Proceedings 9th International Coral Reef Symposium, Bali, Indonesia 23-27 October 2000,Vol. 1.

Coral reef benthic as food and refuge: Diversity, chemistry and complex interactions

E. Cruz-Rivera1 and V.J. Paul1,2 ABSTRACT Benthic filamentous cyanobacteria are common in coral reefs, but their ecological roles are poorly known. We combined surveys of cyanobacteria-associated fauna with feeding preference experiments to evaluate the functions of benthic cyanobacteria as food and shelter for marine consumers. Cyanobacterial mats from Guam and Palau yielded 43 invertebrate species. The small sea hare striatus was abundant on cyanobacterial mats, and only fed on cyanobacteria in multiple-choice experiments. In contrast, feeding experiments with urchins and fishes showed that these macrograzers preferred algae as food and did not consume either of two cyanobacteria offered. Extracts from the cyanobacterium majuscula stimulated feeding by sea hares but deterred feeding by urchins. Thus, some small coral reef grazers use cyanobacteria that are chemically-defended from macrograzers as food and refuge. Cyanobacteria could indirectly influence local biodiversity by affecting the distribution of cyanobacteria-dwelling organisms.

Keywords Algal-herbivore interactions, Chemical differently as food by macro- and mesoconsumers?, and defenses, Cyanobacteria, Lyngbya, Mesograzers, Sea 3) Do cyanobacterial metabolites play a role in these hares interactions?

Introduction Materials and Methods Studies of algal-herbivore interactions have offered Field surveys and collections were conducted at Piti important information on the roles of eukaryotic Reef in Guam (130 30’N, 1440 45’ E) during July 1999 macroalgae as food and shelter for marine consumers. and at three different sites (Lighthouse Channel, Oolong Complex interactions develop around chemically- Channel, and Short Drop Off) at the Republic of Palau (70 defended seaweeds that deter larger consumers such as 30’ N, 1340 30’ E) during April of 1999 and 2000. fish and urchins (macrograzers) while providing safe Surveys of cyanobacteria-associated fauna were habitats for small herbivorous crustaceans, polychaetes, conducted using snorkel or SCUBA by collecting ten and gastropods (mesograzers) adapted to the chemical individual mats of each cyanobacterium. Mats were defenses of the algae (Hay 1992, Hay and Steinberg 1992, sealed inside plastic bags underwater, brought to the Paul et al. 2001). laboratory, and inspected under a dissection microscope at Large benthic filamentous cyanobacteria are common low magnification. were counted, sorted into and often abundant in some coral reefs (Thacker and Paul species, and densities recorded as number of individuals in press). Because of their biomass and productivity, per gram wet mass of cyanobacteria. To eliminate excess benthic cyanobacteria have the potential to be important water, cyanobacterial mats were either spun in a salad food sources for coral reef grazers and to be shelters for centrifuge or pressed gently on absorbent paper, small animals, thus playing important roles in marine depending on the structure and cohesiveness of the mats. communities in a fashion similar to that of eukaryotic Our surveys included only organisms that could algae. Like many eukaryotic algae, some marine potentially be mesograzers (Paul et al. 2001) and cyanobacteria have deterrent chemicals that defend them therefore, did not include smaller consumers such as from macroherbivores (Pennings et al. 1997, Nagle and copepods, ostracods, small nematodes, or foraminiferans, Paul 1998, 1999) but, some smaller consumers appear that were sometimes abundant. adapted to feed on these cyanobacteria (Paul and Cyanobacteria were identified to genus under a Pennings 1991, Pennings and Paul 1993, Nagle et al. compound microscope following the taxonomic system of 1998). However, the relative use of cyanobacteria by Desikachary (1959). Faunal surveys were conducted on diverse marine consumers is poorly known. four cyanobacterial species (, L. In this study, we assess the role of benthic bouillonii, Symploca sp., and Oscillatoria sp.) The cyanobacteria as food and shelter for marine consumers. Symploca from Piti forms prostrate golden yellow mats on We combine observations on the distribution of sand and coral rubble, with densely packed upright cyanobacterial epifauna with experiments on feeding filaments only a few cm high. The Oscillatoria forms behavior of diverse marine grazers to draw parallels thin dark brown mats on sand and rubble that spread as between the ecologies of eukaryotic algae and thin slimy films over the substrate. cyanobacteria in coral reefs. In particular, we ask 1) Do The three cyanobacteria surveyed in Palau included an small benthic organisms find shelter within coral reef unidentified bright red Lyngbya sp. and two unidentified benthic cyanobacteria?, 2) Are these cyanobacteria treated Symploca spp. The Lyngbya forms upright feathery

