Aspects of the larval biology of the sea anemones Anthopleura elegantissima and A. artemisia

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1 Invertebrate Biology 121(3): American Microscopical Society, Inc. Aspects of the larval biology of the sea anemones Anthopleura elegantissima and A. artemisia Virginia M. Weis,a E. Alan Verde, Alena Pribyl, and Jodi A. Schwarz Department of Zoology, Oregon State University, Corvallis, OR , USA Abstract. We investigated several aspects of the larval biology of the anemone Anthopleura elegantissima, which harbors algal symbionts from two different taxa, and the non-symbiotic A. artemisiu. From a 7-year study, we report variable spawning and fertilization success of A. elegantissima in the laboratory. We examined the dynamics of symbiosis onset in larvae of A. elegantissima. Zoochlorellae, freshly isolated from an adult host, were taken up and retained during the larval feeding process, as has been described previously for zooxanthellae. In addition, larvae infected with zooxanthellae remained more highly infected in high-light conditions, compared to larvae with zoochlorellae, which remained more highly infected in low-light conditions. These results parallel the differential distribution of the algal types observed in adult anemones in the field and their differential tolerances to light and temperature. We report on numerous failed attempts to induce settlement and metamorphosis of larvae of A. eleguntissima, using a variety of substrates and chemical inducers. We also describe a novel change in morphology of some older planulae, in which large bulges, resembling tentacles, develop around the mouth. Finally, we provide the first description of planulae of A. artemisia and report on attempts to infect this non-symbiotic species with xooxanthellae and zoochlorellae. Additional key words: Cnidaria, Anthozoa, Actiniaria, larval settlement, melainorphic induccr, symbiosis, zoochlorellae, zooxanthellae The 4 species of the sea anemone genus Anthopleura found along the western coast of North America- A. ekgantissima (BRANDT 18%), A. soh (PEARSE & FRANCIS 2000), A. xanthogrammica (BIIANDT I835), and A. avtemisia (DANA 1846)-are conspicuous members of rocky intertidal communities. A. elegantissima in particular, which is common along the coast from Alaska to Baja California, can form clonal aggregations that occupy large areas within the mid-intertidal zone, to the exclusion of other competitors (Dayton 1971; Sebens 1981a; Fitt et al. 1982). Despite their importance in these communities, examination of the sexual reproductive cycle and life histories of these anemones has been restricted to a handful of studies. Ford (1964), Jennison (1979), and Sebens (198 1 b) described gametogenesis and an annual reproductive cycle in A. elegantissima and A. xanthogrammica that started with gonad development (both species are dioecious) in the late fall and ended with spawning in Author for correspondence. Ph: Fax: weisv@mail.science.orst.edu Present address: Center for Liinnology, University of Colorado, Boulder, CO 80309, USA the late summer. Siebert (1 974) and Smith (I 986) examined laboratory-induced spawning, embryological development, and morphology and behavior of planktotrophic planula larvae of A. elegantissima and A. xanthogrammica. Finally, Sebens ( a, 1982) described recruitment of larvae from both species into mussel beds adjacent to sites with adult anemones. No life history studies have focused on either A. sola or A. artemisia. A. elegantissima and A. xunthogmmmica form symbioses with two different types of unicellular algae (Muscatine ; Muller-Parker & Davy ), dinoflagellates from the genus Symhiodinium (zooxanthellae, LaJeunesse & Trench 2000) and one or more taxonomically undescribed chlorophytes (zoochlorelhe). As in other cnidarian-algal symbioses, the algae provide the anemones with photosynthetically fixed organic carbon and the host anemone in turn supplies the algae with inorganic nutrients, a stable environment, and a refuge from herbivory (reviewed in Muscatine 1990; Muscatine & Weis 1992; Muller-Parker & D Elia 1997). Anemones of both species containing these different algal types are differentially distributed along a latitudinal and tidal-height gradient, with

2 Larval biology of Anthopleura eleguntissimu 191 anemones in more northerly and lower intertidal locations containing predominantly zoochlorellae and anemones in more southerly and higher intertidal locations containing predominantly zooxanthellae (Bates 2000; Secord & Augustine 2000). This distribution of algal types has been attributed to differential physiological tolerance of the algae to temperature and light, with zooxanthellae well adapted to relatively high-light and warm conditions compared to zoochlorellae, which are better adapted to cooler, lower-light environments (Verde & McCloskey 1996a,b, 2001, in press; Saunders & Muller-Parker 1997). A. soh, a newly described, solitary sister species to A. eleguntissima (Francis 1979; McFadden et al. 1997; PEARSE & FRANCIS 2000), is restricted to latitudes below the likely southern limit of zoochlorellae (Secord & Augustine 2000) and is symbiotic only with zooxanthellae. A. artemisia is a non-symbiotic species (Hand 1955; PEARSE & FRANCIS 2000). Recent molecular phylogenetic studies have grouped A. elegantissima, A. sola, and A. xunthogrummica into an eastern Pacific clade, whereas