chemosensory and feeding responses of the nudibranch aeolidia papillosa to the symbiotic sea anemone...

Invertebrate Biology 121(2): 1 IS-125. 0 2002 American Microscopical Society, Inc.

Chemosensory and feeding responses of the nudibranch Aeolidia papillosa to the symbiotic sea anemone Anthopleura elegantissirnu

Barbara Emerson Seavy and Giskle Muller-Parkera

Department of Biology and Shannon Point Marine Center, Western Washington University, Anacortes, WA 98221, USA

Abstract. The aeolid nudibranch Aeolidia papillosa is an important predator on the sea anemone Anthopleura elegantissima, a host to two kinds of endosymbiotic algae: zooxanthellae and zoochlorellae. The possible influence of the algae on the nudibranch’s predatory response to this anemone was examined in a laboratory study. In chemosensory experiments, the nudibranch detected and chose anemone scent over a seawater control, but in both chemosensory and feeding experiments showed no preference for zooxanthellate or zoochlorellate anemones. Ingestive conditioning on zooxanthellate or zoochlorellate anemones had no effect on choice of these two anemone types in chemosensory experiments. Comparisons of the productivity and photosynthetic pigments of algae obtained from nudibranch feces and from anemones show that both algae survive passage through the nudibranch gut. The productivity of fecal zooxanthellae was 1.6X greater than that of zooxanthellae freshly isolated from anemones, although the chlorophyll a content of fecal zooxanthellae was reduced. The productivity and amount of pigments were the same for zoochlorellae in nudibranch feces and freshly isolated from anemones. Comparing fecal and isolated algae, there was no significant difference in the percentage of zooxanthellae in the process of cell division. However, the percentage of dividing cells was 2.6X higher in fecal than in freshly isolated zoochlorellae (18% and 6.9% respectively). Al- though the endosymbiotic algae do not make their host more or less attractive to the nudibranch, this predator may play an important role in maintaining the symbiotic relationship of Antho- pleura elegantissima with zooxanthellae and zoochlorellae by providing viable algae in its feces as a source for the anemone host.

Additional key words: algal symbiosis, zooxanthellae, zoochlorellae

The temperate nudibranch mollusc Aeolidia papillosa (LINNAEUS 1761) is a formidable predator of sea anemones, consuming 50 to 100% of its body weight daily (Ricketts et al. 1985) and influencing the distri- butional patterns of its prey (Harris & Howe 1979; Harris 1986). This aeolid is found in the intertidal zone of both Atlantic and Pacific Oceans (Swennen 1961; Behrens 1991) and subtidally to 870 m (McDonald 1983). It feeds on a variety of anemones (Stehouwer 19.52; Yarnall 1972; Ottaway 1976), 28 prey species worldwide according to McDonald & Nybakken (1978-79). The nudibranch locates prey anemones by chemical scent (Stehouwer 1952; Haaften & Verwey 1960) and, in laboratory experiments, chooses among anemone species (Russell 1942; Stehouwer 1952; Har- ris 1986).

A preferred prey species is the sea anemone Antho-

a Author for correspondence. E-mail: [email protected]

pleura elegantissima (BRANDT 183.5) (Harris 1973; Waters 1973; Edmunds et al. 1974, 1976), an ecolog- ically important member of the rocky intertidal corn- munity of the Pacific coast of North America (Hand 1955; Fitt et al. 1982). This anemone is host to two kinds of endosymbiotic algae, zooxanthellae and zoochlorellae (Muscatine 197 1). Zooxanthellae in A. elegantissima in the Puget Sound region of Washing- ton State have been identified as the dinophyte Sym- hiodinium muscatineii (LaJeunesse & Trench 2000). The zoochlorellae are unidentified green chlorophytes. Individuals of A. elegantissima may contain zooxanthellae, zoochlorellae, or both; some anemones (aposymbiotic) lack symbionts. The distribution of zoochlorellate anemones is restricted to northern latitudes (Secord & Augustine 2000). All of these anemone types are found in the northern Puget Sound region in close proximity to one another (Engebretson & Muller- Parker 1999).

