NEMATOCYST REPLACEMENT IN THE AIPTASIA PALLIDA

FOLLOWING BY 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.

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ABSTRACT

Author: Lucas Jennings

Title: Nematocyst Replacement in the Sea Anemone Aiptasia pallida Following Predation by : 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 , 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.

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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 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.

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Artificial Predation ...... 24 Shrimp Predation ...... 26 Artifical vs. shrimp predation...... 26 Discussion ...... 29 Conclusions ...... 36 References ...... 49

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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), shell- dimorphism 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 (Slattery et al. 2001), and the increase in defensive nematocysts in hard coral (Gotchfeld 2004).

PRODUCTION OF NEW DEFENSIVE STRUCTURES

The 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 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).

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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 (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).

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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 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 p- mastigophores, 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 p- mastigophores 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).

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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.

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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.

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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.

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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.

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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 sub- samples 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 p- mastigophores 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:

-1 mg protein mL = 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 p- mastigophores 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 p- mastigophores 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 p- mastigophores 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 p- mastigophores (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 small-

27 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 shrimp- damaged 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 p- mastigophores (<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 p- mastigophores of this size are found only in the mesenterial filaments and the column walls of A. pallida. Further, Carlgren and Hedgpeth (1952) reported microbasic p- mastigophores 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 p- mastigophore 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 p- mastigophores 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 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 p- mastigophores) 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 p- mastigophores, 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

Group Nematocyst type* N Length µm ** Length µm 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 ANOVA Wilks' Dependent Sum of Mean Body Region Lambda df p Variable* Squares df Square F p Whole anemone 0.190 8 <0.001 LN 0.316 2 0.158 2.849 0.071 MN 0.395 2 0.197 5.120 0.011 SN 1.879 2 0.939 25.026 <0.001 A 1.723 2 0.862 15.924 <0.001 Tentacles 0.343 6 <0.001 MN 0.395 2 1.5E+09 7.683 0.002 SN 1.879 2 7.2E+09 14.581 <0.001 A 1.723 2 2E+08 2.692 0.081 Column 0.771 6 <0.001 LN 0.316 2 0.129 2.093 0.138 SN 1.879 2 0.790 13.242 <0.001 A 1.723 2 0.440 8.214 0.001 Tentacle proportions 0.454 6 <0.001 LN 0.316 2 0.084 16.020 <0.001 SN 1.879 2 0.126 14.337 <0.001 A 1.723 2 0.006 0.640 0.533 Column proportions 0.378 6 <0.001 LN 0.316 2 0.160 14.925 <0.001 SN 1.879 2 0.115 12.604 <0.001 A 1.723 2 0.014 5.910 0.006 Table 2. Summary of MANOVA and ANOVA statistics. *LN: Large nematocysts, MN: Medium nematocysts, SN: Small nematocysts, A: Amastigophores

38

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; 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.

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 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. 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 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. UNID: unidentified nematocysts.

48

REFERENCES

Agrawal, A. A., and R. Karban. 1999. Why induced defenses may be favored over

constitutive strategies in plants. Pages 45-61 in R. Tollrian and C. D. Harvell,

editors. The ecology and evolution of inducible defenses. Princeton University

Press, Princeton, New Jersey, USA

Anderson, P. and C. Bouchard 2009. The regulation of discharge. Toxicon

54: 1046-1053

Bigger, C.H. 1988. The role of nematocysts in anthozoan aggression. In The biology of

nematocysts. Edited by D.A. Hessinger and H.M. Lenhoff. Academic Press, Inc.,

San Diego. pp. 295–308.

Blanquet, R. 1968. Properties and composition of the nematocyst toxin of the sea

anemone, Aiptasia pallida. Comp. Biochem. Physiol. 25: 893-896.

Carlgren, O. and J. W. Hedgpeth. 1952. Actiniaria, Zoanthiaria and Ceriantharia from

shallow water in northwestern Gulf of Mexico. Publ. Inst. Mar. Sci. (Univ.

Texas).

