed from the bowl and placed in a holding container outside of the
incubator. The water in the bowl was changed to remove excess anemone mucus and the
bowl with the damaged anemone was placed back in the incubator and allowed to recover
for 10 days prior to processing. This was repeated for 12 additional combinations of
shrimp and anemones (N= 13). The remaining 14 anemones from each of the two
beakers served as the control group and were carefully placed in the remaining bowls.
19
Both experiments ran for a total of 10 days. During this time, there was no
observable difference between artificial damaged anemones and shrimp-damaged
anemones. After ten days all experimental, including control anemones had a pale brown
coloration that differed than that of a freshly collected individual. After 10 days each
anemone was carefully removed from each of the glass bowls and placed in a 1:1 solution
of 80% MgCl2 to saltwater for 40 minutes. Using a scalpel, the anemones were dissected
into two regions; the tentacular crown and the body column by cutting the anemone just
under the tentacles. Acontia were not separated from the column samples. Each portion
of the anemone was homogenized in 1 ml of RODI water using a Potter-Elvehjem tissue
grinder with a PTFE pestle attached to a drill. Samples were homogenized at a medium
drill speed. After homogenization, 0.5 ml was separated for protein analysis and the
remaining 0.5 ml was used for nematocyst counts. All samples were frozen at -80 oC
until the samples were analyzed.
NEMATOCYST COUNTS
Homogenates were thawed and vortexed prior to counting and 11 µl of suspended
sample was placed on a hemocytometer. Using an EVOS microscope, photomicrographs
were taken of the entire hemocytometer counting grid at 100x magnification. Eight subsamples were prepared from both the tentacular crown and the body column. Samples
were vortexed between subsampling. Using ImageJ image processing software,
nematocysts were counted and identified according to Mariscal (1974). Microbasic pmastigophores were separated into three groups based on capsule length: large (>39 µm),
medium (39- 20 µm) and small (<20 µm).
Microbasic amastigophores were also
20
counted. During the counts, if the size or type of any nematocysts could not be
determined, it was scored as unidentified (UNID) and included in the total nematocyst
counts. Broken capsules were rare but not counted. Using the following equation, the
nematocysts counts for the eight sub-samples were averaged and converted to total
number of nematocysts using a hemocytometer conversion factor:
Total nematocyst (per type) = (Subsample average/(9.0 × 10-4)) X total sample volume
This was performed for all nematocyst types and for each body region (column and
tentacles). The total number of nematocysts of each nematocyst type from the tentacle
samples was added to the total number of nematocysts of each nematocyst type from the
column samples to yield total number of nematocysts for the whole anemone.
Using a spectrophotometer, the absorbance peaks of the second portion of the
homogenate were measured. Absorbance was measured at wavelengths of 260 nm, 280
nm and 750 nm. Absorbance at 750nm was used as a turbidity correction. Using the
following equation adapted from Layne (1957), the absorbance peaks were used to
determine protein concentration:
mg protein mL-1 = 1.55(A280 – A750) – 0.76 (A260 – A750)
A dilution factor was then calculated using the following formula:
Dilution factor = ml aliquot/(ml aliquot + mL dilution)
Total protein was then calculated using the following formula:
21
Total protein= mg protein ml-1/dilution factor × total volume
To calculate whole anemone protein, the total protein from the tentacle samples
was added to the total protein from the column samples. The nematocyst counts were
expressed as a ratio of number of nematocyst per milligram of protein by dividing the
total number of nematocysts per type by the total protein. Using SPSS analytical
software (IBM), one-way MANOVAs were performed on the tentacle data, the column
data and the whole anemone data. Post-hoc pairwise comparisons were performed using
the Tukey procedure. Based on a Shapiro-Wilk test for normality the whole anemone
data and column sample data violated the assumption of normality for a one-way
MANOVA therefore; the data was transformed using a log-transformation. The tentacle
data were found to be normally distributed so no transformations were performed.
