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Home , Chromis chromis, Chromodoris, Discodorididae, Discodoris, Doris verrucosa, Hypselodoris

/. Moll Stud. (1997), 63,431-439 © The Malacological Society of London 1997

MORPHOLOGICAL AND CHEMICAL OF THE MEDITERRANEAN INDECORA ON THE IRCINIA VARIABILIS AND IRCINIA FASCICULATA Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021 A. MARIN1*, M.D. LOPEZ BELLUGA1, G. SCOGNAMIGLIO2 and G. CIMINO2 'Departamento de Ecologia e Hidrologia, Facultad de Biologla, Universidad de Murcia, E-30100 Murcia, Spain. 2Consiglio Nazionale delle Ricerche, Istituto per la Chimica di Molecole di Interesse Biologico, 1-80072-Arco Felice (Na), Naples, Italy (Received 15 March 1996; accepted 27 January 1997)

ABSTRACT supported by recording the induction of feeding- deterrence in the fresh-water fish Carassius auratus The nudibranch Discodoris indecora shows a perfect and in the marine fish chromis and Sparus camouflage on its prey, the dictyoceratid auratus. Ircinia variabilis. Shape and colour of the nudibranch are remarkably similar to that of the sponge which is widespread in the shallow waters of the Mediter- ranean Sea. The metabolite pattern of /. variabilis is INTRODUCTION dominated by unusual molecules, the sesterter- penoids palinurin and variabilin, containing 25 In the evolution of opisthobranch gastropods carbons and displaying at the ends a ^-substituted and especially of nudibranchs, the loss of the furan ring and a tetronic acid. However, a chemical shell has been compensated by the develop- polymorphism has been recently described for /. vari- ment of a wide range of defensive mechanisms. abilis. Some specimens contain either palinurin or Some nudibranchs that feed on cnidarians are variabilin whereas others possess a mixture of both known to store nematocysts within cnidophage sestertepenoids. During the study of Mediterranean cells and use them for their own defence sponges, a linear sesterterpenoid related to palinurin (Thompson, 1960; Greenwood & Mariscal, and variabilin was found in Ircinia fasciculata, that from a taxonomic point of view is closely relatedto 1984). Nudibranchs also retain pigments and Ircinia variabilis. Discodoris indecora shows a typical zooxanthellae from the diet, so they may be defence behaviour when it is disturbed, secreting a camouflaged on the food source (Marin & Ros, copious white slime that contains large amounts of 1991). Certain nudibranchs contain repugnant palinurin and variabilin. Most likely the is able substances often obtained from dietary to transfer the sponge metabolites from the digestive sponges. Harris (1971) suggested that this glands to glands. Electron micrographs of the 'physiological exchange' fits the classical con- nudibranch mantle showed the presence of unicell- cept of symbiotic association. ular glands characterized by a cylindrical vacuole with a big nucleus on the basis. The glands were con- Many chemical studies have been performed centrated in the most conspicuous tubercles of the on Mediterranean dorid nudibranchs. In almost dorsal mantle. Probably, the sponge metabolites are all cases, the diet of sponges has been proved by stored in these glands. the comparison of the metabolic patterns dis- Comparative chemical analysis of mucous secre- played by the nudibranch-sponge pair (Cimino tions, dorsal tubercles, mantle, foot, digestive glands & Sodano, 1994). Nudibranchs belonging to the and hepatopancreas of D. indecora confirmed this Family Chromodoridae ( hypothesis. Palinurin and fasciculation were, of spp., spp and spp.) course, present in the digestive glands but they were display very similar morphological features. also found in the mucus and in the mantle sections However, it seems that the genus Hypselodoris enriched by the above described unicellular glands. spp., with the exception of H. orsini (Cimino, The defensive role of palinurin and variabilin, Fontana, Gimenez, Marin, Mollo, Trivellone & strongly suggested by their anatomical location, was Zubia, 1993), select sponges containing fura- nosesquiterpenoids (Garcfa-G6mez, Cimino & • To whom correipcndcnce ihould be jcnL (Fajc 34-68-363963; E- i kk@f) Medina, 1990; Avila, Cimino, Fontana, Gav- 432 A. MARIN, M.D. LOPEZ BELLUGA, G. SCOGNAMIGLIO & G. CIMINO agnin, Ortea & Trivellone, 1991), whereas the Bergh, 1881 in order to: clarify its feeding genus Chromodoris spp. select sponges possess- habits; investigate the transfer of dietary ing diterpenoids (Molinski, Faulkner, He, Van metabolites to mantle glands; place Discodoris Duyne & Clardy, 1986; Gavagnin, Vardaro, indecora in a hypothetical evolutionary Avila, Cimino & Ortea, 1992) and, finally, scenario of dorid nudibranchs. Cadlina species, possessing a more complex (Andersen, Pika, Dumdei & Burgoyne, 1993), can eat different kinds of sponges. The transfer of the sponge metabolites from the MATERIALS AND METHODS digestive glands to some dorsal formations Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021 (Mantle Dermal Formation) has been previ- Collection of molluscs ously suggested (Avila, Cimino, Fontana, Gav- The nudibranch Discodoris indecora was collected on agnin, Ortea & Trivellone, 1991) but only the sponges lrcinia variabihs (Schulze, 1879) and /. recently has it been rigorously proved fasciculata (Pallas, 1766) which were removed from (Fontana, Gim6nez, Marin, Mollo, Cimino, shallow rocks (1-2 m depth) during April to Septem- 1994). In the case of H. orsirti, the transfer of ber 1993 and 1994 in Spain (Mazarr6n and Torre- the dietary components is also accompanied by vieja, Murcia) and Italy (Naples and Taranto). A population of D. indecora was also found upon /. chemical transformation of the extremely reac- fasciculata. Nudibranchs and sponges were frozen as tive main sponge metabolite, scalaradial, first in soon as possible after collection. Some nudibranchs deoxoscalarin and then in 6-ketodeoxoscalaria were disturbed in order to obtain defensive secre- (Cimino, Fontana, Gimdnez, Marin, Mollo, tions. Trivellone & Zubia, 1993).