Carroll, D. J. and S. C. Kempf. 1990. Laboratory culture of the Aeolid nudibranch

Berghia verrucicornis (Mollusca, Opisthobranchia): some aspects of its

development and life history. Biol. Bull. 179: 243-253.

49

Chornesky, E. 1983. Induced development of sweeper tentacles on the reef coral

Agaricia agaricites: a response to direct competition. Biol. Bull. 165: 569-581.

Clavico, E. E. G., B. A. P. Da Gama, A. R. Soares, K. M. Cassiano and R. C. Pereira.

2013. Interaction of chemical and structural components providing defences to

sea pansies Renilla reniformis and Renilla muelleri. Mar. Biol. Res. 9: 285-292.

Clayton, W. S. 1986. Factors affecting zooplankton feeding by the sea anemone

Aiptasia pallida. Helgolander Meeresun. 40: 83-90.

Clayton W. S. and H. R. Lasker. 1985. Individual and population growth in the

asexually reproducing anemone Aiptasia pallida Verrill. J. Exp. Mar. Biol. Ecol.

90: 249-258.

Cook, C. B., C. F. D’Elia and G. Muller-Parker. 1988. Host feeding and nutrient

sufficiency for zooxanthellae in the sea anemone Aiptasia pallida. Mar. Biol. 98:

253-262.

Daly, M. 2003. The anatomy, terminology, and homology of acrorhagia and

pseudoacrorhagi in sea anemones. Zool. Verh. Leiden. 345: 89-101.

Davy, S. K. and C. B. Cook 2001. The relationship between nitrogen status and carbon

flux in the zooxanthellate sea anemone Aiptasia pallida (Verrill). Mar. Biol.

139: 999-1005.

DeWitt, T. J., A. Sih and D. S. Wilson. 1998. Costs and limits of phenotypic plasticity.

Trends Ecol. Evol. 13: 77-81.

Edmunds, M., G. W. Potts and V. L. Waters. 1976. Defensive behaviour of sea

anemones in response to predation by the opisthobranch mollusc Aeolidia

papillosa (L.). J. Mar. Biol. Assoc. UK. 56: 65-83. 50

Fautin, D.G. – 1988. Importance of nematocysts to actinian . In: D.A.

Hessinger and H.M. Lenhoff (eds.). The Biology of Nematocysts. pp. 487-500.

Academic Press, San Diego

Gochfeld, D. J. 2004. Predation-induced morphological and behavioral defenses in a

hard coral: implications for foraging behavior of coral-feeding butterflyfishes.

Mar. Ecol. Prog. Ser. 267: 145-158

Goulet T. L., C. Cook, and D. Goulet. 2005. Effect of elevated temperature and light

levels on the photosynthesis of different host-symbiont combinations in the

Aiptasia pallida/Symbiodiniumsymbiosis. Limnol Oceanogr. 50: 1490–1498

Hambleton, E. A., A. Guse, and J. R. Pringle. 2014. Similar specificities of symbiont

uptake by adults and larvae in an anemone model system for coral biology. J.

Exp. Biol. 217: 1613-1619.

Harris, L. and N. Howe. 1979. An analysis of the defensive mechanisms observed in the

anemone Anthopleura elegantissima in response to its nudibranch predator

Aeolidia papillosa. Biol. Bull. 157: 138-152.

Harvell, D. C. 1984. Predator-induced defense in a marine bryozoan. Science. 224:

1375-1359.

Harvell, D. C. 1986. The ecology and evolution of inducible defenses in a marine

bryozoan: cues, costs, and consequences. Am. Nat. 128: 810-823.

Harvell, D. C. 1990. The ecology and evolution of inducible defenses. Q. Rev. Biol. 65:

323-340.

51

Hessinger, D. A. and H. M. Lenhoff. 1973. Assay and properties of the hemolysis

activity of pure venom from the nematocysts of the acontia of the sea anemone

Aiptasia pallida. Arch. Biochem. Biophys. 159: 629-638.

Howe, N. R. and Harris, L. G. 1978. Transfer of the sea anemone pheromone

anthopleurine, by the nudibranch Aeolidia papillosa. J. Chem. Ecol. 4: 551-561.