All nematocysts groups including unidentified nematocysts were summed to yield
total generic nematocyst per anemone. This data was found to be normal using a
Shapiro-Wilk test. Using SPSS a one-way ANOVA was performed on the total
nematocyst data. Post-hoc pairwise comparisons were performed using the Tukey
procedure. Using the total nematocyst value, the data were then converted into
proportions for each nematocyst type for the column and tentacle data. The data was
found to be normal using a Shapiro-Wilk test. Using SPSS, one-way MANOVAs were
performed on the tentacle proportion data and the column proportion data. Post-hoc
pairwise comparisons were performed using the Tukey procedure.
22
RESULTS
DETERMINATION OF AIPTASIA PALLIDA CNIDOM
A total of 10 microbasic p-mastigophores of each size class were measured for all
five anemones (N=150). The range of length for the large sized microbasic pmastigophores was 39 - 62 µm with a median length of 51.0 ± 5.6 µm (Table 1; ± based
one standard deviation). The range of length for the medium sized microbasic pmastigophores was 21 - 39 µm with a median length of 24.0 ± 2.3 µm. The range of
length for the small sized microbasic p-mastigophores was 11 - 19 µm with an average
length of 17.0 ± 3.7 µm. Based on a Kruskal-Wallis test, these groups listed above were
significantly different from each other (p<0.001).
EFFECTS OF TREATMENT TYPE ON NEMATOCYSTS
Overall nematocysts abundances and proportions differed significantly among the
treatments (shrimp predation, artificial predation, and controls) as measured by effects on
either portions of the A. pallida body or the entire organism (Table 2). ANOVAs were
used to assess the effects of treatments on individual nematocyst types. Tentacular crown
samples showed significant differences among treatments in the observed number of
medium nematocysts (F 2, 37= 7.683; p =0.002; Table 2) and small nematocysts (F 2, 37 =
14.581; p <0.005) but not among the amastigophores (F 2, 37 = 2.692; p =0.081). The
23
body column samples showed significant differences among the small nematocysts (F 2,
37
= 13.242; p <0.001) and amastigophores (F 2, 37 = 8.214; p =0.001) but not among the
large nematocysts (F 2, 37 = 2.093; p =0.138). For the whole anemone data, there were
significant differences among the medium (F 2, 37= 5.120; p =0.011) small (F 2, 37 =
25.026; p <0.001) and amastigophores (F 2, 37 = 15.924; p <0.001) nematocyst types but
not among the large nematocysts. Based on a one-way ANOVA, significant differences
were found among the total nematocyst for each treatment type (F2, 37=17.504; p=<0.001)
For the proportion data, significant differences among shrimp, artificial predation,
and control treatments were found in both the tentacle and column data. There were
significant differences among the treatments in the observed proportion of medium (F 2, 37
= 16.020; p <0.001) and small (F 2, 37 = 14.337; p <0.001) sized nematocysts (relative to
total observed nematocysts) in the tentacle samples. For the body column data, the
treatments differed in the proportion of large nematocysts (F 2, 37 = 14.925; p <0.001),
small nematocysts (F 2, 37 = 12.604; p <0.001) and amastigophores (F 2, 37 = 5.910; p
=0.006) observed. When considering the whole anemone, treatments differed in the
proportion of e among the large (F2, 37= 7.497; p=0.002), medium (F2, 37= 3.570; p=0.038)
and small (F2, 37=16.005; p<0.001) sized microbasic p-mastigophore size classes, but not
in the proportion of amastigophores (F2, 37=0.679; p=0.51).
ARTIFICIAL PREDATION
Anemones damaged with forceps had significantly more small-sized microbasic
p-mastigophores than control anemones in the tentacle samples (Tukey HSD: p=0.001)
24
(Figure 5). This size class accounted for more than 50% of all nematocysts found in
forceps damaged anemones in both the tentacle (Figure 5) and column samples (Figure
6).
In the tentacle samples, anemones with forceps damage had approximately twice
the amount of small sized microbasic p-mastigophores than control anemones (65,982
and 30,531 mean nematocyst per mg protein respectively; Figure 5). However there was
no difference in the medium sized microbasic p-mastigophore (Tukey HSD: p= 0.580)
and amastigophore (Tukey HSD: p=1.000) nematocyst groups compared to control
anemones. Forceps damaged anemones had proportionately more small sized microbasic
p-mastigophores (Tukey HSD: p<0.001) and significantly less medium sized microbasic
p-mastigophores (Tukey HSD: p<0.001) than control samples.
