MARINE ECOLOGY PROGRESS SERIES Vol. 51: 195-199, 1989 Published January 16 Mar. Ecol. Prog. Ser. NOTE Larvae of the sea hare Aplysia californica settle and metamorphose on an assortment of macroalgal species Joseph R. Pawlik* Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California, 92093-0208, USA ABSTRACT: Larvae of the sea hare Aplysia californica Marine molluscs of the genus Aplysia are among the (Molluscs: Opisthobranchia) spend several weeks feeding in most intensively studied animals on earth and have the plankton pnor to settlement and metamorphosis Previous been the subject of research on development, growth work indicated that metamorphosis was tnggered by only one (or at most a few) algal species. However, in the present and energetics, circadian rhythms, and the neural basis laboratory study, a mean of 30 % or more of the larvae of this for learning, memory and behavior (reviewed in Kan- sea hare metamorphosed in response to 10 of 18 species of del 1979, Carefoot 1987).Research interest in one of the intertidal macroalgae (9 red. 7 brown, 2 green). Metamor- best studied sea hares, A. califol-nica, from the coast of phosis was greatest in response to the red algae Rhodymenja californica, Corallina officinalis, Plocamjurn cartilagineurn California, prompted the laboratory cultivation of the and Laurencia pacifjca. Juveniles of A. californica that had planktotrophic larvae of these animals through metamorphosed on the last 2 species grazed on them and metamorphosis (Kriegstein et al. 1974). In the field, began to grow, whereas juveniles on the other species tended young recruits of these herbivorous snails were most to crawl off the alga and around the assay dish. Of the 8 algae frequently found eating red algae, primarily of the least preferred, only 1 was red, the remainder brown or green. For larvae of A. californica, metamorphosis on a relatively genus Laurencia (Kupfermann & Carew 1974). wide spectrum of algal species may be more efficacious than Moreover, several putatively defensive halogenated metamorphosis on any one alga, because juvenile sea hares natural products isolated from adult A. californica were can readily crawl to nearby algal species that they prefer to found to be derivatives of metabolites of L. pacifjca eat after they have metamorphosed on an alga that is not the~r (Stallard & Faulkner 1974), suggesting some depend- preferred food. ence of the molluscs on this alga. Most marine invertebrates pass through a planktonic Larval settlement of Aplysia californica was reported larval stage prior to settlement and metamorphosis, to be highly substrate-specific, and chemical inducers and the recruitment of their larvae is important in were believed to be responsible for substrate choice structuring benthic marine communities (Keough & (reviewed in Carefoot 1987). Kriegstein et al. (1974) Downes 1982, Keough 1983, Gaines & Roughgarden found that laboratory-reared larvae would settle and 1985). Larval settlement is largely regarded as a metamorphose on Laurencia pacifica, but not on response to complex, and often highly specific, species of Plocarnium, Polysiphonia, Daysia, Chondrus environmental stimuli (Burke 1983, Crisp 1984), espe- or Ulva. Subsequently, Capo et al. (1979) reported that cially chemical cues (reviewed in Hadfield 1986, Paw- 2 red algae from the New England coast, Neoagar- lik & Faulkner 1986). However, few naturally-occurring dheilla baileyl and Gracilaria sp., would also induce chemical inducers of larval settlement and metamor- settlement and metamorphosis of A. californica. The phosis have been isolated and identified (Kato et al. study described herein was initiated in an effort to 1975, Cuomo 1985, Pawlik 1986). isolate and identify compounds from L. pacifica that induced metamorphosis of A. californica. It was disco- californica ' Present address: Friday Harbor Laboratories, University of vered, however, that metamorphosis of A. Washington, 620 University Road, Friday Harbor, Washing- was much less specific than had been previously re- ton 98250, USA ported. O Inter-Research/Printed in F R. Germany 196 Mar. Ecol. Prog. Se Materials and methods. All larvae used in experi- therein, were examined under a dissecting microscope ments were the progeny of 2 specimens of Aplysia to determine the number of larvae that had undergone californica that had been cultured as larvae in the lab, metamorphosis. Juveniles had lost their velar cilia and had metamorphosed upon exposure to branchlets of the rudiments of their velar lobes had grown anteriorly Laurencia pacifica, and had been reared from juveniles to extend beyond the shell when crawling (see Kneg- to reproductive adults on a diet of Plocamium car- stein 1977, for drawings and details). Unmetamor- tilagineum