COMMENTARY Chemical or nematocyst - based defence in the nudibranch Cratena peregrina ? – A reply to B . K . Penney Arnaldo Marin

Aguado & Marin (2007) analysed the interaction between Cratena peregrina (Gmelin, 1791) and predatory fish in laboratory and field assays, using both live aeolids and artificial models. Penney’s comment (2009) on the article reopens the old question of the role of nematocyst-based defence in nudibranchs. Penney suggests that the defensive mechanism of C. peregrina is chemical because the nematocysts used in assays could be unarmed. Penney argues that (1) Aguado & Marin (2007) obtained nematocysts by macerating Eudendrium hydroids with a mortar and pestle, and this cannot be considered equivalent to kleptocnidae isolated from C. peregrina; (2) the method by which the authors attempted to incorporate nematocysts into the test food is unclear; and (3) regardless of how nematocysts were added to the artificial food models, it is possible that no functional nematocysts would remain by the time fish encountered them. The main purpose of our article was to demonstrate that fish learn to avoid the warning coloration pattern of C. peregrina, due to associating bad taste or unpleasant experience with the colour pattern. Nematocysts are contained in the defensive exudates and in the external part of the body of C. peregrina, which can only be regarded as circumstantial evidence that these defensive cells or the molecules that they contain represent a deterrent for predators. Of course, the article does not provide experimental evidences that the source of deterrence is the nematocysts, but there is no proof, either, that chemical defences play an active role. If we support the general idea that a positive result for deterrence alone cannot be taken as evidence for a nematocyst-based defence, the same argument must apply to chemical-based defence. We must classify nematocyst-based defences as a special form of chemical defences because nematocysts contain harmful molecules. From this point of view, C. peregrina transfers chemicals from food to the cnidosacs at the tip of the ceras. There is no evidence that C. peregrina contains secondary metabolites other than the nematocyst toxins. Chemical analysis supports the general hypothesis that the nematocysts are the main weapons. Cimino et al. (1980) studied three species of the hydroid Eudendrium (E. rameum, E. racemosum, E. ramosum) in the Bay of Naples (Italy), which are prey of C. peregrina. These authors found the same pathway of polyhydroxylated steroids (Cholest-4-en-4, 16b, 18, 22R-tetrol-3-one 16,18-diacetate) in the hydroids and in the nudibranchs. Unfortunately these steroids do not play a defensive role in hydroids or nudibranchs. Some aeolids contain pigments and secondary metabolites from their prey, which probably have no chemical defence role either (e.g. Goodwin & Fox, 1955; McBeth, 1972: the compounds are ubiquitous and conserved amongst numerous non-deterrent species; Cimino et al., 1980: the compounds are located within internalized structures and thus not quickly encountered by predators). Sequestered compounds can occur at concentrations lower than threshold feeding deterrent levels (Karuso, 1987; Rogers & Paul, 1991). Thus, metabolite accumulations within opisthobranchs may represent a mechanism to store toxic chemicals with impunity for subsequent elimination, and not necessarily to defend the organism (Pennings & Paul, 1993). Penney suggests that because E. carneum from North American possesses chemical extracts that deter feeding by pinfish, extracts from Eudendrium employed with models of C. peregrina could be unpalatable because they contain chemicals and not active nematocysts. Stachowicz & Lindquist (2000) indicated that crude extracts from Eudendrium deterred feeding, ‘demonstrating’ that defensive chemistry plays a significant role in the unpalatability of these species. To test their hypothesis that unpalatable secondary metabolites represent alternative antipredator defences for hydroids, they compared the palatability of intact polyps with those in which the nematocysts had been chemically stimulated to discharge. Hydroid polyps defended solely by nematocysts should become palatable when their nematocysts are discharged before offering them to predators, whereas the palatability of chemically defended hydroids should not change after treatment. However, neither the lipophilic (DCM þ butanol soluble compounds) nor the watersoluble fractions of Eudendrium deterred feeding. These authors suggested that there is an additive or synergistic effect among compounds in the two fractions, or that the deterrent compounds decomposed during the partitioning of the crude extract. In my opinion these results do not demonstrated that defensive chemistry plays a significant role in the unpalatability because toxins contained in nematocysts could also act as a chemical deterrence. If there truly are active chemicals in Eudendrium, the lipophilic and the water-soluble fractions must be clearly deterrent to fish, but this is not the case. Secondary metabolites contained in the discharged nematocysts could be responsible for fish rejection. The biosynthesis of defensive metabolites is costly for organisms. It is not surprising that species showing de novo biosynthesis of defensive metabolites use these metabolites for other purposes. This is the case in the nudibranch Tethys fimbria, which synthesizes prostaglandin derivates (Marı́n, Di Marzo & Cimino, 1991). The structural variety of the lactones and the data on their distribution in the body of T. fimbria suggest a range of different biological functions: to contract smooth muscle fibres, defence allomones of the ceratal secretion, and in basic physiological functions (e.g. ion regulation, renal function and reproductive biology). These arguments do not mean that other aeolidoidean nudibranchs should also contain defensive metabolites. The aeolid nudibranch Phyllodesmium guamensis feeds on the soft corals Sinularia maxima and S. polydactyla. Phyllodesmium guamensis sequesters a diet-derived diterpene 11b-acetoxypukalide selectively within various body parts. Levels were highest in the cerata, with moderate and low to non-existent levels in the mantle and viscera, respectively. Laboratory tests showed that 11b-acetoxypukalide deters feeding by the puffer fish Canthigaster solandri at a given concentration (0.5% dry mass), while feeding field experiments with Correspondence: A. Marin; e-mail: [email protected]