In the column samples, forceps damaged anemones showed no significant
difference in large sized microbasic p-mastigophores (Tukey HSD: p= 0.225; Figure 6)
but had significantly more small sized microbasic p-mastigophores (Tukey HSD:
p=0.006) and amastigophore nematocysts (Tukey HSD: p=0.011) than controls. Forceps
damaged anemones in column samples also had proportionately more small microbasic
p-mastigophores (Tukey HSD: p=0.009) and amastigophores (Tukey HSD: p= 0.006)
than control anemones but showed no significant difference in the proportion of large
nematocysts (Tukey HSD: p= 0.488).
For the whole anemone data, forceps damaged anemones had significantly more
small sized microbasic p-mastigophores than controls (Tukey HSD: p<0.001) but
25
differences in the other nematocyst groups were not significant (Figure 7). Artificially
damaged anemones had significantly more total nematocyst than controls (Tukey HSD:
p=0.017; Figure 8).
SHRIMP PREDATION
Anemones attacked by shrimp had fewer nematocysts in all nematocyst groups
for tentacle (Figure 5), column (Figure 6) and whole anemone data (Figure 7) compared
to controls with the exception of large microbasic p-mastigophores in column samples
(not significant: Tukey HSD p>0.05).
In the tentacle samples anemones attacked by shrimp had significantly fewer
medium-sized microbasic p-mastigophores (Tukey HSD: p=0.001) with no significant
difference in the other nematocyst groups when compared to control anemones (Figure
5). For the tentacle proportion data, there were no significant differences in nematocyst
size classes between anemones damaged by shrimp and controls (Figure 9).
In column samples, anemones attacked by shrimp had nearly half the amount of
small sized microbasic p-mastigophores as controls (17,440 and 35,435 nematocysts per
mg protein respectively) but this difference was not significant (figure 6). For the
column proportion data, no significant differences were seen between anemones attacked
by shrimp and controls (Figure 10).
For the whole anemone data, anemones attacked by shrimp had significantly
fewer small size microbasic p-mastigophores (Tukey HSD: p= 0.018) and microbasic
26
amastigophores (Tukey HSD: p<0.001) than controls (Figure 7). Shrimp-damaged
anemones had significantly few total nematocyst than controls (Tukey HSD: p=0.010).
ARTIFICIAL VS. SHRIMP PREDATION
In the tentacle samples, artificially damaged anemones had significantly more
medium (Tukey HSD: p=0.024) and small (Tukey HSD: p<0.001) microbasic pmastigophores than those predated by shrimp, but there was no difference in
amastigophores (Tukey HSD: p=0.124) (Figure 5). In column samples, artificially
damaged anemones had significantly more small-sized microbasic p-mastigophores
(Tukey HSD: p<0.001) and amastigophores (Tukey HSD: p=0.001) but there was no
different in large-sized microbasic p-mastigophores (Tukey HSD: p=0.960) (Figure 6).
Considering the whole anemone data, artificially damaged anemones had significantly
more medium microbasic p-mastigophores (Tukey HSD: p=0.028), small microbasic pmastigophores (Tukey HSD: p<0.001) and amastigophores (Tukey HSD: p=0.006) but
there was no difference in large microbasic p-mastigophores (Tukey HSD: p=0.758)
(Figure 7).