and L. pacifica. Small pieces of egg mass phosed larvae were distinguished by the presence of were placed in aerated beakers containing l pm-fil- velar cilia, whether crawling or not. Larvae and juve- tered, natural seawater (hereafter referred to as 'sea- niles were removed as they were counted. Metamor- water') until hatching occurred. Larvae were then phosed larvae were often difficult to locate, particularly transferred to 2 1 Fernbach flasks filled to the neck with on highly branched algae, and data were discarded if seawater containing 40mg 1-' each of the antibiotics less than 15 of 20 larvae or juveniles were recovered streptomycin sulfate and sodium penicillinG (Sigma per dish. Chemical Co., St. Louis, Missouri) and 104 cells ml-' of The intertidal macroalgae used in the assays were the green flagellate Pavlova lutheri. Larval entrapment collected from Casa Cove and Dike Rock, La Jolla, at the air/water interface was prevented by spreading California, in Apnl and May 1987. Branchlets of algae flakes of cetyl alcohol (l-hexadecanol, Sigma) on the were added to assay dishes within 24 h of collection. water surface. Flasks were kept at 20°C in a chamber Only branchlets free of epiphytes were used in assays 35 cm beneath 2 continuously-illuminated 60 W fluo- and all associated fauna were removed prior to their rescent lights. transfer to assay dishes. Algal identifications were Every 4 d, culture vessels were cleaned. Contents of based on Abbott & Hollenberg (1976). Algae used the flask were poured through a series of mesh screens were: Codium fragile and Ulva sp. (Chlorophyta); Col- so as to retain the larvae while eliminating larger and pomenia sin uosa, Dictyopteris undula ta, Enderachne smaller particles. The flasks were then scrubbed with binghamiae, Pach ydictyon coriaceum, Pelvetia fas- hot, fresh water, rinsed in seawater, and reconstituted tiqiata, Sarqassum muticum and Zonaria farlowii as before. Cultures were stirred with a jet of 25ml of (Phaeophyta); and Callophyllis violacea, Centroceras seawater once each day. Using these techniques, lar- clavulatum, Chondria californica, Corallina officinalis, vae exhibited growth rates comparable to those Gigartina canaliculata, Laurencia pacifica, Plocamium observed in other studies (Kriegstein et al. 1974, Paige carhlagineum, Pterocladia capillacea and Rhodymenia 1986). californica (Rhodophyta). Larvae were competent to metamorphose approxi- Preliminary experiments were performed in an mately 35 d after hatching and were used in assays 40 attempt to characterize the chemical inducers of to 60d after hatching. The development of 4 to 6 red metamorphosis of Aplysia californica. Branchlets of spots on the perivisceral membrane of the larvae did Plocamium cartrlagineum and Laurencia pacifica were not prove to be a reliable indicator of larval maturity, as separately frozen and freeze-dried, and a portion of the detailed by Kriegstein (1977), because larvae lacking freeze-dned algae was sequentially extracted in dis- perivisceral spots frequently underwent normal settle- tilled organic solvents (hexanes, diethyl ether, ethyl ment and metamorphosis. Twenty larvae were trans- acetate and methanol, in that order). The resulting ferred to each glass assay dish (8 cm diam., 4 cm high) extracts of each alga were combined and coated onto a containing 60 to 70 m1 of seawater coated with flakes of disk of filter paper (Whatman, 4 cm diameter). Disks cetyl alcohol. Branchlets of intertidal algae having and extracted algae were placed under a vacuum to roughly equivalent surface areas were added to each remove all traces of solvents. Assays were run as before dish (no alga was added to the control dish). The water with living, freeze-dried and freeze-dried and surface in each dish was examined under a dissecting extracted algae, and algal extracts on filter paper microscope, and the larvae trapped in the air/water Freeze-dried and freeze-dned and extracted algae interface were freed by hitting them with drops of were first rehydrated in seawater under a vacuum. seawater. Dishes were covered with white paper (to All assays were run with 3 replicates and the mean decrease light levels and prevent algal branchlets from percentage of larval response for each assay deter- forming bubbles and floating to the surface) and placed mined. Differences in mean percentage metamorphosis under the same lighting and temperature conditions were tested with l-way analysis of variance (ANOVA) that had been employed in larval culture. After 2d, performed on arcsin-transformed data. The Tukey test dishes were cleaned in the same manner as larval was applied a posteriori to determine which treatments culture vessels, and the dishes reconstituted as they resulted in