116 Seavy & Muller-Parker

if prey selection on the basis of endosymbiont com- plement occurs, this will influence the relative population abundance of anemone types and their respec- tive contributions to gross primary production within the intertidal habitat (Fitt et al. 1982; Verde & Mc- Closkey 1996). Preferential predation on a given anemone type might also provide a benefit to anemones belonging to the less preferred type. Experiments with one major predator on this anemone provide ev- idence of predation refuge for zoochlorellate anemones. The tidepool sculpin Clinocottus globiceps is a visual predator (Yoshiyama et al. 1996) that nips off anemone tentacles and shows clear preference for zooxanthellate anemones (Augustine & Muller-Parker 1998). This fish digests zooxanthellae but not zoochlorellae.

This study parallels that of Augustine & Muller- Parker (1998), in which the role of endosymbiotic algae in predator-prey interactions is evaluated with A. papillosa, a predator that, as noted above, detects its anemone prey by chemosensory means. The nudibranch predator also differs from the sculpin predator in that both symbiotic algae ingested with anemone tissues are photosynthetically competent within the cerata (McFarland & Muller-Parker 1993). The nudibranch might select between zooxanthellate and zoochlorellate anemones if the algal types differ in their nutritional contributions during passage through the nudibranch gut. The first part of this study examines the ability of the nudibranch to choose between anemone types on the basis of chemical scent alone. We fed nudibranchs on an exclusive diet of zooxanthellate or zoochlorellate anemones and then tested the effect of this ingestive conditioning on choice between symbiotic anemones. The second part examines whether nudibranchs feed selectively on one or more types when provided a choice among zooxanthellate, zoochlorellate, and aposymbiotic anemones. We also examined how nudibranch predation affects the symbiotic algae by comparing the productivity, chlorophyll content, and percent dividing cells of fecal algae with that of algae freshly isolated from anemone hosts.

Methods

Collection of nudibranchs and sea anemones

Specimens of Aeolidia papillosa and Anthopleura elegantissirnu were collected as needed between No- vember 1997 and May 1998 from 4 sites in northern Puget Sound, Washington: Anaco Beach (48"29'N, 122'42'W) and Shannon Point (48"30'N, 122'41 'W), Fidalgo island; Eagle Cove, San Juan island (48"27'N, 123"02'W); and Admiralty Inlet, Whidbey Island (48'08'N, 122'36'W). The ambient seawater temper-

ature ranged from a mean of 7.1"C in January to a mean of 10.4"C in May. individuals of the three anemone types (zooxanthellate, zoochlorellate, aposymbiotic) were confirmed by microscopic examination of ex- cised tentacles. Tentacles containing 290% of one alga, or no endosymbionts, were used to assign individuals to one of these types. All anemones were maintained in ambient seawater in flow-through sea tables and were fed freshly hatched brine-shrimp nau- plii (Artemia franciscana) weekly. Anemones used as chemical scent in the chemosensory experimental apparatus were maintained in the holding boxes for the duration of each experiment under similar conditions.

A. papillosa was found in the intertidal zone close to large populations of zooxanthellate anemones, which was the most abundant anemone type at the collection sites (unpubl. obs.). Three collections of nudibranchs were used in the experiments (groups I-iil, Table 1). Initial wet weight and length were recorded for each individual and thereafter every 7 to 10 days. Each nudibranch was placed in an individual plastic mesh basket maintained in ambient seawater in a flow- through sea table. Nudibranchs were allowed to feed ad libitum on zooxanthellate anemones, except for those used to investigate the role of ingestive conditioning on prey choice. After experiments were com- pleted, remaining anemones and nudibranchs were re- turned to their collection sites.

Experimental protocols

Each chemosensory and feeding experiment consisted of a number of trials during which individual nudibranchs were tested for prey choice. Identical apparatuses were placed in adjacent sea tables allowing 2 trials to be run simultaneously. Each sea table was fitted with an overhead light bank 1.2 m long of full- spectrum lights providing 110 pmol photons m s-' (12:12 light cycle). All trials were initiated between 1000 and 1830 h.

Anemones used in experiments had an oral disk di- ameter of 0.5-1.0 cm and were not fed for at least 24 h before a trial. Nudibranchs were selected randomly and were not fed for 3 days before testing. After each trial the apparatuses were scrubbed, cleaned with Mi- cro-9O0, and rinsed thoroughly with RO (reverse os- mosis) water.

Chemosensory experiments

To determine if chemical scent from anemones containing zooxanthellae, zoochlorellae, or no algae affects prey choice by A. pupillosa (Table l), we ran 3 cheinosensory experiments. The first experiment tested nudibranch choice between anemones and seawater;

Nudibranch predation on a symbiotic sea anemone 117

Table 1. Three groups of the nudibranch Aeolicfia papillosa and the experiments in which they were used. Sample statistics are mean 5 standard deviation. Weights and lengths are those at the time of the trials. Anemones, 4 of each type (AN); seawater (SW). Types of the sea anemone Anthopleura elegantissirnu: Zooxanthellate (ZX); zoochlorellate (ZC); aposyrn- biotic (APO).