For the tentacle proportion data, artificially damaged anemones had significantly
more medium microbasic p-mastigophores (Tukey HSD: p=0.003; Figure 9), and
significantly fewer small microbasic p-mastigophores (Tukey HSD: p=0.004) but there
was no difference in amastigophores (Tukey HSD: p=0.699). The column proportion
data showed that artificially damaged anemones had significantly fewer large size
microbasic p-mastigophores (Tukey HSD: p<0.001; Figure 10), significantly more small27
sized microbasic p-mastigophores (Tukey HSD: p<0.001) and no difference in
amastigophores (Tukey HSD: p=0.624). Artificially damaged anemones had
significantly more total nematocysts than shrimp-damaged anemones (Tukey HSD:
p<0.001)
28
DISCUSSION
The aim of this study was to determine if Aiptasia pallida exhibits an inducible
defense in response to predation by Lysmata wurdemanni by increasing the number of
defensive nematocysts. This was not supported by the results of this study. Instead,
digital micrographs for column samples of L. wurdemanni damaged anemones had
roughly the same amount of large sized (defensive) microbasic p-mastigophores as
forceps damaged anemones (xˉ = 24,580 and 24,661 nematocysts per mg protein
respectively). Both groups appeared to have fewer defensive nematocysts than control
anemones (Figure 7), but, these differences were not significant. In terms of
proportional changes, artificially damaged anemones had significantly fewer defensive
nematocysts than both controls and shrimp-damaged anemones, however shrimpdamaged anemones and control anemones had no significant difference in defensive
nematocysts (Figures 8 and 9).
Even though the results of this study did not detect differences in the numbers of
large sized microbasic p-mastigophores following natural and artificial tissue damage,
they do support the idea that nematocyst production is plastic in A. pallida. Artificially
damaged anemones showed a large increase in small-sized microbasic p-mastigophores
in both body regions and had significantly more total nematocyst than both shrimp
29
damaged anemones and controls. Shrimp-damaged anemones had fewer over all
nematocysts than either artificially damaged or controls anemones; however, significant
differences were only seen in medium sized microbasic p-mastigophores, amastigophores
and in total overall nematocyst. These results indicate that different types of tissue
damage can produce different cnidom complements.
The cnidom of Aiptasia pallida is relatively simple, being made up of microbasic
p-mastigophores of varying sizes, microbasic amastigophores, basitrichs and spirocysts.
Large microbasic p-mastigophores (>39 µm) were found only in column samples while
medium sized (20-39 µm) microbasic p-mastigophores were only found in tentacle
samples. The column samples included acontia. In this study, small sized microbasic pmastigophores (<20 µm) were found in both the tentacles and the column, which differs
from the finding of Carlgren and Hedgpeth (1952) that reported that microbasic pmastigophores of this size are found only in the mesenterial filaments and the column
walls of A. pallida. Further, Carlgren and Hedgpeth (1952) reported microbasic pmastigophores with a size range of 29.6-39.5 m in the mesenterial filaments which also
differs from this study in that nematocysts of this size were only found in the tentacular
crown. Basitrichs and spirocysts occur throughout the body of A. pallida (Carlgren and
Hedgpeth 1952), but they are more transparent than other types of nematocysts. Given
this, they were likely to be overlooked in the digital micrographs employed in this
studyand therefore were not quantified.
Little is known about the functional roles of the different microbasic pmastigophore size classes in Aiptasia pallida. Microbasic mastigophores are penetrant
30
nematocysts capable of piercing the exoskeletons of crustaceans (Phelan and Blanquet
1985). The large microbasic p-mastigophores in A. pallida are found on acontial threads.
These threads are extensions of mesenterial filaments located within the column walls
and are only expelled when the anemone is agitated, which suggests a defensive role
(Shick 1991; Marino et al. 2008). During this study, individual Lysmata wurdemanni fed
on A. pallida by attacking the tentacles. When L. wurdemanni came in contact with
acontial threads that contain the large p-mastigophres, there was an instantaneous
reaction that suggestedimmediate recognition and avoidance. However, after initial
contact and retreat, L. wurdemanni continued to attack the anemone again and fed until
the anemone was consumed. As stated above, medium sized microbasic pmastigophores were only found in the tentacles and one possible role for medium sized
nematocysts could be prey capture. The observation that they appeared to do little to
deter predation by L. wurdemanni on the tentacle crown would suggest that they have
less of a defensive function than the large nematocysts of the acontia. It is difficult to
determine the role of small sized microbasic p-mastigophores because they are located in
the tentacles or on the oral disc, within the mesenterial filaments and along the body wall.