The main biological role of these sponge Transmission electron microscopy metabolites sequestered by chromodorid gastropods is the protection against potential Nudibranchs were narcotized in 7.5% MgCl2 and predators. A more complex habit is shown by pieces of mantle were excised and fixed in for 1 h in species (family ), analo- cold (1-4°C) 2.5% Millonig's phosphate-buffered glutaraldehyde (pH 7.2-8.2). Fixation was followed gous to chromodorid gastropods, transfer by a 24 h wash in 2.5% NaHCO and then post-fixed sponge compounds to the mantle and into the 3 in 2% OsO4 in 1.25% NaHCO3 for 1 h. The tissues mucus, whereas spp. is able were dehydrated in an ethanol series and embedded to biosynthesize 'de novo' icthyodeterrent in Epon. Ultrathin sections were stained with uranyl molecules (Cimino, De Rosa, De Stefano, acetate and lead citrate. Sodano & Villani, 1983). Strangely enough, the Mediterranean Doripsilla aereolata contains in its glands the same mixture of Extraction and isolation of metabolites sesquiterpenoids esters found in Dendrodoris, Frozen were carefully dissected to remove but notal metabolites are quite different mantle, foot, digestive gland and reproductive (Okuda, Scheuer, Hochlowski, Walker, & organs. These were extracted with acetone. The Faulkner, 1983). sponges were cut into small pieces and then extracted with acetone. The extraction was repeated three From an evolutionary point of view, dorid times. The solvent was then removed in vacua and the gastropods with notal spicules are situated aqueous residues were extracted with ether and then between non spiculated nudibranchs and the with n-butanol. Each extract was chromatographed ancestral shelled forms. Until now, only a few on silica gel plates (Merck F^) in chloroform- studies have been performed on Mediterranean methanol (95:5). The chromatograms were checked molluscs with notal spicules. verrucosa for fluorescent compounds by exposure under u.v. contains some unusual diacylglycerols, whereas light and furans by spraying with Ehrlich reagent. atromaculata (Bergh, 1880) even Compounds from the crude extract were purified by though its digestive gland contains some silica gel chromatography and structurally elucidated by proton nuclear magnetic resonance ('H-NMR) dietaric polyacetylenes from the sponge Pet- spectrometry. rosia ficiformis, is not able to transfer these compounds to the mantle (Cimino, Gavagnin, Sodano, Puliti, Mattia & Mazzarella, 1988; PaJatabdity and toxicity bwassays Castiello, Cimino, De Rosa, De Stefano & Sodano, 1980). Discodoris indecora is similar to Ichthyotoxicity assays were conducted with the fish Gambusia affinis, Chromis chromis and Spams P. atromaculata, a selective predator of auratus, following the procedure described by sponges. Gunthorpe & Cameron (1987), and using the toxicity We have investigated Discodoris indecora ranking denned by Coll et al. (1982). DEFENCE ALLOMONES OF DISCODORIS INDECORA 433 RESULTS which lies inside the cell (Fig. 2). At the distal tip of the gland, the cytoplasm possesses ovoid Defensive behaviour vacuoles which coalesce towards the cell The body colour of D. indecora is golden- membrane (Fig. 2). In addition, smooth muscle brown with yellow and opaque white pigment fibres pass through and surrounding the con- on the top of many of the mantle tubercles. The nective matrix. mantle is covered with, large, and rounded tubercles. In the field, the foot and body are Allomone distribution close to the sponge where the colour and shape Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021 of the mantles give good camouflage. When Comparison between thin layer chromatogra- D. indecora is handled roughly the dorsal tuber- phies and 'H-MNR spectra carried out with cles discharge a copious opalescent white secre- extracts from Discodoris indecora and fascicu- tion. lation, variabilin and palinurin (Fig. 3) isolated from Ircinia fasciculata turned out be similar (Fig. 4). All dissected nudibranchs contained fasciculatin, palinurin and variabilin or a mix- Transmission electron microscopy ture of variabilin and palinurin. This metabolic The dorsal tubercles originate from an expan- variation was present at both localities studied. sion of connective tissue where unicellular The existence of variabilin and fasciculatin in glands are located. In transverse section, these Discodoris indecora confirms that it feeds on /. cells are narrow and filled with an elongated variabilis and /. fasciculata since these sesterter- central vacuole (Fig. 1). The base of the cells penes only are described in these sponges has a very large and marginal nucleus. The (Cafieri, Fattorusso & Santacroce, 1972). greater part of the basal cytoplasm, surround- Acetone extracts from mucus secretion ing the basal nucleus, is filledwit h small vesicles showed in thin layer chromatography the and sparse endoplasmic reticulum. The central presence of a large quantity of the three sestert- cytoplasm appears to be filledwit h longitudinal erpenes. Composition and concentration vacuoles surrounding the vacuolic channel depended on diet of the nudibranch. The origin