Number of Number of nudibranchs

Nudibranch nudibranchs making a Number of Nudibranch Nudibranch Experiment group‘ tested choice trials weight (g) length (cm)

C hernosensor y AN vs S W I11 18 13 17 10.6 -C 2.08 7.1 t 0.65 zx vs ZCh I 18 28 34 19.3 t 4.08 9.2 5 0.95 ZX vs APO 111 12 16 17 10.8 5 2.02 6.9 5 0.64

One I 12 9 12 20.3 -C 4.48 9.3 -C 1.30 Two I 1 18 16 18 11.8 -C 3.29 7.7 -C 0.68

Feeding Choice

‘I Collection dates (1997-1998): Group I: Nov 11 to Jan 30; Group 11: Mar 19 to Apr 2; Group 111: Apr 2 to Apr 17. ‘’ Nudibranchs subjected to ingestive conditioning before trials.

anemone scent was provided by a single group of 12 anemones (4 of each type). A second experiment evaluated the effect of the most recent feeding experiences on nudibranch choice between scents from zooxanthellate and zoochlorellate anemones; nudibranchs were maintained on a diet of either zooxanthellate or zoochlorellate anemones before testing (ingestive conditioning). A third experiment tested nudibranch choice between scents from zooxanthellate and aposymbiotic anemones.

The chemosensory apparatus (Fig. 1) was modified from that used by Smith (1976-77) in tests of anemone scent with the wentletrap snail Epitonium tincturn. Be- cause A . pupillosa explores its surroundings (Waters 1973; Edmunds et al. 1976; our unpubl. obs.), we con- sidered Smith’s ( 1976-77) design particularly appro- priate as it allowed full exploration of the arena with no barriers to interfere with behavior as occurs with the use of a standard Y-tube (Harris 1973; our unpubl. obs.). Each clear Plexiglas chemosensory apparatus consisted of two main parts: a large arena in which individual nudibranchs were tested, and two holding boxes containing anemones (or one with no anemones) used as chemical scent point sources (Fig. la). A pas- sageway (I .5 cm diam.) connected the arena to each catch-chamber, allowing seawater to flow into the arena and the nudibranch to enter the catch-chamber (Fig. 1). A Y-shaped stream of seawater from the two point sources converged in the center of the arena, -12 cm from the front (Fig. la) and exited through holes in the back of the arena. The shape of the flow was checked with food coloring before trials. Seawater flow into the catch-chambers and the arena was adjusted to a rate of 150-160 ml min-’. Seawater entering the anemone holding boxes was directed over the

anemones by a Plexiglas baffle with screened holes near the bottom (Fig. lb).

Anemones of equal size (N = 11 or 12) were allowed to settle in a holding box for at least one day before trials. Anemones in the left and right holding boxes were alternated by type to avoid possible left- right bias in prey selection by the nudibranchs. Trials were begun when at least % of the anemones in each holding box had expanded tentacles and exposed oral disks. To initiate a trial, a nudibranch was placed on a roughened Plexiglas plate (9.5 cm diam.), which was then placed into the arena. The head of the nudibranch was positioned at the convergence point of the two streams of seawater flowing from the anemone holding boxes (Fig. la). Each nudibranch was given up to 2 h to make a choice. A choice was scored if the nudibranch inserted its head, including at least both rhin- ophores, into a catch-chamber. The total elapsed time to a choice and the search behavior of the nudibranch were recorded.

To test for ingestive conditioning, nudibranchs were divided into 2 subgroups with nudibranchs in each group averaging 10 g. All nudibranchs in both subgroups were tested twice, once after each diet. In one subgroup, nudibranchs were initially fed ad libitum on zooxanthellate anemones for 7 days, not fed for 3 days, tested, then fed Loochlorellate anemones ad Zi- bitum, similarly fasted, and tested. The other subgroup began their cycle with a diet of zoochlorellate anemones.