Based on these locations, a suggested function of these small nematocysts could be prey
manipulation, as they are found both externally and internally; however, this could not be
verified.
The average photosynthetic active radiation (PAR) measurement of the incubator
used was 76.2 µmol m-2 s-1. Goulet et al. (2005) found that Aiptasia pallida collected
from Key Largo, Florida had a compensation irradiance of 36.5 µmol m-2 s-1. This
31
suggests that the symbiotic zooxanthellae may have provided energy for regrowth of
tissues lost to the damage inflicted upon the anemones. It has been shown that, under
optimal conditions, 90-99% of total carbon fixed by photosynthesis via zooxanthellae can
be translocated to the host (Starzak et al 2014). However, carbon translocation estimates
for A. pallida are far less, at around 20% (Davy and Cook 2001), and given the irradiance
levels used in the present study, translocation may have been less than that. Organic
acids, glucose, and glycerol are some of the carbon products that can be translocated by
zooxanthellae to the host (Whitehead and Douglas 2003). While these photosynthetic
products are important for energy and growth, they lack important nutrient elements,
particularly nitrogen that is required for protein synthesis. Phelan and Blanquet (1985)
found that the thread and capsule of acontial nematocysts (large microbasic pmastigophores) in A. pallida are made of collagen proteins, with glycine, proline and
hydroxyproline being major amino acid components. The venom within these
nematocysts is also comprised of peptides and proteins, containing mostly glutamic acid
(Blanquet 1967; Phelan and Blanquet 1985). It is likely that the threads and venom of
medium sized and small sized microbasic p-mastigophores in A. pallida are made of the
same proteins; however, this has not been reported in the literature. In any case, nitrogen
would be needed for both venom and structural components for newly synthesized
nematocysts.
The current study used 1 µm filtered, UV sterilized saltwater taken from saltwater
wells with no nutrients added. The water was changed every other day, the experimental
bowls were cleaned of algae when needed and the anemones were not fed for a total of 17
32
days. Cook et al. (1988) reports that the effects of nutrient limitation in the zooxanthellae
of Aiptasia pallida can be seen 10 to 30 days after the last feeding. During this time, both
the numbers of zooxanthellae and host protein content drop considerably (Cook et al.
1988). All anemones in this study showed a decrease in color during the experiment
which suggests a loss in zooxanthellae density.
The nitrogen content of the seawater used in these experiments was not measured
during the experiment, and it is not certain if nitrogen limitation was a factor. There are
two ways in which a symbiotic host can obtain nitrogen compounds: through direct
feeding or through translocation of nitrogen compounds from the zooxanthellae to the
host through absorption of nitrogenous compounds from sea water (Muller-Parker and
Davy 2001). Lipschultz and Cook (2002) found little evidence of translocation of
ammonium nitrogen from zooxanthellae to host in A. pallida and found that ammonium
assimilation in the body column of aposymbiotic A. pallida was low. They suggest that
the host might play an important role in the assimilation of ammonium and stated that
host feeding might be more important for nitrogen in column tissues because nitrogen
was not transferred from the tentacle down to the column (Lipschultz and Cook 2002).
The large increase seen in artificially damage anemones may be due to sufficient nitrogen
storage or recycling within host tissues; however, this cannot be determined from the
current study.
One explanation as to why shrimp-damaged anemones had fewer nematocysts if
all types compared to artificially damaged or control anemones is that there is an
inducible defense occurring. There is little information about the energy costs needed to
33
produce nematocysts of different size and function. One possibility is that the production
of large sized microbasic p-mastigophores require more energy and protein-nitrogen than
the production of other nematocysts found in the A. pallida cnidom. Large sized
microbasic p-mastigophores have an average length of 51 µm and medium and small
sized microbasic p-mastigophores have an average length of only 24 and 16 µm
respectively. Large sized microbasic p-mastigophores likely have a longer thread and
contain a larger volume of venom than the other types. If shrimp-damaged anemones are
allocating energy to healing and to the production of large sized defensive nematocysts,
then there is less energy to allocate to the production of other nematocysts types which
could explain why this group has the significantly less total nematocysts groups (Figure
8). In plants, it has been shown that resource availability can alter plastic responses
where limited resources often result in a limited expression of the plastic trait (Kohyama
1987; Ruiz et al. 2006; Ward et al. 2012).