A. artemisia groups with other conspecifics in a distinct western Pacific clade, despite its occurrence in the eastern Pacific (Geller & Walton 2001). Larvae of A. ekgnntissima and A. xanthogrummica are oval planulae, pm long and 100 p,m wide with a conspicuous apical tuft of numerous elongate cilia present at the aboral pole (Siebert 1974). They swim actively in the water column (Siebert 1974), feed by extrusion of a mucous thread that traps food (Siebert 1974; Schwarz et al. 2002), and persist in culture for as long as 3 months (Smith 1986). Despite attempts by many workers to induce settlement by the addition of various natural substrates, such as mussel shells, rocks, and macrophytes, to larval cultures (Siebert 1974; Smith I986), no larvae have ever been observed to undergo settlement and metamorphosis. Gametes and larvae of A. elegantissima and A. xunthogrammica lack symbiotic algae (Siebert 1974). These anemones must therefore acquire symbionts from the environment during their development. We were initially interested in larvae of A. elegantissima as a model for the study of the onset of symbiosis between sexually produced cnidarian offspring and their symbiotic zooxanthellae. Symbiosis onset in A. elegantissima can begin in the larval phase during the feeding process, whereby zooxanthellae, freshly isolated from adults, are taken in along with food (Schwarz et al. 2002). Algae are phagocytized by host gastrodermal cells and ultimately persist in the gastrodermal tissue. A similar process of algal acquisition (infection) by feeding during the larval stage has been described in the anemone Aiptasia tagetes (Riggs 1988) and the scleractinian coral Fungia scuturia (Krupp 1983; Schwarz et al. 1999). What started as the search for an ideal model for the study of cnidarian larval biology and symbiosis onset has instead developed into a 7-year love/hate relationship with larvae of A. elegantissima. Despite our best efforts, answers to key questions surrounding developmental processes, such as spawning and fertilization patterns and settlement and metamorphosis, remain elusive. Here we report results from studies on symbiosis onset, specifically in the examination of infection dynamics of the larvae by both zooxanthellae and zoochlorellae and in the differential infection by zooxanthellae and zoochlorellae as a function of light regime. We describe a morphological change in some mature larvae that may represent the development of tentacles before settlement. Further, we provide the first description of larvae of A. artemisia and the response of this non-symbiotic species to exposure to zooxanthellae and zoochlorellae. We also report on some key failures in our examinations of larval A. eleguntissima. We describe variability in spawning and fertilization success through the study period, that has hampered our ability to continue studies of larvae. In addition, we describe our numerous failed attempts to induce settlement and metamorphosis of thcsc larvae, with the aim of preventing repetition of thesc approaches by other researchers in future studies. Methods Animal maintenance, gamete collection, and larval cultures Adults of Anthopleura elegantissima, to be used as broodstock and as sources of freshly isolated algae, were collected at various times from 1990 to 2001, from the following locations along the Pacific coast of the U.S.: Santa Cruz, California; Strawberry Hill, Neptune Beach, Seal Rock, and Boiler Bay, Oregon; and Swirl Rocks, Washington. Zooxanthellat-e animals were collected from all locations, but all zoochlorellate animals were collected at Swirl Rocks. Experimcnts in 1995 and 1996 were performed at Long Marinc Laboratory (University of California Santa Cruz) in Santa Crux, California; those in were performed at Hatfield Marine Science Center (Oregon State University) in Newport, Oregon; and those in the summer of 2000 were performed at Walla Walla College Marine Station, Anacortes, Washington. All foreign material adhering to the anemones (e.g., fragments of shell and macroalgae) was removed after collection and individual anemones were allowed to attach to bricks (3-10 anemones of one clone per brick). The bricks were submerged in outdoor seawater tanks ex-

3 192 Weis, Verde, Pribyl, & Schwarx posed to ambient sunlight. Animals were fed previously frozen brine shrimp or minced mussel 1-3 times a week. Techniques moditied from Smith (1986) and Schwarz et al. (2002) were used to induce spawning. In the morning, bricks with the anemones were placed into open aquaria containing ambient, static seawater and exposed to ambient sunlight for a period of time without changing the seawater. Water temperature was not controlled and rose during this exposure. On overcast or foggy days, the seawater in the containers was replaced with cold ambient seawater at dusk. On sunny days, the anemones were exposed for 4-6 h before the water was changed. Within 12 h after a water change, the anemones released gametes. When spawning occurred, thousands to hundreds of thousands of negatively buoyant eggs settled on the oral disc or on the bottom of the aquaria and were subsequently collected with a large plastic pipette (turkey bastes) and placed into a large glass container with seawater. A small amount of sperm suspension was similarly collected and added to the eggs for fertilization. The mixture was maintained in a cold room at 14 C (in California and Oregon), or in 10-liter containers suspended in indoor tanks of flow-through ambient seawater at C (in Washington). At 24 h after fertilization, planulae were filtered out of the seawater using a 50-pm mesh screen and placed into fresh seawater in glass or plastic containers. For the California and Oregon experiments, larvae were kept in the incubators mentioned above in a 12-h: 12-h light/ dark regime at an irradiance of -40 pmoles of quanta/ m2/sec. For the Washington experiments, larvae were exposed in outdoor tanks to 55% ambient sunlight using neutral density screens placed over the containers of larvae. Tn all locations, every 2 days, the seawater was changed throughout the experiments. Larvae kept in unfiltered seawater remained aposymbiotic throughout their -30-day lifespan unless experimentally infected (see below). Field collection of gametes of A. artemisia At Strawberry Hill and Neptune Beach, Oregon during a low tide on 26 June 1998, numerous individuals of A. artemisia were observed spawning. Eggs and sperms were collected with a Pasteur pipette and mixed in several 15-ml conical tubes. The tubes were transported back to the laboratory and the developing planulae were maintained in the same fashion as larvae of A. elegantissirnu. Preparation of freshly isolated zooxanthellae and zoochlorellae Algae were isolated from adults of A. elegantissima (see Schwarz et al. 1999, 2002). Adult anemones were cut in half transversely and the basal half was discarded. The oral half was minced with a razor blade, homogenized in filtered seawater (FSW) with a groundglass tissue grinder, and centrifuged at 4000 g for 5 min to separate the algal cells from the animal tissue. A thin animal pellet fraction, layered above the algal pellet and consisting mostly of nematocysts, was carefully removed and discarded. The algal pellet was resuspended in FSW and re-pelleted 3 times to partially clean the algae of contaminating animal debris. The final pellet was resuspended in FSW and this dense suspension was passed through a 1 SO-Fm mesh screen to break up large algal clumps. Algal isolates were used within I h of preparation. In numerous preliminary experiments, we attempted to quantify the number of algae that we added to larvae. After the 3 rinses in FSW (described above), both zooxanthellae and zoochlorellae were highly clumped and contaminated with animal debris, making quantification impossible. Additional seawater rinses did not improve results. In an attempt to obtain more uniform suspensions, after the rinses, the algal suspension was placed in a syringe and forced through a metal SO-pm mesh screen. Algae in the resulting suspension were no longer clumped and could be quantified; however, subsequent infection was severely reduced (data not shown), presumably because the algae were compromised during preparation. We therefore decided to provide unquantified, high concentrations of algae to the larvae (see below) and obtained repeatable infection results. Infection of A. elegantissima and A. artemisia with algal isolates In order to compare the abilities of the two algal types to infect A. elegantissima larvae, we measured the percentage of larvae infected with zooxanthella and zoochlorella isolates and the den\ity of algae in larvae through time. For each experiment, - 10,000 4-day-old larvae were divided between two gla\s or plastic containers. Zooxanthella isolates were added to one container and zoochlorella isolates to the other. Larvae of A. elegantissima acquire algae during feeding when algal cells enter through the mouth with food (Schwarz et al. 2002). Therefore, several drops of homogenized brine shrimps (Artemiu sp.) or mussel tissue (Mytilus sp.) were added to the bowls to stimulate a feeding response. After 5-6 hours, algal iwlates were still present in large quantities at the bottom of the dish. Larvae were then concentrated with a SO-pm mesh screen, placed into clean filtered seawater, and returned to incubators (in CA and OR, see above) on a 12-h:12-h light/dark regime at 40 ymol quanta/m2/

4 Larval biology of Anthopleura elegantissirnu I93 Table 1. Natural and artificial substrates used in attempts to induce settlement and metamorphosis in larvae of AnthopZeuru eleguntissima. Substrates Age of larvae Location ol (days) Symbiotic state trcatment Live mussels, shells, and shell debris (MytiZus) 16, 18 aposymbiotic, zooxanthellate, CA, OR Miscellaneous rocks collected from intertidal 16, 18 zoochlorellate aposymbiotic, zooxanthellate, CA, OR Adult anemone 16, 18 zoochlorellate aposymbiotic, zooxanthellate, OR xoochlorellate Plastic Petri dishes roughened with sandpaper" 13 aposymbiotic WA Plastic Petri dishes scored with scalpel" 13 aposymbiotic WA Plastic Petri dishes with light and dark formica tiles 13 aposymbiotic WA on bottom" Plastic Petri dishes with adult anemone (zooxanthel- 13 aposymbiotic WA late and zoochlorellate) in dish" * All Petri dishes and tiles were seasoned in unfiltered running seawater for 10 days before use. sec, or to outdoor seawater tanks in 55% ambient light (in WA, see above). For some experiments performed in Oregon in August 1998, symbiotic larvae were incubated at 120 pmol quanta/m2/sec (see Results). Infections of larvae of A. arternisia with algae isolated from A. elegantissima were performed as for larvae of A. eleguntissima in Oregon, except that unclumped algae, prepared by