Plankton Benthos Res 14(2): 131–134, 2019 Plankton & Benthos Research © The Japanese Association of Benthology Note Larval settlement of an algivorous snail species () induced by symbiotic sea urchins and conspecific snails

Luna Yamamori* & Makoto Kato

Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606–8501, Japan Received 5 January 2019; Accepted 5 February 2019 Responsible Editor: Shigeaki Kojima doi: 10.3800/pbr.14.131

Abstract: The rock-boring sea urchin, Echinostrephus molaris, excavates soft rocks, and the pits are later used by non-boring sea urchins such as Echinometra mathaei and Anthocidaris crassispina. The pits used by these non-boring sea urchins are also characteristically inhabited by a limpet-like algivorous trochid snail, Broderipia iridescens (Gas- tropoda, Trochidae). To determine how this unique symbiotic association is maintained, we observed reproduction and larval development of the snail, and performed assays to detect what induced settlement in the snail larvae. As candi- dates of settlement inducers, a habitat biofilm plate, the host and the non-host sea urchins, and conspecific snails were kept in separate glass bowls, and newly hatched B. iridescens veliger larvae were introduced to each bowl. Sixty to 80% of larvae settled in bowls that contained the non-boring host sea urchins and conspecific snails. On the other hand, only approximately 8% of the larvae settled in bowls containing the boring non-host sea urchins. This is the first report demonstrating that larval settlement of an algivore is induced not by the habitat biofilms, but by the symbiotic hosts.

Key words: Echinoid, Fossarininae, intertidal, settlement, veliger

Marine intertidal rock reefs are inhabited by a diverse molaris (Blainville 1825), and the other two are non-boring range of sessile and creeping organisms, most of which species, Echinometra mathaei (Blainville 1825) and Antho- colonize the reefs through larval settlement. Pelagic larvae cidaris crassispina (Agassiz 1863), both of which use the generally settle in specific microhabitats by sinking in re- pits made by the boring sea urchins. Interestingly, B. iri- sponse to specific hydrodynamic conditions, such as turbu- descens snails are symbiotic only with the two non-boring lence (Fuchs et al. 2004), and are attracted by specific cues sea urchin species, which provide the snail with a safe emitted from substrates, foods, hosts, conspecifics, and po- space protected from predatory fishes and crabs by their tential mates (Larsson & Jonsson 2006). For example, the stout spines (Yamamori & Kato 2017). Based on our direct larvae of molluscan algivores are attracted by chemical observations, the snail is considered to be commensal with cues emitted by food algae (Kay 2002). This settlement be- the sea urchin. Although B. iridescens snails are obligate havior is excellently tuned, particularly in sessile, parasitic, symbionts of these sea urchin species, they are still micro- and symbiotic organisms, which must pinpoint suitable mi- algal grazers. The exact cue used by the snail larvae to crohabitats within their short larval periods (Crisp 1974). settle in habitats occupied by non-boring urchins remains Among algivorous trochid snails with spirally coiled top unknown. shells, Broderipia iridescens (Broderip 1834) is unique be- To identify the settling cue of B. iridescens larvae, we cause it has a flat, zygomorphic, limpet-shaped shell. In conducted laboratory experiments in which B. iridescens subtidal to intertidal rock reefs in the temperate zone of larvae were introduced into containers with and without the Japanese Archipelago, there are three dominant sea habitat biofilms, three sea urchin species, and adult B. urchin species: one is a boring species, Echinostrephus iridescens snails. Although the experimental system was simple, the results clearly revealed that larval settlement * Corresponding author: Luna Yamamori; E-mail, strobilation980@ was induced by the host sea urchins and by conspecific gmail.com snails. 132 L. Yamamori & M. Kato

Animals used in this study were collected in rocky sub- kinds of sea urchins, and adult B. iridescens snails were tidal zones around the Seto Marine Biological Laboratory respectively introduced into glass bowls containing 1 L fil- (SMBL) of Kyoto University at Shirahama, Wakayama tered seawater (for habitat biofilm plates and sea urchins, Prefecture (33°69′51″N, 135°33′58″E) in July 2017. To ob- one plate or individual was introduced per bowl, and for tain B. iridescens larvae, we collected 50 B. iridescens adult B. iridescens, five individuals were introduced per snails with shell length of 4.5–8.1 mm from pits of non- bowl). We introduced 30 B. iridescens veliger larvae that boring sea urchins. The snails were kept together in a glass had hatched within the previous 12 h into each glass bowl. bowl (15 cm in diameter) with 1 L of filtered seawater at The same number of larvae were also introduced into a 24°C, which was similar to the surface water temperature bowl without any candidate settlement inducer (control). of their natural habitat. After one night of culturing, 19 egg The bowls were examined every 12 h, and the number of strings were obtained. Each egg string contained 25–40 larvae that settled were counted. During the experiment, larvae. All egg strings were collectively cultured in a new the light-dark conditions were in accordance with nature. glass bowl with filtered seawater, and the culture water Settlement was confirmed by the disappearance of the ve- was exchanged every 12 h. After 48 to 60 h of culturing, lum. The above-mentioned protocol was repeated three veliger larvae hatched out of the egg strings. As a candi- times using different plates/individuals of candidate settle- date settlement inducer, we prepared substrate replicas of ment inducers, and Dunnett’s tests were conducted to de- the snails’ habitat. Thin plastic plates (2×5×0.03 cm) were termine if there were significant differences between each kept in a flow of natural seawater of B. iridescens habitat experimental group and the control. for two weeks to allow the formation of a biofilm contain- B. iridescens is gonochoristic. Our observations indi- ing prey microalgae. The microalgae were checked under cate that a mature male B. iridescens snail (shell length an optical microscope, and confirmed to be similar to the >4 mm) mounts a female and mates using its copulato- algal flora in sea urchin pits in terms of both species com- ry organ on the right anterior side. Females expel float- position and density. The main component of microalgae ing fertilized egg strings, in which cleavage proceeds, was Cocconeis sp., the main food source of B. iridescens and trochophore larvae grow (Fig. 1a). Trochophore lar- snails. For other candidate settlement inducers, we collect- vae metamorphose into pre-torsional veliger larvae, which ed three species of sea urchins; the non-boring host sea continuously rotate in egg membranes through the cili- urchins E. mathaei and A. crassispina, and the non-host ary motion of the velum (Fig. 1b). On the third day after boring sea urchin E. molaris. All sea urchins and snails spawning, the rotational speed of veliger larvae increased, were kept for 12 h to ensure that they excreted all of their eventually breaking the egg membrane, allowing the lar- intestinal contents. vae to swim out of the broken membranes. The hatched Larval settlement assays were conducted to detect which veliger larvae have spiral protoconchs (Fig. 1c), and can factor induces settlement of B. iridescens larvae, i.e., habi- swim freely. Approximately three days after hatching, the tat biofilms containing prey algae, host and non-host sea larvae used in our study approached the bottom of the urchins, or conspecifics. The habitat biofilm plate, three glass bowl by waving their velum, and settled on substrata