Karban, R., and I. T. Baldwin. 1997. Induced responses to herbivory. University of

Chicago Press, Chicago, Illinois, USA.

Kass-Simon, G. and A. A. Scappaticci, Jr. 2002. The behavioral and developmental

physiology of nematocysts. Can. J. Zoolog. 80: 1772-1794.

Kohyama, T. 1987. Significance of architecture and allometry in saplings. Funct. Ecol.

1: 399-404.

Lages, B. G., B. G. Fleury, C. E. L. Ferreira and R. C. Pereira. 2006. Chemical defense

of an exotic coral as invasion strategy. J. Exp. Biol. 328: 127-135.

Lages, B. G., B. G. Fleury, A. M. C. Hovell, C. M. Rezende, A. C. Pinto and J. C. Creed.

2012. Proximity to competitors changes secondary metabolites of non-indigenous

cup corals, Tubastraea spp., in the southwest Atlantic. Mar. Biol. 159: 1551-

1559.

Layne, E. Spectrophotometric and turbidimetric methods for measuring proteins.

Methods in Enzymology 3: 447-455. 1957.

Lehnert, E. M., M. S. Burriesci and J. R. Pringle. 2012. Developing the anemone

Aiptasia as a tractable model for cnidarian- symbiosis: the

transcriptome of aposymbiotic A. pallida. BMC Genomics. 13: 1-10.

52

Lipschultz, F. and C. B. Cook. 2002. Uptake and assimilation of 15N by the symbiotic

sea anemones Bartholomea annulata and Aiptasia pallida: conservation versus

recycling of nitrogen. Mar. Biol. 140: 489-502.

Lively, C. M. 1986a. Predator-induced shell dimorphism in the acorn barnacle

Chthamalus anisopoma. Evolution. 67: 858-864.

Lively, C. M. 1986b. Competition, comparative life histories and maintenance of shell

dimorphism in a barnacle. Ecology. 67: 858-864.

Marino, A., R. Morabito, T. Pizzata and G. La Spada. 2008. Effect of crude venom from

nematocysts of Pelagia noctiluca (Scyphozoa) on discharge of acontia of

Calliactis parasitica (). Chem. Ecol. 24: 9-17.

Mariscal, R. N. 1974. Nematocysts. In Coelenterate Biology: Reviews and New

Perspectives, (eds. L. Muscatine & H. M. Lenhoff), pp. 129-179. New York,

London: Academic Press.

Muller-Parker, G. and S. K. Davy. 2001. Temperate and tropical algal-sea anemone

symbioses. Invertebr. Biol. 120: 104-123.

Ӧstman, C., J. R. Kultima, C. Roat, and K. Rundblom. 2010. Acontia and mesentery

nematocyst of the sea anemone Metridium senile (Linnaeus, 1761) (:

Anthozoa). Sci. Mar. 74: 483-497.

Phelan, M. A. and R. S. Blanquet. 1985. Characterization of nematocysts proteins from

the sea anemones Aiptasia pallida and Pachycerianthus torreyi (Cnidaria:

Anthozoa). Comp. Biochem. Physiol. 81: 661-666.

53

Rhyne, A. L., J. Lin and K. J. Deal. 2004. Biological control of aquarium pest anemone

Aiptasia pallida Verrill by peppermint shrimp Lysmata Risso. J. Shellfish Res.

23: 227-229.

Ruiz-R, N., D. Ward and D. Saltz. 2006. Population differentiation and the effects of

herbivory and sand compaction on the subterranean growth of a desert lily. J.

Hered. 97: 409-416.

Ruppert, E. E. and R. S. Fox. 1988. Seashore Animals of the Southeast: A Guide to

Common Shallow-Water Invertebrates of the Southeastern Atlantic Coast. Univ.

of South Carolina Press, 429 p.

Schlesinger, A., E. Kramarsky-Winter, H. Rosenfeld, R. Armoza-Zvoloni and Y. Loya.

2010. Sexual Plasticity and Self-Fertilization in the Sea Anemone Aiptasia

diaphana. PLoS ONE. 5: 1-7.