Shrimp-damaged anemones exhibited a greater acontial response during predation
than the artificially damaged anemones, which may have led to a decrease in the numbers
of large nematocysts. Nematocysts are used one time, and have to be replaced after
firing. During shrimp predation, L. wurdemanni was seen attacking, retreating and then
returning again to feed. During each attack, they came into contact with acontial threads
which would cause large nematocyst to fire. Unfortunately, numbers of large
nematocysts pre and post attack could not be quantified and any attempt to do so would
have caused further artificial damage which might have hindered the comparison between
natural and artificial predation.
34
Defensive responses to natural predation generally require a cue given off by the
predator itself, as in Chornesky (1983). Following recovery, shrimp-damaged anemones
showed no significant difference in the number of large defensive nematocysts when
compared to controls. This indicates that if large amounts of defensive nematocysts were
used during shrimp attacks, these anemones were able to synthesize them to levels similar
to that of an undamaged anemone grown under the same conditions. Shrimp-damaged
anemones had significantly fewer medium-sized nematocysts than both controls and
artificially damaged anemones when comparing the whole anemone data and
significantly fewer small-sized nematocysts in the column.
During the recovery period, there was no visible difference between anemones
that had been artificially damaged and those that were shrimp-damaged, and yet
differences were seen with regards to nematocyst number and type. Although artificial
damage was conducted in a similar manner to that of shrimp-damage (e.g. tentacle
damage), one possible explanation is that the type of damage inflicted on the anemone
varied somewhat. During artificial damage, tentacles were removed at the junction of the
tentacle and the oral disc. During this process, the oral disc likely experienced damage.
In contrast, during a shrimp attack, the anemones pulled the tentacles down and slightly
inflated the column. As the shrimp fed, they removed tissue from the tentacles and the
column just under the tentacles.
35
CONCLUSIONS
Whether nematocyst production in Aiptasia pallida is an inducible defense or not
is still unclear. What is clear is that the cnidom of A. pallida exhibits phenotypic
plasticity. The cnidom of each treatment group showed variation. Shrimp-damaged
anemones had less nematocysts than either artificially damaged or controls anemones;
however, significant differences were only seen in medium sized microbasic pmastigophores, amastigophores and total number of nematocysts ratherthan in the large
sized defensive nematocysts. Artificially damaged anemones had twice the number of
small sized microbasic p-mastigophores than controls possibly suggesting a shift in
nematocyst production to enhance feeding.
This experiment was not designed to test the effects of nitrogen assimilation yet in
the tentacle samples, artificially damaged anemones were capable of producing as many
total nematocysts as controls and significantly more small sized nematocysts. It is clear
that more work is needed on nitrogen assimilation and distribution in Aiptasia pallida and
how nitrogen limitation effects the production of protein rich structures such as
nematocysts. It is also clear that other types of defensive mechanisms, such as chemical
cues associated with the presence of a predator need to be explored
36
TABLES
Nematocyst type*
N
Length µm **
Length µm
Group
designations
LN
50
51.0
39-62
>39 µm
MN
50
24.0
21-29
20-39 µm
SM
50
17
11-19
<20 µm
Table 1. Group designations for the different sized microbasic p-mastigophores found in
Aiptasia pallida. Groups were found to be significantly different with a Kruskal-Wallis
Test (p<0.001).