1 Figure 1. Discodoris indecora. 1, Drawing of the notal tubercle structure observed in the electron microscope; 2, Lateral view of Discodoris indecora. Abbreviations: CV, central vacuole; N, nucleus; M, connective tissues; E, epidemis. 43* A. MARIN, M.D. LOPEZ BELLUGA, G. SCOGNAMIGLIO & G. CIMINO Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021

Figure 2. Discodoris indccora. A. Longitudinal section through apex of the dorsal papillae showing unicell- ular glands filled with an empty vacuole (x 3000). B. Longitudinal section through base of the dorsal papillae (x 3000). Abbreviations: CV, central vacuole; DV, apical vesicles; ER, endoplasmic reticulum; F, smooth muscle; M, connective tissues; N, nucleus. of the secretion is apparently in unicellular branch. The acetone extracts from the dorsal glands contained in the dorsal papillae of the papillae, previously isolated from the nudi- mantle. This statement is based on chemical branch, contained large amounts of the analysis of the dissected parts of the nudi- sesquiterterpenes. DEFENCE ALLOMONES OF DISCODORIS INDECORA 435

OH

o Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021

o 2. Variabilin

O 3. Palinurin Figure 3. Sponge metabobtes sequestered by Discodoris indecora.

Palatabilily and toxicity bioassays Discodoris indecora appears to be well-adapted to chemical variability of Ircinia species. These Variabilin was active in the feeding inhibition linear furanosesterterpenes can vary in the bioassay with the fresh water fish Carassius 2 same location and sponge population. The auratus at a concentration of 300 jig.cm~ but it function of the defensive mucus deserves was not toxic to Gambusia affinis at a concen- further investigations. We need to know if the tration of 10 ppm. Antifeedant assays con- individual metabolites have the same chemo- ducted in the marine fishes Chromis chromis tactic effects that the mixture found in the and Sparus auratus induced similar antifeedant 2 defensive mucus. behaviour at 300 ng.crrr as in Carassius aura- tus. Discodoris indecora from the Spanish coasts was always collected with /. variabilis. Of course, knowing the polymorphism of /. vari- abilis, the main sponge metabolite was either DISCUSSION variabilin or palinurin or a mixture of the two sesterterpenoids. Generally, the metabolite The presence of variabilin and palinurin in D. pattern of the gastropod was identical to that of indecora confirms the nudibranch-sponge asso- the associated sponge but sometimes both ciation. Three species of the genus Ircinia have sesterterpenoids were found in a nudibranch been recorded in the with a living upon a sponge containing only one similar structural and chemical pattern: Ircinia sesterterpenoid. This proves the ability of the oros, I. variabilis, and /. fasciculata. However, nudibranch to move from a specimen of /. vari- the furanoid sesterterpenes fasciculatin and abilis to another. variabilin have only been isolated from the Specimens of D. indecora maintained in sponge /. fasciculata and /. variabilis respec- aquaria were observed grazing upon Ircinia tively (Cafieri, Fattomsso & Santacroce, 1972). variabilis. While in the aquarium this prey- 436 A. MARIN, M.D. LOPEZ BELLUGA, G. SCOGNAMIGLIO & G. CIMINO Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021