forcing them through a 50-pm mesh screen, were used to infect the larvae. Immediately after feeding, and approximately every 2 days thereafter, at least 25 larvae were examined from each of the larval containers. Larvae suspended in FSW were dispensed onto a glass microscope slide. A cover slip was placed on the drop and pressed onto the slide with a pencil eraser. This pressure was sufficient to squash the larvae without completely dispersing their contents. Depending on the experiment, the number of larvae infected and/or the number of algal cells per larva were quantified for each sample. Attempts to induce settlement and metamorphosis of A. elegantissima using substrates and metamorphic inducers Several substrates (Table 1) and compounds (Table 2) were added to cultures of both symbiotic and aposymbiotic larvae of A. elegantissima in an attempt to initiate settlement and metamorphosis. For the experiments with natural substrates in California and Oregon, hundreds of larvae were added to glass bowls containing one of the substrates listed in Table 1. The bowls were incubated as described above for larval cultures and were monitored daily under a dissecting microscope for evidence of settlement. For the experiments in Washington, larvae were added to plastic Petri dishes. All Petri dishes were conditioned in unfiltered running seawater for 10 days before larvae were added. Groups of larvae for each treatment were subdivided into 8 replicates; 4 replicates were placed in a refrigerated incubator and 4 in an outdoor flow-through seawater tank at 55% ambient sunlight. The dishes were examined daily for - 15 days under a dissecting microscope for evidence of larval settlement and metamorphosis. Larvae were exposed to several compounds known to elicit settlement and metamorphosis in various marine invertebrates, including some cnidarian species (see references in Table 2). Approximately 15 larvac were placed in each well of 24-well culture plates in FSW (5 ml per well) with one of the compounds for the duration of the monitoring period (n = 8 wells for each compound at each concentration). Controls were set up under identical conditions without a chemical inducer. Plates were incubated as described for larval cultures. The plates were examined daily during the monitoring period (see Table 2), under a dissecting microscope, for evidence of larval settlement and metamorphosis. Statistical analyses All data were checked for normality and homogeneity of variances (Cochran's method, Winer 1971) before conducting statistical analysis. If data were nonnormal or heterogeneous, the data were log-transformed before analysis. All percentage data were square-root arcsine transformed before analysis. Analysis of variance (2-way ANOVA), post-hoc Tukey (HSD) test, and linear regression were performed using the statistical package STATISTICA\WD (Statsoft, Inc.).

5 194 Weis, Verde, Pribyl, & Schwarz Table 2. Metamorphic inducers used in attempts to induce settlement and metamorphosis in larvae of Anthopleura elegantissima. Duration Age of of moni- Location Concentration of larvae toring Symbiotic of Metamorphic inducer inducer (days) (days) state* treatment Reference Metamorphosin A (peptide: DNPGLW) 12-0-tetradecanoyl- phorbol- 13-acetate (TPA) 1, 2, 5, 10, 20 mm 14 5 no record CA 11 3 APO, ZX OR Leitz et al Henning et al. 1996, I998 Phorbol 1 2,13- dibutyrate (PDBu) 11 3 APO, ZX OR Henning et al. 1996, 1998 NH,CI 1s IS APO, ZX, WA zc Coon ct al KCI y-aminobutyric acid (GABA) 3,4--di hydroxyphenylalanine (DOPA) 10-2, 10 7, 10 4, 10-5, 10-6, 10 7, M 10 2, 10-1, 10 4, 10-5, 106, 10-7, M :+ APO = aposymbiotic, ZX = zooxanthcllate, ZC = xoochlorellate APO, ZX, WA ZC APO, ZX, WA zc IS 1s APO, ZX, WA zc e.g., Pechcnik & Gee 1993; Bryan et al. 1997; Biggers & Laufcr 1999 e.g., Morse 1985; Bryan et al e.g., Morse 1985; Bryan et al Results Spawning and fertilization Observations of spawning and fertilization of gametes of Anthapleuua elegantissirna were recorded through the spring and summer of (Table 3). Spawning and fertilization occurred as early as April I in one year to as late as September 4 in another. There was a high variability in both spawning and fertilization success throughout the monitoring period. Between 1995 and 1998, 82% of the attempts to induce spawning were successful (does not include spontaneous spawning). In contrast, from 1999 through 2001, just 69% of attempts yielded gametes, and 4 of these 9 events resulted in release by only one gender, usually males (6 of 7 such instances). Similarly, in , when both sexes spawned (both spontaneous or induced), gametes were successfully fertilized 100% of the time. In contrast, gametes generated from were successfully fertilized in only 20% (1 of 5) of events in which both sexes released gametes. Infection of A. elegantissirna larvae with zooxanthellae and zoochlorellae In 1999 in Oregon and in 2000 in Washington, we examined the dynamics of infection by zooxanthellac and zoochlorellae in larvae of A. elegantissirna, in numerous trials using larvae of different ages and from different parents. When unquantified algae were provided at saturating concentrations, % of larvae acquired algae on day 0 (selected data shown in Fig. I). In most experiments, the percentage of infected larvae decreased over time in both zooxanthellate and zoochlorellate populations to as low as 25% by days post-infection. In all infection experiments, zoochlorellae infected a higher percentage of larvae (usually >90%) on day 0 than did zooxanthellae (60-90%). Algal infection was strongly influenced by different light regimes (Fig. 1). Zoochlorellae appear to persist in larvae incubated in relatively low-light conditions and zooxanthellae persist in those incubated in relatively high-light conditions. In a low-light environment of 40 pmol quanta/m2/sec (OR, June 1998), lar-