The results were analyzed using a 2 X 2 chi-square contingency table with expected frequencies of 3 . 0 (Zar 1996). Fisher’s exact test (Zar 1996) was used on all contingency tables having expected values -3 .0 . The log-likelihood G-test (Zar 1996) for goodness of

Seavy & Muller-Parker 118

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Fig. 1. Apparatus used for chemosensory experiments (modified from Smith 1976-77).

a. Top view: A nudibranch in the main arena moves up the stream of seawater flowing from a catch- chamber and indicates a choice by entering it. Holding boxes contained anemones (or seawater alone) to provide chemical scent point sources.

b. Side view: Seawater flows by gravity from a head tank through the anemone holding boxes. Anemone chemical scent is carried into the arena through the catch-chambers and exits via holes in the back of the arena and out a drain at the opposite end of the sea table.

Common Head Tank - - - Bank of Full-spectrum Lights

H 6 cm Anemone

Holdlng boxes

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Nudibranch predation on a symbiotic sea anemone I19

fit was used to analyze nudibranch choices in response to anemones vs. seawater and in response to zooxanthellate vs. aposymbiotic anemones.

Feeding experiments

Two similar experiments were used to determine if choices made solely on a chemosensory basis were consistent with choices made when nudibranchs could eat their prey as well (Table 1). In Experiment One, each of 12 nudibranchs from the second chemosensory experiment (zooxanthellate vs. zoochlorellate anemones) was tested once in a 24-h trial. In Experiment Two, each of 18 freshly collected nudibranchs was tested once in a 24-h trial.

Each feeding apparatus consisted of a clear Plexi- glas circular arena (44 cm diam.) with a mesh wall (15 cm high) within which the nudibranch was provided free access to its anemone prey. A separate baffle of PVC pipe (2.2 cm diam., 52 cm long) with holes (3 mm diam.) along its length was placed on the bottom of the sea table -50 cm from the arena to provide a unidirectional flow of seawater across the sea table. Although the overall flow was unidirectional, the mesh wall of the arena created a mixed flow within the arena (as shown by dye checks).

Anemones (N = 12, 4 of each type) were offered to individual nudibranchs in each trial. Pairs of zooxanthellate, zoochlorellate, or aposymbiotic anemones were allowed to settle on roughened Plexiglas plates. Plates of anemones were placed at 6 equidistant points in a circle, with anemone types alternating. Anemone pairs were rotated clockwise by one position before each trial to control for any effect of spatial orientation. Each nudibranch was placed on a roughened plastic plate in the center of the arena with its head directed toward the seawater inflow and was observed until it began eating its first anemone, which occurred within 3-21 min. We recorded the initial anemone type chosen and its position in the arena, as well as the number, type, and position of additional prey selec- tions made during the 24-h trial period, although the sequence of the latter was not known. We also recorded whether each anemone was consumed entirely or partially.

The log-likelihood G-test was used to analyze the feeding response of nudibranchs to anemone type by comparing the initial and subsequent choices made by each nudibranch, and by comparing the total number of anemones consumed in a 24-h period with expected values for equal choice. This test was also used to check for possible effects of anemone position in the arena.

Viability of algal endosymbionts in nudibranch feces

Viability of algal endosymbionts after passage through the nudibranch gut was assessed because digestibility of algal symbionts might influence prey choice. The productivity, chlorophyll content, and percent dividing cells of intact zooxanthellae and zoochlorellae obtained from nudibranch feces were compared with that of algae isolated directly from the anemones maintained in the holding boxes.

Nudibranchs were fed an exclusive diet of zooxanthellate or zoochlorellate anemones for 4 to 6 days before collection of feces. Nudibranch baskets were cleaned the day before fecal collection to ensure a fresh (<24-h) fecal sample. Fecal pellets from individual nudibranchs were collected by pipette, washed 3 or 4 times by centrifugation (5 min at 4000 rpm), and re-suspended in fresh 5-pm filtered seawater. Fecal algal suspensions were filtered through 20-pm Nitex mesh to remove animal debris and diluted with filtered seawater to yield final cell concentrations of 7-9 X lo5 cells ml-I. For comparison to fecal algae, zooxanthellae and zoochlorellae were isolated directly from anemones by homogenizing tissues of individuals of each type in filtered seawater using a 60-ml glass tis- sue grinder with a motorized Teflon pestle. Isolated algal samples were prepared as described for fecal algal samples.