1 University of Guam Marine Laboratory, UOG Station, Mangilao, GU 96923, USA 2 [email protected]

colonies about 10 cm tall on rocks and dead coral. (N=11) in Guam. Ropes were left for 1-3 hr in the field. Symploca sp. 1 was found mostly on the underside of Because these assays ran for such a short period and corals (although it also occurs in the open) forming small because fish consumption was so high, controls for discreet reddish orange colonies about 3-5 cm high, autogenic changes in mass were not used. However, we resembling the shape of Schizothrix mexicana. Symploca scored these assays conservatively by classifying algae sp. 2 forms prostrate, sometimes extensive, mats with only as eaten (> 50% mass lost) or not eaten (<50% mass filaments around 5 cm high. The mats are delicate, with a lost), and analyzed the data using contingency table cotton-like appearance and light pink shades (towards the analyses. Ropes in which none of the 7 algal species was tops of the mats) over a white background. Lyngbya sp. consumed by 50% or more did not offer information on and Symploca sp. 2 were collected in both April of 1999 fish feeding preferences and were not used in the and 2000 from the same sites. Data from faunal surveys analyses. were seldom distributed normally and variances were Consumers could avoid cyanobacteria due to the rarely homogeneous, thus, most analyses were performed presence of defensive secondary metabolites. We tested either by Kruskal-Wallis or Mann-Whitney U tests (Palau this by comparing the palatability of crude extracts from 2000 surveys), but ANOVA was utilized when the common cyanobacterium Lyngbya majuscula offered appropriate. to sea hares and urchins. We hypothesized that extracts Multiple choice feeding assays were conducted with would not deter the sea hares but would deter the urchins. the most abundant organism found on cyanobacterial mats Freeze-dried L. majuscula was extracted in 1:1 ethyl in Guam and Palau, the small sea hare (Opisthobranchia) acetate/methanol. The crude extract was diluted in ethyl (previously Stylocheilus longicauda ether and coated at natural concentration (per dry mass) – see Rudman 1999). In Guam, two small (20-30 mm) onto freeze-dried powdered Enteromorpha, using a rotary sea hares were placed in round dishes (16 cm diameter, 6 evaporator to eliminate the organic solvent. Enough ether cm height) filled with sea water and offered a was used to completely cover the freeze-dried algae in the simultaneous choice among five eukaryotic macroalgae flask. This provided our treatment food. Control food (the green algae Enteromorpha clathrata and Caulerpa was prepared by treating Enteromorpha with ether alone. racemosa, the brown algae Padina tenuis and Sargassum The foods were then used to prepare agar-based artificial polycystum, and the red alga Acantophora spicifera) and diets following methods in Hay et al. (1998). Our two common cyanobacteria (Lyngbya majuscula and L. standard recipe contained 2g of freeze-dried algae, 0.36g bouillonii). Similarly sized pieces of each alga were spun of agar, and 18 ml of distilled water. Pair-wise choice in a salad centrifuge to remove excess water and weighed assays were performed by simultaneously offering grazers at the start of the assays. Algal pieces differed in weight food strips containing freeze-dried Enteromorpha either (ca.150-2000 mg) because of differences in algal with or without L. majuscula extracts. Three small sea densities, but because the pieces were of similar size the hares, or one urchin, were placed in replicate containers animals had similar likelihood of encountering each alga. (n=15 and 12, respectively) similar to those described Ten replicate experimental containers received sea hares previously. Replicates in which no food was consumed and algae while another seven had algae without sea hares were not used in the analyses because they provided no and served as controls for changes in algal mass unrelated information on consumer feeding preference. Sea hares to consumption. After allowing animals to feed for 1.5 and urchins were allowed to feed on these artificial diets days, algae were weighed again (after removing excess for 2 and 3 days, respectively. Data from these assays water) and the amount consumed was calculated and were analyzed with two-tailed t- tests. corrected for autogenic changes in algal mass (Peterson and Renaud 1989). Algae that were not consumed and Results grew appear as negative values in the results. Organisms A diverse fauna of 43 species was associated for these and other feeding assays (see below) were with the benthic cyanobacteria (Figs. 1-3). Gastropods, collected from sites at Piti Reef, Pago Bay, and Tumon crustaceans, and polychaetes dominated in numbers and Bay in Guam. Data from these multiple-choice feeding probably in species diversity as well. The total number of assays were analyzed using Friedman’s tests (see species is probably an underestimate because the taxo- Stachowicz and Hay 1999). nomy of such groups as cephalaspidean snails and Feeding assays with the common urchin Echinometra polychaetes is poorly understood. Overall, the most mathaei followed a similar laboratory protocol to that for abundant cyanobacteria-associated animals were the small Stylocheilus, but used larger algal pieces (1.5-5.0 g). sea hare Stylocheilus striatus, the cephalaspidean snails Single urchins were placed in round flow-through tanks Diniatys dentifera and Ventomnestia villica, and a small (25 cm diameter, 25 cm height) and allowed to feed for unidentified rissoellid snail (Jeffreysilla sp.). All these two days. Feeding assays with fishes were conducted in animals were observed in more than one cyanobacterial the field using algal pieces suspended on plastic ropes. species from Guam and/or Palau (e.g., Stylocheilus, Figs. Each replicate rope had one piece of each alga or 1-3). Other gastropods observed included the snails cyanobacterium (3-11 g) threaded at the base into the rope Cerithium zebrum (=Bittium zebrum) and C. punctatum and arranged haphazardly within replicates. Ropes were (=C. alveolus), and the cephalaspideans Haminoea ovalis, taken to the field and attached by one end to dead corals H. nigropunctatus and an undescribed Diniatys sp. The or rocks. Small floats kept the ropes upright while crustacean fauna was dominated by amphipods (Ampithoe attached to the bottom. Two separate assays were sp.) and tanaids (Leptochelia cf. dubia). Portunid crabs conducted at Western Shoals (N=12) and Cocos Lagoon