6 Larval biology of Anthoplettra elegantissirnu I95 Table 3. tanks. Spawning and fertilization of Anthopleura elegantissirnu. Gametes from adults maintained in outdoor seawater 1998 I Spawning Induced or Date of attempt Location spontaneous Males Females Fertilization 199s June 21 CA Induced Y N July 3 CA Spontaneous Y Y Ye5 July 18 CA Spontaneous Y Y Yes August 24 CA Induced N N August 27 CA Induced N N 1996 April 1 April 2 CA CA Spontaneous Spontaneous Y Y Y Y Yes Ye4 June 24 CA Induced Y N July 2 CA Spontaneous Y Y Ye5 July 22 CA Induced Y Y Ye May 28 OR Induced Y Y Ye5 September 4 OR Induced Y Y Ye4 June 5 OR Induced Y Y Ye5 June 24 OR Induced Y N July 7 OR Induced Y Y Ye\ August 12 OR Induced Y Y Yc5 June 17 OR Induced N N Junc 24 OR Induced Y N August 3 OR Induced Y Y N 0 August 20 OR Induced Y Y N 0 early June OR lnduced Y N late June OR Induced Y N July 10 WA Induced Y Y Ye5 July 29 WA Induced N Y August 13 WA Induced N N August 22 WA Induced N N August 23 WA Induced N N September 3 WA Induced Y Y NO 200 I August 3 OR Induced Y Y NO vae infected with zoochlorellae remained >90% infected for the duration of the 16-day experiment whereas the proportion of larvae infected with zooxanthellae decreased dramatically from 65% to just 23%, at a rate of -3.6%/day (Fig. 1A). A similar experiment, performed in a moderate-light regime of I20 kmol quanta/m2/sec (OR, August 1998) resulted in decreasing levels of infection for both zoochlorellate (-2.5Wday) and zooxanthellate (-3.7Wday) larvae (Fig. IS), rates similar to those shown by zooxanthellate larvae at 40 kmol quanta/m2/sec. Finally, in a third experiment in a relatively high-light regime of 55% ambient light (WA, July 2000), proportions of larvae infected with Loochlorellae decreased through time at -4.1 %/day, whereas proportions of zooxanthellate larvae decreased at only - I.4%/day (Fig. IC). For some infection experiments, the density of algae per larva was monitored through time, in addition to infection percentage. Although the average densities varied between experiments, the density pattern was always the same and is illustrated (Fig. 2) for one experiment (OR, August 1998; same larvae a\ in Fig. IB). The average number of algae per larva immediately after feeding was highly variable for both roochlorellae and zooxanthellae, but zoochlorellae were always present at a higher density than were zooxanthellae. For example, for the experiment shown in Fig. 2, the average number of zoochlorellae per larva on day 0 was 70 (40% of larvae contained over 100 algal cells), significantly higher than the average of just 20 algae per larva for zooxanthellate larvae (Tukey, p <.OO 1 ; Fig. 2A,B). Average densities of both algal types decreased dramatically starting 1 day after infection, such that by day 3, most larvae contained <I0 algal cells per larva regardless of algal type (significantly lower than day 0 for both algal types, Tukey, p <.001; Fig. 2A,C). Algal numbers continued to decrease gradually (but not significantly) throughout the 14-day experiment to values of 1 or 2 algal cells per larva for both algal types (Fig. 2A).

7 I96 + zooxanthellae 0 zoochlorellae Weis, Verde, Pribyl, & Schwarz A -+ zooxanthellae -0- zoochlorellae OR, 40 pmol quanta/m*/sec A/ = Q) U. 40 yl 20 1-, OR, 120 pmol quanta/mz/sec = o -B( - z Q O b 0 O b Q WA, 55% ambient sunlight Days after infection Fig. 1. Infection of larvae of Anthopleura elegantissima with moxanthellae or zoochlorellae in 3 different larval cohorts exposed to different light regimes. Larvae from: (A) Oregon in June 1998; (B) Oregon in August 1998; and (C) Washington in July Percentage of infection decreases through time regardless of symbiont type; however, zoochlorellae persist in larvae in low light and zooxanthellae persist in larvae in high light. Larvae were 4 days old when infected with algal isolates. Each point represents counts of larvae. A. artemisia larvae and infection with zooxanthellae and zoochlorellae Larval development in A. artemisia was essentially identical to that of A. elegantissima. Within one day of fertilization, barrel-shaped planulae, with an apical C.cI c ti a Days after infection Day 0 t5 zooxanthellae zoochlorellae 1 I I >I >I00 Number of algae / larva Fig. 2. Density of zooxanthellae and zoochlorellae in a group of larvae of Anthopleuru elegantissima through time. (A) Changes in average number of zooxanthellae and zoochlorellae per larva over time, showing an initial sharp decrease in density of algae in both zooxanthellate and xoochlorellate larvae and subsequently a gradual decline for the duration of the experiment. Points represent mean t SD, n = 25 larvae counted. Frequency distribution of number of zooxanthellae and zoochlorellae per larva at day 0 (B) and day 3 (C) showing a shift from high percentages of' larvae possessing many algae to most larvae possessing <10 algae. *** = Density of zoochlorellae different from density of zooxanthellae at p <.001 (Tukey).