For productivity measurements, 0.5-ml subsamples of each fecal and isolated algal sample were dispensed into clear 7-ml glass scintillation vials and incubated with I4C (see Augustine & Muller-Parker 1998) within 4 h of collection. Samples were incubated for 0.5 h at ambient seawater temperature (14°C) under a mean ir- radiance of 142 "_ 13 pmol photons m-z s- l in a water- cooled light incubation apparatus equipped with tung- sten-halogen lights. Photosynthetic rates were calculated as in McFarland & Muller-Parker ( 1993). A time-zero vial for each sample was acidified immedi- ately following I4C addition to check for the efficiency of removal of inorganic C from samples. Algal Sam- ples incubated in dark vials were used to correct for any heterotrophic fixation of I4C-bicarbonate.

For photosynthetic pigments, 3 4-ml subsamples of each fecal and isolated algal sample were filtered under gentle vacuum onto GF/C filters, placed in foil packets, and frozen until analyzed. Thawed filters containing zooxanthellae were homogenized in 100% ac- etone and filters containing zoochlorellae were homogenized in 100% methanol, which is a better solvent for chlorophyll b. Samples were analyzed as described in Augustine & Muller-Parker (1 998). The equations of Jeffrey & Humphrey (1975) were used to


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ZX Diet ZC Diet

Paired Choice Fig. 2. Number of nudibranchs, Aeolidia papillosa, choosing zooxanthellate (ZX), zoochlorellate (ZC), or aposymbiotic (APO) anemones in paired choice trials in chemosensory experiments. Ingestive conditioning consisted of a controlled diet of either zooxanthellate (ZX) or zoochlorellate (ZC) anemones before the trials. The nudibranchs were tested twice, once after each diet.

determine the chl a and chl c content of zooxanthellae, and the equations of Holden (1 976) were used to determine the chl a and chl b content of zoochlorellae.

Cell counts and percent dividing algal cells were determined from analysis of frozen fecal and isolated algal samples. Cell densities were estimated from the mean of 12 to 36 field counts using a hemacytometer at 400X magnification. The number of algae in the process of dividing (with a complete division furrow) was recorded as a percent of the total number of cells. At least 1000 algal cells were counted in each sample.

All comparisons between fecal and freshly isolated algal samples were analyzed by two-sample t-tests using Statistix 4.1 (Analytical Software) with (Y < .05. Replicate subsamples obtained in the productivity and pigment analyses were averaged to provide one value per sample before statistical analysis. Data for percent dividing cells were transformed (arcsine) before analysis. Degrees of freedom were adjusted to correct for heterogeneous variance in the chlorophyll c data only.

Results Chemosensory experiments

In trials presenting a choice between anemone scent (from combined anemone types) and seawater, 11 nu-

dibranchs chose anemone scent while 2 selected plain seawater. This difference is significant (G = 6.86, P < .0 1 , N = 13) and shows that Aeolidia pupillosa can detect and choose anemone chemical scent from a dis- tance. The nudibranch selects its prey solely on the basis of the anemone scent and not on the basis of the symbiotic condition of the anemone. Nudibranchs maintained on controlled diets of zooxanthellate anemones and switched to controlled diets of zoochlorellate anemones (and vice versa) were tested twice (once after each diet) and showed no significant difference in choice between zooxanthellate and zoochlorellate anemone scents, regardless of their most recent dietary experience ( x2 = 0.11, P = .74, Fig. 2). Moreover, nudibranchs did not choose between zooxanthellate and aposymbiotic anemones (G = 1.01, P = .31, Fig. 2).

Although nudibranch choice was not affected by anemone type in the chemosensory experiments, the behavior of nudibranchs offered the choice of LOOX-

anthellate and zoochlorellate anemone scents differed from that of nudibranchs offered all other paired choices. Nudibranchs exposed to scents from zooxanthellate and zoochlorellate anemones checked both openings to the catch-chambers more often than when presented with other choices. Only 36% of the nudibranchs checked both anemone and plain seawater scents, whereas 64% checked both zooxanthellate and Looch- lorellate anemone-scent point sources before making a choice. The nudibranchs investigated both moxan- thellate and zoochlorellate anemone scents significantly more often than they investigated only one of the two scents (G = 3.94, P = .04, N = 26). Nudibranchs provided a choice between zooxanthellate and aposymbiotic anemones and between anemones and seawater did not investigate both sources more often than they investigated a single source before making a choice (G = 0, P = .99, N = 16 and G = 1.97, P = .18, N = 13, respectively). The amount of time for nudibranchs to make their choices ranged from 2 to 120 min; 60% of all nudibranchs made their choice within the first 0.5 h of the 2-h trials.