(Thalamita corrugata and Thalamita integra) were also more diverse epifauna than the other three cyanobacteria common in mats of Lyngbya majuscula from Guam, sampled in Guam (Fig. 1). This species had significantly suggesting that predators could also find refuge among more Stylocheilus striatus (p<0.001, Kruskal-Wallis) and cyanobacteria. amphipods (p=0.018, Kruskal-Wallis) than at least two of Small tube-building alpheid shrimp (Alpheus sp.) were the other cyanobacteria sampled, a significantly higher only collected in Palau, where they occurred on both number of cephalaspideans (p=0.003, Kruskal-Wallis) Lyngbya and Symploca (Figs. 2-3). However, we also and tanaids (p=0.035, Kruskal-Wallis) than at least one of observed the congeneric Alpheus frontalis, a large tube- the other cyanobacteria surveyed and showed a strong, building shrimp common on L. bouillonii in Guam and though not significant, trend (p=0.083, Kruskal-Wallis) Palau, but it was not included in our surveys because the towards higher density of snails than all other shrimp avoided collection (and thus, positive cyanobacteria (Fig. 1). In contrast, the congeneric identification). Other taxa found among cyanobacterial Lyngbya bouillonii had the lowest numbers and mats included large nematodes, nemerteans, oligochaetes, diversity of epifauna of all the cyanobacteria sampled cumaceans, nudibranchs, caprellid amphipods, non- (Fig. 1). Interspecific differences in the abundances of sea alpheid shrimps, hermit crabs, majid crabs, galatheid hares, tanaids, polychaetes, cephalaspideans, and alpheid crabs, isopods, and pycnogonids. shrimps were also found for the cyanobacteria surveyed in Palau in both years (Figs. 2-3).