8 Larval biology of Anthopleura elegantissirnu 197 tuft like that of A. elegantissirnu, were actively swimming in the water column. By day 4, larvae had developed a mouth. In order to determine if this nonsymbiotic species was capable of becoming infected in the larval stage with zooxanthellae and/or zoochlorellae, we conducted infection experiments like those described for A. elegantissirnu, providing saturating concentrations of zooxanthellae or noochlorellae isolated from adults of A. eleguntissimu. We monitored the larvae on days 0, 2, and 4 only (n = 25 larvae counted daily for each algal type). On day 0, 64% of larvae incubated with zooxanthellae acquired algae whereas only 20% incubated with zoochlorellae acquired algae. By day 2, no larvae incubated with LOoxanthellae contained algal cells and only 12% of those exposed to zoochlorellae still contained any algal cells. By day 4, larvae containing roochlorellae decreased again to 4%. Regardless of algal type or day, the number of algal cells per larva always remained <10 (data not shown). We did not determine whether algal cells were phagocytized by gastrodermal cells or merely remained inside the gastric cavity without entering host cells. Fig. 3. Larvae of Anthopleura elegantissirnu displaying a lobed morphology. This morphology was observed in only a few groups of larvae, 2-3 weeks old, and occurred regardless of symbiotic state (see text for details). (A) Planula (aposymbiotic) beginning to change shape, still barrelshaped but small bulges at oral end around mouth are evident. (B) Larva (zoochlorellate) with more distinct oral bulges and a shortened oral-aboral axis, causing the larva to take on a rounded appearance. (C) Larva (aposymbiotic) with fully-developed lobed morphology, with highly pronounced bulges around the mouth. Open block arrows in all three panels indicate bulges emerging from oral end around mouth. Apical tuft (AT) is evident in larvae in A and C. ZC denotes zoochlorellae. Scale bars, 50 pm. Experiments using substrates and compounds to induce settlement and metamorphosis in A. elegantissirnu larvae No definitive settlement or metamorphosis was observed by larvae placed in any of the treatments tried (Tables 1, 2). Some larvae placed in containers with mussels and intertidal rocks (Table 1) did display some early signs of settlement behavior. Larvae were seen attaching to rocks and shells. They would remain for minutes to hours but would be missing later when checked again. Larvae placed with these natural substrates were extremely difficult to follow, because of their small size (<200 pm) and the complexity of the substrate. In all cases, within I to 2 days of placing the larvae in these containers, they disappeared. Larvae placed with artificial substrates (Table 1) or in metamorphic inducers (Table 2) never displayed any settlement behavior and remained actively swimming in the water column for the duration of the exposures. Changes in morphology in older larvae Larvae of A. elegantissirnu from three different spawns (CA, April 1996; OR, September 1997; and OR, June 1998) displayed marked changes in morphology at 2-3 weeks of age. Multiple bulges were observed emerging from the oral end (Fig. 3A,B). Over time, these bulges became pronounced and took on the look of developing tentacles (Fig. 3C). At the same time, in many larvae, the oral-aboral axis became

9 198 Weis, Verde, Pribyl, & Schwarz shortened, causing the larvae to lose their barrel shape and take on a rounded shape (Fig. 3B). The apical tuft remained present at the aboral end (Fig. 3A,C). The change to a lobed morphology occurred synchronously in larvae from the same spawn in different bowls, and occurred in aposymbiotic, zooxanthellate, and zoochlorellate larvae. In all three groups of larvae in which the lobes were observed, the majority of the larvae assumed this morphology. However not all larval groups that reached 2-3 weeks of age underwent a change to this morphology. Some groups persisted as barrel-shaped planulae for the duration of their lives (-1 month). Discussion Spawning and fertilization Spawning in Anthopleura elegantissirnu was observed or induced far earlier in the season in this study than had been reported previously, either in field studies from Central California (Ford 1964; Jennison 1979) or from Washington (Sebens 1981b). Spawning was reported in late summer or early fall, whereas we observed spontaneous spawning as early as April in California one year, and repeatedly induced spawning in June and July in (Table 3). We report high variability in experimental spawning and fertilization success (Table 3) that we cannot explain by any changes in experimental technique. Likewise, spawning and fertilization variability was not correlated with a potentially aging broodstock. Some of the animals that spawned (both spontaneously and induced) in Santa Cruz in 1995 and I996 had been in seawater tables for several years. Further, animals were newly collected from the field before the failed spawning attempts of 24 June 1999, 12 August 1999, and late June 2000 in Oregon and that of 13 August 2000 in Washington. Spawning did not appear to be related to the presence of other spawning conspecifics. There were occasions when anemones spawned when alone in a tank and conversely, there were occasions when a single anemone spawned in a tank with other anemones (data not shown). These data are consistent with other studies by Smith ( 1986) that found no correlation between spawning and presence of other spawning conspecifics. This study provides little