**LN: Large nematocysts, MN: Medium nematocysts, SN: Small nematocys
**Median length
37
MANOVA
Wilks'
Dependent Sum of
Lambda
Variable* Squares df
Body Region
df
p
Whole anemone
0.190 8 <0.001
LN
0.316 2
MN
0.395 2
SN
1.879 2
A
1.723 2
Tentacles
0.343 6 <0.001
MN
0.395 2
SN
1.879 2
A
1.723 2
Column
0.771 6 <0.001
LN
0.316 2
SN
1.879 2
A
1.723 2
Tentacle proportions 0.454 6 <0.001
LN
0.316 2
SN
1.879 2
A
1.723 2
Column proportions 0.378 6 <0.001
LN
0.316 2
SN
1.879 2
A
1.723 2
ANOVA
Mean
Square
0.158
0.197
0.939
0.862
1.5E+09
7.2E+09
2E+08
0.129
0.790
0.440
0.084
0.126
0.006
0.160
0.115
0.014
F
2.849
5.120
25.026
15.924
7.683
14.581
2.692
2.093
13.242
8.214
16.020
14.337
0.640
14.925
12.604
5.910
Table 2. Summary of MANOVA and ANOVA statistics.
*LN: Large nematocysts, MN: Medium nematocysts, SN: Small nematocysts, A:
Amastigophores
38
p
0.071
0.011
<0.001
<0.001
0.002
<0.001
0.081
0.138
<0.001
0.001
<0.001
<0.001
0.533
<0.001
<0.001
0.006
FIGURES
Figure 1. Illustration showing the production of the induced spines in Membranipora
membranacea. Inset shows spine detail. Figure from Harvell 1990.
39
Figure 2. Conical (left) and bent (right) forms of Chthamalus anisopoma showing the
location of the apertures. The bent form of C. anisopoma develops in response chemical
cues given off by Acanthina aangelica, a common barnacle predator. Figure from Lively
1986a.
40
Figure 3. Treatments performed on Agaricia agaricites to determine cues associated with
the formation of induced sweeper tentacles. Only treamtents where animate damage was
present resulted in sweeper tentacle formation. Animate damage herein is defined as
artifical damage. Figure taken Chornesky 1983.
41
Figure 4. Anatomy of a sea anemone. Note the location of the acontia, cinclides and
acrorhagus. Figure from Shick 1991.
42
Figure 5. Means of the different type nematocysts per milligram of protein for each
treatment type for tentacle data. Large, medium and small denote the size classes of
microbasic p-mastigophores. Statistically similar groups within each nematocyst type (p
< 0.05; post-hoc Tukey test) are indicated with the same letter while those that are
significantly different (p< 0.05) are indicated by different letters. Error bars are ± one
standard error.
43
Figure 6. Means of each nematocyst type per milligram of protein for each treatment
type for column samples. Large, medium and small denote the size classes of microbasic
p-mastigophores. Statistically similar groups within each nematocyst type (p< 0.05; posthoc Tukey test) are indicated with the same letter while those that are
significantly different (p < 0.05) are indicated by different letters. Error bars are ± one
standard error.
44
Figure 7. Means of each nematocyst type per milligram of protein for each treatment
type for whole anemone data. Large, medium and small denote the size classes of
microbasic p-mastigophores. Statistically similar groups within each nematocyst type
(p<0.05; post-hoc Tukey test) are indicated with the same letter while those that are
significantly different (p < 0.05) are indicated by different letters. Error bars are ± one
standard error.
45
Figure 8. Means of the total nematocysts per milligram of protein. Statistically similar
groups within each nematocyst type (p < 0.05; post-hoc Tukey test) are indicated with the
same letter while those that are significantly different (p < 0.05) are indicated by different
letters. Error bars are ± one standard error.
46
Figure 9. Nematocyst proportions for each nematocyst type in percentage for tentacle
samples. Large, medium and small denote the size classes of microbasic pmastigophores. Statistically similar groups within each nematocyst type (p < 0.05; posthoc Tukey test) are indicated with the same letter while those that are
significantly different (p < 0.05) are indicated by different letters. Error bars are ± one
standard error. UNID: unidentified nematocysts.
47
Figure 10. Nematocyst proportions for each nematocyst type in percentage for column
smaples. Large, medium and small denote the size classes of microbasic pmastigophores. Statistically similar groups within each nematocyst type (p < 0.05; posthoc Tukey test) are indicated with the same letter while those that are
significantly different (p < 0.05) are indicated by different letters. Error bars are ± one
standard error. UNID: unidentified nematocysts.
48
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