ppm

ppm l ppm Figure 4. Comparative 'H-NMR spectra of Ircinia and Discodoris indecora. 'H-NMR spectra of palinurin from Discododis indecora (a) and Ircinia variabilis (c); 'H-NMR spectra of variabilin from the nudibranch (b) and the sponge (d). predator relationship was clear, it was not sponge's. To clarify whether these metabolites observed in the field. The sponges collected can play a defensive role, the mucous secretion with the nudibranch in the field did not show was chemically analyzed. Every population of external signs of grazing activity. In this respect, D. indecora secreted either fasciculatin or it is interesting that Discodoris indecora grazes variabilin or a mixture of fasciculatin and vari- exclusively in the inner part of the sponge. This abilin according to the chemical composition of feeding behaviour may be associated to the the digestive glands. The ichthyodeterrence of cryptic coloration of this species on the genus fasciculatin and variabilin was characterized by Ircinia. The nudibranch's camouflage could not laboratory bioassays. be functional upon grazed patches because the The taxonomic distribution of chemical brown colour of the sponge is due to presence defence in dorid nudibranchs suggests that the of symbiotic cyanophyceae located in the early adaptive radiation of the suborder ectosomal layer of the sponge; the tissues involved toxic resistance to chemical defence of without cyanophyceae are whitish-yellow. This sponges (Fig. 5). The ancestral dorid possibly behaviour could imply mutalistic relationship found an alternative means of protection in terms of the persistence of the predator-prey because the storage of tissues of the sponge association in the way that the nudibranch gains with toxic metabolites within the digestive a cryptic substratum and the sponge maintains gland probably reduced predation. The dorids external surface photosynthetically active. subsequently evolved a variety of dorsal glands Cattaneo-Vietti et al. (1990) indicate that with the development of an efficient mecha- Discodoris indecora species are always located nism for the separation, mobilization, storage on shallow rocky bottoms. This bathymetric and delivery of the harmful sponge metabolites distribution is due to the symbiotic association (Faulkner & Ghiselin, 1983). Yet another stage of the sponge Ircinia fasciculata and /. variabilis in the evolution of chemical defence has been with the cyanobacterial symbionts (Neveux et described in Hypselodoris webbi and H. vil- al. 1988). Light limits the top depth where the lafranca (Garcfa-G6mez et al. 1990; Avila, algal symbiont can live and thus indirectly the Cimino, Fontana, Gavagnin, Ortea, Trivellone, DEFENCE ALLOMONES OF DISCODORIS INDECORA 437

Dendrodoris origin; bfosyntfiesfeed" Doripsllla ; bJotrpnsfofmotion of

teontfrtfes orfgN host" sponge? Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021

sesterterpendfcfe ; origin; host sponges

Doris verrucosa Piatydoris argo cte n<>vo- Peltodoris afromacutafa apongiyofous nudfcffachs wtthout clefensfve gtanas origin: host sponges*

Figure 5. Defense mechanisms and evolutionary ecology of Mediterranean dorid nudibranchs. MDFs, mantle dermal formations.