insight into the observed variability in spawning and fertilization; the work was not originally designed to track these parameters. We did not monitor gonad size or percent fertility of the broodstock, nor did we track spontaneous spawning events in the field. A more systematic field study, performed over a longer period of time, would be required to definitively demonstrate changes in reproductive success over time for these long-lived animals. Other studies have yielded little information about the variability of sexual reproduction in A. elegantissirnu. Previous studies have reported yearly cycles of gonad growth starting in late fall and ending with spawning, as evidenced by decreased gonad size in late summer to early fall (Ford 1964; Jennison 1979; Sebens 198 lb). Actual spawning or fertilization rate has never been monitored. There is one hint from field studies of larval recruitment in A. elegantissirnu and A. xunthogrammica that production of larvae may be episodic (Sebens 1981a, 1982). A large, isolated larval recruitment event occurred in one year and was not repeated in subsequent years at the same site. This could be evidence for strong spawning and Fertilization in that one year, but could also have been due to various physical or biotic conditions that favored settlement. Infection of larval A. elegantissirna with zooxanthellae and zoochlorellae This study demonstrates the ability of larvae of A. eleguntissirna to acquire zoochlorel lae, freshly isolated from an adult, during feeding, in the same manner as larvae acquire zooxanthellae (Schwarz et al. 2002). The proportion of larvae infected with either algal type declined over time in most experiments (Fig. I). No comparable data have been reported lor other symbiotic cnidarian planulae. These declines in percent infection may be due to differential mortality of symbiotic compared to non-symbiotic larvae or, alternatively, to expulsion or digestion of' algae in some symbiotic larvae over time. Zooxanthellate larval cultures remained relatively stable in high-light treatments compared to zoochlorellate cultures, which were stable in low-light treatmcnts (Fig. I). These data are consistent with the variety of' studies that have shown differential habitat distribution of A. elegantissirnu and A. xanthogrurnmica harboring the two algal types (Bates 2000; Secord & Augustine 2000) and differential tolerance of the two algal types to temperature and light in adults of A. eleganl (Verde & McCloskey 1996a,b, 2001, in press; Saunders & Muller-Parker 1997). Zooxanthellae have been shown to out-perform zoochlorellae, as measurcd by growth rates and photosynthetic productivity, in warmer, higher-light environments, and have been hypothesized to outcompete zoochlorellae in anemones found in these habitats. The opposite has been found in anemones in cooler, lower-light environments where zoochlorellae perform better than zooxanthellae. Our study suggests that this differential environmental tol-

10 Larval biology of Anthopleura elegantissima 199 erance of the symbionts may be manifest as early as the larval stage of these anemones. The temporal profiles of algal density in A. elegantissima larvae were very similar between algal types and between different experiments. The high numbers of algae in larvae at day 0 are likely a count of algae in the gastric cavity, taken in during feeding and not phagocytized by the cells of the larval gastroderm (Fig. 2). The much lower numbers of algae on subsequent days likely represent algae that have been phagocytized by gastrodermal cells. This interpretation is supported by previous work in which transmission electron micrographs of A. elegantissima larvae 4 days after infection showed zooxanthellae only in gastroderma1 cells and none in the gastric cavity, although it is possible that algal cells remaining in the gastric cavity could be washed away during fixation (Schwarz et al. 2002). Zoochlorellae average 9.6 pn2 in diameter compared to an average of 12.5 km for zooxanthellae in A. eleguntissima (Verde & McCloskey 1996b). Therefore, the significantly higher densities of zoochlorellae compared to 7,ooxanthellae in larvae at day 0 could be due to the gastric cavity being able to accommodate a greater number of smaller zoochlorellae than larger zooxanthellae. By several days after infection, algal density stabilized at -1-2 algal cells per larva (Fig. 2) and was consistent between experiments and across all light regimes (data not shown). In contrast, when experimentally infected with zooxanthellae, larvae of the scleractinian coral Fungia scutaria, which are comparable in size to larvae of A. elegan an average of algal cells per larva (Weis et al. 200 I). Whether this discrepancy in algal density between F. scutaria and A. elegarztissima is a real biological difference or reflects sub-optimal culturing conditions of A. elegantissima is not known. Riggs (1988) did not quantify algal density in experimentally infected larvae of Aiptasia tugetes. To our knowledge, no other infection studies have been performed on larvae from any other species of cnidarian. There is no information on the mode of acquisition of symbionts by larvae of A. elegantissima or any other cnidarian, in the field, therefore it is difficult to know how relevant the artificial conditions created in this study are to the biology of symbiosis onset. In the laboratory, larvae of A. elegantissirnu can acquire zooxanthellae from black egesta, which contain high concentrations of zooxanthellae, released from adult anemones (Schwarz et al. 2002). Another