Schlichting, C.D. 1986. The evolution of phenotypic plasticity in plants. Annu. Rev. Ecol.

Syst. 17:667-693.

Sebens, K. P. and J. S. Miles. 1988. Sweeper Tentacles in a Gorgonian Octocoral:

Morphological Modifications for interference Competition. Biol. Bull. 175: 378-

387.

Sefton, N., and Webster, S. 1986. Caribbean reef invertebrates. Sea Challengers,

California, Monterey.

Shick, J. M. 1991. A Functional Biology of Sea Anemones. Chapman and Hall, London

and other cities. 395 pp.

54

Slattery, M., J. Starmer and V. J. Paul. 2001. Temporal and spatial variation in defense

metabolites of the tropical Pacific soft corals Sinularia maxima and S.

polydactyla. Mar. Biol. 138: 1183-1193.

Stachowicz, J. J. and N. Lindquist. 1997. Chemical defense among hydroids on pelagic

Sargassum: predator deterrence and absorption of solar UV radiation by

secondary metabolites. Mar. Ecol. Prog. Ser. 155: 115-126.

Starzak, D. E., R. G. Quinnell, M. R. Nitschke and S. K. Davy. 2014. The influence of

symbiont type on photosynthetic carbon flux in a model cnidarian-dinoflagellate

symbiosis. Mar. Biol. 161: 711-724.

Stephenson, T. A. and A. Stephenson. 1952. Life between tide-marks in North America:

II. Northern Florida and the Carolinas. J. Ecol. 40: 1-49.

Sterns, S. C. 1989. The Evolutionary Significance of Phenotypic Plasticity. Bioscience.

39: 436-445.

Sultan, S. E. 1987. Evolutionary implications of phenotypic plasticity in plants. Evol.

Biol. 21: 127–176.

Swanson, R., and O. Hoegh-Guldberg. 1998. Amino acid synthesis in the symbiotic sea

anemone Aiptasia pulchella. Mar. Biol. 131: 83-93.

Thorington, G., V. McAuley and D. Hessinger. 2010. Effects of satiation and starvation

on nematocyst discharge, prey killing, and ingestion in two species of sea

anemone. Biol. Bull. 219: 122-131.

Tollrian, R. and C. D. Harvell, 1999. The ecology and evolution of inducible defenses.

Princeton University Press, Princeton, New Jersey, USA.

55

Turner, V. L. G., S. M. Lynch, L. Paterson, J.L. Leon-Cortes and J. P. Thorpe. 2003.

Aggression as a function of genetic relatedness in the sea anemone Actinia equina

(Anthozoa: Actiniaria). Mar. Ecol. Prog. Ser. 247: 85-92.

Trussell, G.C. 1996. Phenotypic plasticity in an intertidal snail: The role of a common

crab predator. Evolution 50: 448-454.

Trussell, G.C., M.O. Nicklin. 2002. Cue sensitivity, inducible defense, and trade-offs in

a marine snail. Ecology 83: 1635-1647.

Ward, D., M. K. Shrestha and A. Golan-Goldhirsh. 2012. Evolution and ecology meet

molecular genetics: adaptive phenotypic plasticity in two isolated Negev desert

populations of Acacia raddiana at either end of a rainfall gradient. Ann Bot-

London. 109: 247-255.

Wellington, G. 1980. Reversal of digestive interactions between Pacific reef corals:

mediation by sweeper tentacles. Oecologia. 47: 340-343.

Whitehead, L. F. and A. E. Douglas. 2003. Metabolite comparisons and the identity of

nutrients translocated from symbiotic algae to an animal host. J. Exp. Biol. 206:

3149-3157.

Williams, R. B. 1991. Acrorhagi, catch tentacles and sweeper tentacles: a synopsis of

‘aggression’ of actiniarian and scleractinian Cnidaria. Hydrobiologia. 216/217:

539-545.

Zhang, D., J. Lin, and R. L. Creswell. 1998. Effects of food and temperature on survival

and development in the peppermint shrimp Lysmata wurdemanni. J. World

Aquacult. Soc. 29: 471-476.

56

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