Feeding experiments

The nudibranchs randomly fed on all 3 anemone types, ingesting an average of 3 anemones in each 24- h trial (an average of 2.9 anemoneshudibranch in Ex- periment One and an average of 3.5 anemoneshudibranch in Experiment Two). There were no significant differences in the types of anemones ingested by nudibranchs in Experiment One (G = 0.12, P = .94) or in Experiment Two (G = 0.62, P = .73; Fig. 3). All anemones consumed by nudibranchs were separated


22

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Fig. 3. Number of anemones, Anthopleura elegantissirnu, of each type consumed by nudibranchs, Aeolidia papillosa, during Experiment One and Experiment Two. Zooxanthel- late (ZX); zoochlorellate (ZC); aposymbiotic (APO).

according to type. All anemones belonging to each type were pooled for all 24-h feeding trials for statistical analysis because there was no prey selection by anemone type exhibited by nudibranchs in their initial prey choices (Experiment One: G = 1.52, P = .46, N = 26; Experiment Two: G = 1.14, P = S 6 , N = 56) and in their subsequent prey choices (Experiment One: G = 0.21, P = .90, N = 10; Experiment Two: G = 3.82, P = .16, N = 33).

Potential sources of bias in chemosensory and feeding experiments

Nudi branch choices were analyzed for possible bias in the experiments, including time in captivity, ran- domness of assignment to apparatuses, and the effect of using two apparatuses. Statistical analyses showed that none of these sources contributed any bias to the experimental results (Seavy 1999). There was also no effect of anemone position in the arena on prey choices in the feeding experiments (Experiment One: G = 4.58, P = .46, N = 26 anemones eaten; Experiment Two: G = 4.08, P = .53, N = 56 anemones eaten).

The sizes of the nudibranchs in each group were analyzed by one-way ANOVA. Nudibranchs in group I were significantly larger during the chemosensory trials than nudibranchs in group I11 (groups I vs. I11 respectively, weight: F = 58.78, P < .001; length: F = 58.60, P < .001, N = 68; Table 1). During the feeding choice trials, nudibranchs in group I were also

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zooxanthellae zoochlorellae Fig. 4. Carbon fixation rates and percent dividing cells of zooxanthellae and zoochlorellae isolated from thcir anemone host Anthopleura elegantissirnu (isolated) or obtained from feces of the nudibranch Aeolidia pupillosa (fecal).

A. Carbon fixation rates, mean + SEM. (N = 3 for isolated algae, N = 6 for fecal zooxanthellae, N = 8 for fecal zoochlorellae). ** indicates P < .Ol.

B. Percent dividing cells, mean + SEM. ( N = 3 for isolated algae, N = 6 for fecal zooxanthellae, N = 7 for fecal zoochlorellae). *:I:* indicates P < ,001.

significantly larger than those in group I1 (groups I vs. 11; weight: F = 19.54, P < .001; length: F = 11.10, P < .001, N = 44; Table 1). Despite this significant difference in size, all nudibranchs selected anemone prey randomly in all experiments, indicating that nudibranch size did not influence predation on anemone types.

Productivity, percent dividing cells, and chlorophyll content of algal endosymbionts

Compared to freshly isolated zooxanthellae, those that passed through the nudibranch digestive tract showed significantly higher productivity (t = 3.74, P < . O l ) but this was not the case for zoochlorellae (t = 0.87, P = .40) (Fig. 4A). The percent of dividing cells among fecal zoochlorellae was significantly


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zooxanthellae zoochlorellae Fig. 5. Mean values + SEM for chlorophyll content of zooxanthellae and zoochlorellae isolated from their anemone host Anthopleura eleguntissima (isolated) or obtained from feces of the nudibranch Aeolidia papillosa (fecal). (N = 3 for isolated algae, N = 6 for fecal zooxanthellae, N = 8 for fecal zoochlorellae). ** indicates P < .01.

greater than among freshly isolated zoochlorellae (t = 9.19, P < .001; Fig. 4B), whereas the difference in percent dividing cells among fecal zooxanthellae was not significantly different from that among freshly isolated zooxanthellae (Fig. 4B).

Fecal zooxanthellae contained significantly less chl a (t = 3.43, P < . O l ) and the same amount of chl c (t = 2.04, P = .17) compared to freshly isolated zooxanthellae (Fig. 5). There were no significant differences in chlorophylls u and h of fecal and freshly isolated zoochlorellae.