possible source of high quantities of algae is the feces of the nudibranch Aeolidia papillma, which feeds on adults of A. elegantissirnu, passes the symbionts unharmed through its digestive tract, and releases them in very high concentrations in the fecal pellets (Seavy & Muller-Parker 2002). If either of these sources of algal cells are used by larvae, the high concentrations of cells used in this study could be mimicking these infection modes. Larvae of A. urtemisiu We witnessed a mass spawning of A. artemisia during a low tide in July of 1998 at Strawberry Hill and Neptune Beach, Oregon. Virtually all observed individuals were spawning at both locations we visited. Similar events of single species spawning have been observed for A. elegantissima (E. Sanford, Stanford University, pers. comm.) and for A. xanthogrammica (J. Schwarz, unpubl. obs.). A. artemisia, a nonsymbiotic anemone species (Hand 1955; PEARSE & FRANCIS 2000) residing in a separate west Pacific clade from A. eleguntissima, A. solu, and A. xunthngrammica (Geller & Walton 2001 ), did acquire zooxanthellae and zoochlorellae, but algal cells occurred at low densities and at very low percentages. We did not determine if any algal cells entered host gastrodermal cells or if they remained in the gastric cavity. The initial numbers and densities of algae in the larvae at day 0 were much lower than those of A. eleguntissima. We have hypothesized that algae are initially taken into the gastric cavity in A. elegantissima and the coral F. scutaria as a result of a nonspecific feeding response (Schwarz et al. 1999, 2002). If this were true, then one would predict that larvae of A. artemisia would respond in the same way to algae in the presence of food; initially they would acquire large numbers of algal cells that would later be either expelled or digested. Instead, far fewer larvae of A. artemisia took up algae and far fewer algal cells were acquired per larva than in A. elegantissirnu. This suggests that some specificity is involved in the early stages of symbiont-host contact in A. eleganti.rsimu. Alternatively, reduced uptake rates in A. artemisia could be explained by the use of freshly isolated algae that had been passed through a 50-pm mesh screen, a technique that sometimes reduced infection rates in A. elegantissima larvae (see Methods). Attempts to induce settlement and metamorphosis in A. eleguntissima There is a rich literature on inducers of marine invertebrate larval settlement (see Table 2 for examples of references). Much is understood in some species of invertebrates, including some cnidarians, about the cues and signals, both natural and artificial, that induce settlement. Borrowing from these studies, we tried to induce larvae of A. elegantissima to settle using numerous substrates and chemicals, and Tables 1 and 2

11 200 Weis, Verde, Pribyl, & Schwarz document our failed attempts. Other researchers have attempted settlement studies on A. elegantissirnu, using various natural substrates and larvae of approximately the same age, also without success (Siebert 1974; Smith 1986). Settlement is a complex signaling process that can vary dramatically between groups of invertebrates (Hofmann et al. 1996). It is possible that we did not try the right concentration, duration, or combination of cues that the larvae need to initiate settlement. Furthermore we did not consider environmental parameters, such as light, temperature and even emersion that could be required for settlement. Another possible explanation for the inability to induce settlement is that the larvae used in the studies were too young and not yet competent to settle. It has been hypothesiled that planulae of A. elegantissirnu are longlived planktotrophic larvae capable of long-distance dispersal (Sebens a,b, 1982). Larvae have been kept alive in culture for as long as 3 months (Smith 1986), in our hands, -6 weeks. To date we have been unable to keep large cultures healthy and axenic for long enough to attempt settlement experiments on larvae older than 3 weeks. Changes in morphology of older A. elegantissima larvae Some, but not all, groups of larvae over 2 weeks of age, developed a morphology that consisted of several large protuberances situated around the mouth (Fig. 3). Although these structures resemble tentacles in their location and shape, histological studies are needed to confirm the presence of two cell layers and the gastric cavity. These structures were much larger than the collar described by Siebert (1 974) and observed in our cultures, which consists of a small circular ridge surrounding the mouth of larvae of all ages. Smith ( 1986) does not report any comparable morphological changes in his study of larvae of A. elegantissirnu, and to our knowledge, no similar accounts of this multilobed morphology have been described in other cnidarian planulae. We initially hypothesized that the appearance of the morphology signaled competence to settle; however larvae placed in bowls with natural substrates and the phorbol esters TPA and PDBu (see Table 2) did not settle. Conclusions Our ongoing struggles-to obtain A. elegantissirnu planulae predictably, to infect larvae with a quantified number of algal cells, and to induce larvae to settleillustrate just some of the difficulties encountered in examining the early life history stages of cnidarians and the onset of their symbioses with algae. This ex- plains in part why progress in this area of cnidarian biology and symbiosis lags behind studies on adults. 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