Fig. 2 Mean (+1SE) abundance of eight animal groups Fig. 1 Mean (+1SE) abundance of animals associated associated with three benthic cyanobacteria from Palau in with four benthic cyanobacteria from Guam in July 1999 April 1999 (N=10). Analyses and symbols as in Fig. 1 (N=10). Analyses are by one-way ANOVA or Kruskal- Wallis followed by Tukey’s HSD post hoc tests or Tukey- Sea hares, urchins, and fishes showed different type non-parametric comparisons. Same letter above two patterns of feeding preferences for eukaryotic macroalgae bars indicates statistically equivalent means. versus cyanobacteria (Fig 4). Stylocheilus strongly preferred Lyngbya majuscula and to a lesser extent L. There were noticeable interspecific differences among bouillonii, but the urchin Echinometra mathaei showed cyanobacteria in the numbers and diversity of their significant feeding preferences for some macroalgae associated faunas. Lyngbya majuscula from Guam had a when offered a choice among algae and cyanobacteria

(p<0.001, Friedman’s test, Fig. 4). Grazing was highest tenuis was the species most consistently consumed, but all on the red alga Acantophora, followed by Padina, eukaryotic algae tested were significantly eaten compared Sargassum, and Enteromorpha. to cyanobacteria (p<0.001, Chi-square). In contrast, only Enteromorpha and Caulerpa were significantly consumed at Western Shoals (p<0.001, Chi-square test).

Fig. 3 Mean (+1SE) abundance of five animal groups associated with two benthic cyanobacteria from Palau in April 2000 (N=10). Analyses are from t-tests or Mann- Whitney U tests.

Despite large differences in mean consumption, variance was high and consumption of these four algae was statistically equivalent after post hoc tests. Consumption of the green alga Caulerpa racemosa was very low and statistically equivalent to that of cyanobacteria did not graze on any of the other algae offered (p<0.001, Friedman’s test). The large variance in Caulerpa mass loss during the Stylocheilus assays was caused by some of the algae becoming reproductive and not by sea hares consuming this seaweed. The urchin Echinometra mathaei showed significant feeding preferences for some macroalgae when offered a choice among algae and cyanobacteria (p<0.001, Friedman’s test, Fig. 4). Grazing was highest on the red alga Acantophora, followed by Padina, Sargassum, and Enteromorpha. Despite large differences in mean Fig. 4 Feeding choices of the sea hare Stylocheilus consumption, variance was high and consumption of these striatus, the urchin Echinometra mathaei, and fishes from four algae was statistically equivalent after post hoc tests. two reefs offered five eukaryotic algae and two Consumption of the green alga Caulerpa racemosa was cyanobacteria (Lyngbya majuscula and L. bouillonii). For very low and statistically equivalent to that of fishes, data are the number of replicates in which >50% of cyanobacteria. that alga was eaten. Analyses are by Chi-square or Natural assemblages of fishes from two different reefs Friedman’s tests. Letters above bars indicate significant showed different patterns of feeding among the groupings macroalgae. At Cocos Lagoon the brown alga Padina