Discussion

The chemosensory experiment with sea anemones (Anthopleura elegantissima) vs. plain seawater estab- lished that the nudibranchs (Aeolidia papillosa) re- spond to anemone scent. However, the nudibranchs do not select anemone prey based on their symbiotic condition. These nudibranchs consistently showed no preference for zooxanthellate, zoochlorellate, or aposymbiotic anemones in both chemosensory and feeding experiments (Figs. 2, 3). There are no differences in the nutritional quality of the anemone types, since the biochemical composition of zooxanthellate, zoochlorellate, and aposymbiotic individuals of A. elegantissima is similar (Blevins 1991). Both the chemosen-

sory and feeding experiments provide strong support for our conclusion that prey choice by this nudibranch is not influenced by the symbiotic condition of its prey.

Previous studies have shown that prey choice by A. papillosa can be affected by ingestive conditioning (Hall et al. 1982, 1984); a 7-d unrestricted diet of a single anemone type modified nudibranch response to prey scent. In our study, ingestive conditioning of nudibranchs, fed on an exclusive diet of zooxanthellate anemones and on an exclusive diet of zoochlorellate anemones, had no effect on prey choice. Fasting before feeding trials may alter feeding behavior of predators (Cronin & Hay 1996). In the ingestive choice experiments, nudibranchs were not fed for 3 days before testing, which may have contributed to the observed lack of preference. However, in the 24-h feeding experiments, prey choices were random after the first anemone was consumed. If hunger caused indiscriminate feeding, we might have expected prey choice to be exhibited during consumption of the second or third anemone in a 24-h period.

The chemosensory experiments provided some ev- idence that these nudibranchs could detect differences in chemical scent between zooxanthellate and zoochlorellate anemones. Those nudibranchs that were provided a choice between these two anemone types checked both anemone scents significantly more often than they checked only one scent before making a choice. However, this behavioral response did not influence the choice of prey.

McFarland & Muller-Parker (1 993) found that A. papillosa did not selectively retain one alga over the other in its cerata when switched to a diet of the other type of A. elegantissima. Algal densities dropped to zero after 11 days without access to anemone prey, even when nudibranchs were maintained in the light. Although their study showed that algae remained photosynthetically active in the cerata, McFarland & Mull- er-Parker ( 1 993) concluded that the nature of the symbiotic relationship was at best “primitive” (sensu Kempf 1991) because the algae were not retained. Our results provide further support for this conclusion; the lack of choice of anemone prey according to its symbiotic condition suggests that the presence of algal symbionts is not important to this predator. This nudibranch also ingests and thrives on a variety of non- symbiotic anemone species (Stehouwer 1952; Yarnall 1972; Ottaway 1976; McDonald & Nybakken 1978-

We conclude that neither zooxanthellae nor zoochlorellae in A. elegantissima provide any protection from predation by this nudibranch. This contrasts with the preferential predation on zooxanthellate over zoochlorellate anemones observed for the mosshead

79).


sculpin Clinocottus globiceps by Augustine & Muller- Parker (1998). In that study, zoochlorellae remained viable after passage through the fish gut while zooxanthellae did not. Possibly, the ability to digest the endosymbionts may influence predator choice. As found by McFarland & Muller-Parker (1 993), the feces of nudibranchs fed zooxanthellate and zoochlorellate anemones contained intact and photosynthetically active algae (Figs. 4, 5) . Although we do not know the proportion of algae that remained viable after passage through the nudibranch gut, these results suggest that digestibility of algae is not a basis for prey selection. The influence of dual algal symbionts on predation on A. elegantissima might be further clarified by exam- ining the feeding response and feces of other chemosensory predators of this anemone, such as the seastar Derrnusterias imbricatus (Ottaway 1976; Harris 1986), the wentletrap snail Epitonium tinctus, and the pyc- nogonid Pycnogonum stearnsi (Ottaway 1976).

Conversely, the algal symbionts may benefit from being eaten by A. papillosa and released from the host, as separation from the host can stimulate symbiont cell division. Both zooxanthellae and zoochlorellae ex- pelled from A. elegantissima during 48-h intervals had 3- to 4-fold increases in cell division compared to freshly isolated algae (McCloskey et al. 1996). How- ever, in nudibranch feces deposited during 24-h intervals, percent cell division of algal symbionts differed from that of freshly isolated symbionts only for zoochlorellae. Compared to isolated zoochlorellae, fecal zoochlorellae exhibited a 2.5 X greater percentage of cells in division (Fig. 4B). Whether this increase was due to simple release from the host (as in expulsion) or to stimulation of division during passage through the nudibranch gut is unknown, but fecal zooxanthellae did not display any increase in percent cells dividing. The percentage of dividing zoochlorellae isolated from anemones was 15X greater than that of isolated zooxanthellae (Fig. 4B), consistent with other studies (Verde & McCloskey 1996; Saunders & Muller-Parker 1997).