1991), which accumulate longifolin in mantle ability of food in Discodoris indecora could dermal formations (MDFs) (Fig. 5). The mantle make unnecessary the presence of the mantle dermal formations are globular structures dermal formations as in Hypselodoris spp. located in the ridge of the mantle. Ultrastruc- However, a nudibranch with a food-derived tural observations of mantle dermal formations defensive chemical is dependent upon its food show an accumulation of large vacuolar cells source. A solution to this problem has been not contiguous to the mantle surface. It is also resolved by Dendrodoris limbata, which synthe- noteworthy that these Hypselodoris species of sizes its own chemical defence (Cimino, De aposematic coloration are with rare exception Rosa, De Stefano, De Sodano, Villani, 1983). found in the field upon their food. Hypselodoris The more primitive defensive strategy of the webbi and H. villafranca feed on Dysidea fragis, family Discodoridae may be represented by a sponge of small size with usually a good dis- that only stores a tance between specimens in the field. Apose- mixture of acetylenes from the sponge Petrosia matic coloration and presence of structures for ficiformis in its digestive gland (Castiello et al., storage of defensive chemicals, as mantle der- 1980). Furthermore, the of P. romac- mal formations, may be essential when the ulata notum shows blunt tubercles supported by nudibranch must wander to find new sponges. densely packed calcareous spicules covered by Nudibranch may find advantageous to store the epithelium, but it does not contain defen- chemicals in the mantle dermal formations for sive glands (Fig. 5). In alarm this nudibranch in long periods, to compensate for prolonged alarm could eject digestive contents as a defen- separation from food. Thus, a constant avail- sive secretion. Thus, Discodoris indecora with 438 A. MARIN, M.D. LOPEZ BELLUGA, G. SCOGNAMIGLIO & G. CIMINO defensive dorsal glands and with reduced Peltodoris atromaculata and the sponge Petrosia mechanical defences may be a derived form of ficiformis. Tetrahedrom Letters, 21:5047-5050. the family (Fig. 5). CATTANEO-VIETTI, R., CHEMELLO, R. & GIANNUZZI- A few conclusions may be drawn from the SAVELLI, R. 1991. Atlas of Mediterranean nudi- branchs. Editriche La Conchiglia. Roma. present study. Discodoris indecora shows CIMINO, G., CRISPINO, A. DE STEFANO, S., morphological and chemical camouflage on GAVAGNIN, M. & SODANO, G. 1986. A naturally- several sponges of the genus lrcinia with a occurring analog of methylthioadenosine (MTA) similar structural and chemical pattern. The from the nudibranch mollusc . nudibranch feeds upon these sponges that con- Experientia, 42:1301-1302. tain antifeedant metabolites which are stored CIMINO, G., DE ROSA, S., DE STEFANO, S., DE Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021 within dorsal cells of the mollusc and used for SODANO, G. & VILLANI, G. 1983. Dorid nudibranch their own defence. Defensive glands are elaborates its own chemical defence. Science, 219: restricted to dorsal tubercles where allomones 1237-1238. are stored within unicellular cells. The white CIMINO, G., FONTANA, A., GIMENEZ, F., MARIN, A., slime released in alarm from dorsal tubercles MOLLO, E., TRIVELLONE, E. & ZUBIA, E. 1993. Bio- contains antifeedant allomones similar to those transformation of a dietary sesterpenoid in the Mediterranean nudibranch Hypselodoris orsini. previously described from the sponges that it Experientia, 49: 582-586. feeds on. CIMINO, G., GAVAGNIN, M., SODANO, G., PULITI, R., MATTIA, A. & MAZZARELLA, L. 1988. Verrucosin- A and -B, ichthyotoxic diterpenoic acic glycerides with a new carbon skeleton from the dorid nudi- ACKNOWLEDGEMENTS branch Doris verrucosa. Tetrahedron, 44: 2301- 2310. The authors are grateful to Prof. M. Garcfa for tech- CIMINO, G. & SODANO, G. 1989. The chemical ecol- nical assistance (Servicio Microscopfa Electronica, ogy of Mediterranean opisthobranchs. Chemica Universidad de Murcia). Scripta, 29:389-394. CIMINO, G. & SODANO, G. 1994. Transfer of sponge secondary metabolites to predators. In: Sponges in REFERENCES Time and Space (W.M. van Soest, O.M.G. van Kempen & J.C. Braekman eds.), 459-472. Proceed- ALFANO, G., CIMINO, G. & DE STEFANO, S. 1979. Pal- ings of the 4th international Porifera congress. inurin, a new linear sesterterpene from a marine Amsterdam. Netherland. sponge. Experientia, 35:1136-1137. 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Defensive adaptations in MOLINSKI, T.F., FAULKNER, DJ., HE, C.H., VAN opisthobranchs. Journal of the Marine Biological DUYNE, G.D. & CLARDY, J. 1986. Three new rear- Association of the U.K., 39:123-134. Downloaded from https://academic.oup.com/mollus/article/63/3/431/1073528 by guest on 24 September 2021