Fig. 1. Larval development in B. iridescens, with pictures of the larvae. (a) Events in development: spawning, metamorphosis from trocho- phore to veliger larvae, hatching from eggs, and settlement. (b) Pre-torsional veliger larvae in an egg string. (c) Hatched veliger larva. (d) Settled veliger larva, with cephalic tentacles containing papillae. Scale bar=100 µm Larval settlement of an urchin-associated snail 133 under specific conditions. In bowls containing sea urchins, Induction of larval settlement by adult conspecifics is B. iridescens larvae settled not on the sea urchins them- known to occur in a wide range of taxa, including Cnidar- selves, but on the bottom of the glass bowl. After settle- ia, , Arthropoda, Echinodermata, and Urochor- ment, larvae cast off the velum and developed cephalic data (Burke 1986). In barnacles, the settlement-inducing tentacles with papillae (Fig. 1c). cue is reported to be an α2-macroglobulin-like glycopro- The temporal changes in the cumulative settling rate tein that has a specific sugar chain that is considered to of larvae under each experimental condition are shown be functional (Matsumura et al. 1998, Dreanno et al. in Fig. 2. Larval settlement occurred 60–96 h after hatch- 2006). Settlement induced by conspecifics is considered ing. The final settling rates were as follows: control (B. to be a reliable means to enable gregarious recruitment, iridescens veliger larvae only), 3.3±1.9% (mean±SD); and allows individuals to locate suitable habitats and mates E. mathaei, 74.4±7.3% (D=9.84, Dunnett’s test: p= (Grove & Woodin 1996). 4.26×10−7); A. crassispina, 78.9±7.8% (D=10.5, Dunnett’s Among the diverse range of symbiotic associations re- test: p=2.21×10−7); E. molaris, 7.8±1.1% (D=0.157 Dun- ported in marine ecosystems (e.g., Morton 1989, Anker nett’s test: p=0.878×10−7); adult B. iridescens, 67.8±1.1% et al. 2005), some symbionts locate and reach their hosts (D=8.92, Dunnett’s test: p=1.22×10−6); and habitat bio- using their sensory and locomotory abilities. For example, film plate, 2.2±1.2% (D=0.157, Dunnett’s test: p=0.878). the ectoparasitic pea crab Dissodactylus primitivus (Bou- The settlement rates were significantly higher in cultures vier 1917) is chemically attracted by its host sea urchin with non-boring sea urchins and conspecific snails than in (Bruyn et al. 2011), and a symbiotic shrimp, Gnathophyl- those with boring sea urchins, habitat biofilm plates, and loides mineri (Schmitt 1933), detects its host sea urchin only larvae (control). by both visual and chemical cues (Williamson et al. 2012). Our data suggest that settlement of B. iridescence larvae In contrast to these on-host symbiotic organisms, B. iri- was induced by the presence of symbiotic sea urchins and descens snails inhabit sea urchin pits rather than the sea conspecific individuals. Although the chemical structure urchins themselves, suggesting that there is another cue of the inducers was not determined, the results of our ex- inducing final settlement on algal mats. periment suggest that host sea urchins and conspecifics Because all other sister taxa in the Fossarininae (Trochi- secreted some chemicals that induced the larvae to settle. dae) are free living, the symbiotic association with sea ur- The fact that settlement was induced by non-boring ur- chins must have evolved independently in B. iridescence. chins, but not by boring urchins, suggests that the inducer To understand the evolutionary origin of this symbiotic as- is not a general chemical shared by a range of sea urchin sociation, studies to determine the cues of larval settlement species. In this experiment, because of difficulties with de- in the sister taxa are required. termining the microbial flora, it is not certain that the bio- film used exactly reflected the actual microbial flora of the Acknowledgements habitat of B. iridescens snails. However, considering that B. iridescens snails are algal grazers, the induction of lar- We are grateful to all the staff at the Seto Marine Bi- val settlement by sea urchins is remarkable. This is the first ological Laboratory and Shirahama Aquarium for sup- reported case of the larvae of an algivore being attracted porting our survey. This work was supported by a Grant- not to a food alga itself, but to their symbiotic hosts. in-Aid for JSPS Fellows Grant number 18J22890 to LY, and a Grant-in-Aid for Scientific Research Grant number 15H02420 to MK.

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