diversity of epifauna on cyanobacterial mats and the dependence of some of these animals on cyanobacteria as food suggest that cyanobacteria can potentially enhance local biodiversity patterns in some coral reefs. These “positive” roles of marine cyanobacteria have been largely ignored because cyanobacteria have seldom been treated as normal components of coral reef communities. This is likely because benthic cyano- are mostly noted when they form blooms that adversely affect marine communities (Nagle and Paul 1998), or when associated with diseases in humans (e.g., “swimmer’s itch,” Moore 1984) or corals (Rutzler and Santavy 1983, Feingold 1988). Researcher have also suggested that benthic cyanobacteria may be used as Fig. 5 Feeding by sea hares (Stylocheilus) and urchins indicators of environmental degradation in both (Echinometra) on artificial diets containing (treatment) or freshwater (Perona et al. 1998) and marine benthic lacking (control) extracts from the cyanobacterium habitats (La Pointe 1999). While the view of cyano- Lyngbya majuscula. Both diets were offered simul- bacteria as abnormal nuisances rather than normal taneously. Analyses are by t-tests. components of benthic environments prevails in marine community studies, cyanobacteria are typical members of the benthic community in many reefs around Micronesia A more diverse fish community occurred at Cocos and often occur in densities similar to those of other Lagoon than at Western Shoals during these experiments components of the coral reef biota (Thacker and Paul in and most grazing at this latter site was done by the press). “blacktongue unicornfish” Naso hexacanthus (E. Cruz- Epifaunal diversity varied significantly among the Rivera, personal observation). Despite differences in cyanobacterial species surveyed; the highest number of feeding patterns, none of the two cyanobacteria tested species was associated with Lyngbya majuscula while the were eaten by fishes at either site. lowest numbers were on L. bouillonii. The low diversity Cyanobacterial chemical deterrence explained some of on the latter could be related to the strong association the feeding choices observed. Crude extracts of the between the large alpheid shrimp Alpheus frontalis and L. cyanobacterium Lyngbya majuscula, the preferred food of bouillonii. In Guam, the shrimp is found solely on L. the sea hare (Fig. 4), strongly stimulated feeding by this bouillonii where it builds a tube from the cyanobacterium. mesograzer (Fig. 5). In contrast, the urchin Echinometra Like all snapping shrimp, A. frontalis uses its large claw mathaei was strongly deterred (p<0.001, t-test) by in aggressive encounters towards other animals. This Lyngbya extracts. Thus, our data showed opposite results explanation for the low diversity of epifauna on L. for the meso- and macrograzer feeding on live algae (Fig. bouillonii is consistent with our experimental results that 4) and cyanobacterial extracts (Fig. 5). Stylocheilus readily consumed L. bouillonii (Fig. 4), even though it was never found on this cyanobacterium in the Discussion field (Fig. 1). Lyngbya majuscula extracts stimulated feeding by The role of eukaryotic macroalgae as food and refuge Stylocheilus striatus (Fig. 5) which is consistent with for small herbivores (mesograzers) has been studied previous demonstrations that feeding in this sea hare is extensively in both tropical and temperate marine often not deterred and can be stimulated by secondary systems. Chemically-defended macroalgae can indirectly metabolites from L. majuscula (Nagle et al. 1998). enhance the survival of mesograzers by providing However, the types and yields of secondary metabolites “enemy-free space” and a variety of animals selectively can vary dramatically between populations of L. associate with, and feed on, defended algae, are cryptic on majuscula and the same metabolite can be a feeding defended algal hosts, and behaviorally or physiologically stimulant or deterrent to Stylocheilus depending on its sequester chemicals from the algae and use those concentration (Nagle et al. 1998, Paul et al. 2001). Thus, chemicals as acquired defenses against predators (Hay cyanobacterial grazers might encounter foods that are 1992, Hay and Steinberg 1992, Paul et al. 2001). either stimulatory or deterrent depending on We show that some large benthic cyanobacteria in cyanobacterial secondary metabolites, even when faced coral reefs host a diverse animal fauna (Figs. 1-3) and that with the same species of cyanobacterium. some of these animals selectively consume cyanobacteria Lyngbya majuscula was not readily consumed by that are low preference items for larger grazers (Fig. 4). urchins or fishes (Fig. 4). For the urchin Echinometra We also demonstrate that chemical feeding deterrents mathaei, we demonstrated that L. majuscula extracts were against macrograzers occur in at least some cyanobacteria strongly deterrent (Fig. 5). Other benthic cyanobacteria but that the same chemicals do not deter feeding by a are also chemically defended against macrograzers specialist mesograzer (Fig. 5). Thus, some defended (Pennings et al. 1997, Nagle and Paul 1998, 1999, Paul et benthic cyanobacteria serve as food and shelter for small al. 2001). For example, Nagle and Paul (1999) tested grazers and, in this context, they parallel chemically- extracts of 10 cyanobacterial collections from Guam defended eukaryotic macroalgae in coral reefs. The

against an herbivorous parrotfish and found strong Moore RE (1984) Public health and toxins from marine deterrence in all of the extracts. blue-green algae. In: Ragelis EP (ed) Seafood toxins. Plant and algal chemical diversity have shaped the American Chemical Society Symposium Series 262, evolution of plant-herbivore interactions and associations Washington: 369-376. in both terrestrial and marine systems (Rosenthal and Nagle DG, Camacho FB, Paul VJ (1998) Dietary pre- Berenbaum 1992, Hay and Fenical 1996, Paul et al. ferences of the opisthobranch mollusc Stylocheilus 2001), but until recently the chemical interactions longicauda for secondary metabolites produced by the between benthic cyanobacteria and marine consumers tropical cyanobacterium Lyngbya majuscula. Mar Biol have not been considered (Paul et al. 2001). 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