The productivity of algal syinbionts obtained from nudibranch feces, compared to that of freshly isolated algae, was the same in zoochlorellae and was significantly higher in zooxanthellae (Fig. 4A). We found similar values for carbon fixation rates by zooxanthellae and zoochlorellae, in contrast to the results of Verde & McCloskey (1996), who found that net photosynthesis normalized to algae and whole anemones (A. elegantissima) was 1.4-2.2X greater in zooxanthellae than in zoochlorellae. Our values for carbon fixation rates of both isolated and fecal zooxanthellae (Fig. 4A) are 2-3X lower than rates reported by McFarland & Muller-Parker (1993; 1.9 pg C cell-' h-l)

and Augustine & Muller-Parker (1998; isolated algae: 3.1 pg C cell-' h-I), who used similar methods to mea- sure productivity. However, our values for the productivity of zoochlorellae (Fig. 4A) are comparable to those obtained by McFarland & Muller-Parker ( 1993) and Augustine & Muller-Parker (1998). Although we have no explanation for the lower rates of photosynthesis by zooxanthellae obtained in this study, the find- ing that fecal zooxanthellae had higher photosynthetic rates than freshly isolated zooxanthellae (despite reduced chlorophyll content) supports our conclusion that viable zooxanthellae occur in nudibranch feces. The chlorophyll concentrations of zooxanthellae and zoochlorellae (Fig. 5 ) are similar to concentrations reported by Verde & McCloskey (1996) and Augustine & Muller-Parker (1998) for algae sampled from anemones during the summer.

As first proposed by Parker (1984), predator feces may be an important source of algae for the establish- ment of symbiosis in anemones. In the case of A. papillosa, this source may be significant. For example, a typical anemone collected from the field (1 00 mg pro- tein biomass) contains 1.5 X lo7 zooxanthellae or 4.5 X lo7 zoochlorellae (Engebretson & Muller-Parker 1999). Each nudibranch consumed an average of 3 anemones per day (this study). Assuming all symbiotic algae pass through the gut unharmed, 4.5 X lo7 zooxanthellae and 1.4 X 10* zoochlorellae could be released by a single nudibranch in one day. In contrast, the daily expulsion of zooxanthellae and zoochlorellae from anemones represents 2% of the symbiont population (calculated from data in McCloskey et al. 1996). Thus, 3 anemones would normally release only 9 X lo5 zooxanthellae and 2.7 X lo6 zoochlorellae per day. Athough over time each anemone may contribute a greater number of algae by expelling a small percentage each day, thc nudibranch fecal pcllets provide a highly concentrated source of algae in a package that is readily ingested by anemones (our unpubl. obs.).

Algae are dispersed in the feces of nudibranchs as they move around the intertidal searching for anemones to eat and for mates. The gametes and larvae of A. elegantissima do not contain algal endosymbionts (Siebert 1974). Nudibranch feces might supply symbionts to anemone larvae and to anemones that lack endosymbionts. Although A. pq~il lnsa is not likely to influence the composition of the intertidal anemone population through selective predation on a particular anemone type, it may indirectly affect the distribution of anemone types at a site by dispersing zooxanthellae and zoochlorellae in its feces. The actual fate of these fecal algae is unknown and merits further investiga- tion.

124 Seavy & Muller-Parker

Acknowledgments. We are grateful for a grant from West- ern Washington University for the construction of the apparatuses and to the WWU Scientific Technical Services staff for constructing these. We thank B. Bingham for assistance with statistical analysis and R. Anderson for assistance in methodology and behavioral aspects of this study. We also thank L. Francis for sharing her special insight of anemone behavior, in particular that of Anthopleura elegantissirna, and G. Jensen for his assistance with the initial collection of Aeolidia papillosa. Reviewers and Co-Editor C. Cook helped substantially improve the manuscript. This project was com- pleted in partial fulfillment of the requirements for a MS degree in Biology at Western Washington University.

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