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Coloration and defense in the nudibranch gastropod Hypselodoris fontandraui.

Introduction

Although much controversy remains over the theory of warning signals, the term "aposematism," first used by Poulton (1890), is still widely defined as it was originally proposed--to describe the association between easily detectable conspicuous signals and unprofitability (Wallace, 1867; Darwin, 1871; Poulton, 1890; Cott, 1940; Merilaita and Kaitala, 2002; Mappes et al, 2005). In addition, the similarity of color patterns in groups of species is generally explained in evolutionary terms by the assumption that the predators act as selective agents in promoting similarities between these species (Bates, 1862; Muller, 1879; Fisher, 1930). According to this concept, predators learn to avoid unpalatable prey that use conspicuous signals, and then generalize this avoidance to similar color patterns (Lind-strom et al., 2004). This defensive mechanism in which groups of similarly colored species share the cost of the education of predators is called Mullerian mimicry (Muller, 1879) and it implies aposematism, whereas Batesian mimicry occurs in otherwise undefended species that rely on resembling the unpalatable ones (Bates, 1862).

Edmunds (1991) argued that one or both types of mimicry frequently occur in similarly colored nudibranch species, suggesting that this seems to be the strongest support for aposematism in this group of molluscs, because the widespread occurrence of mimicry in those species is almost impossible to explain unless some of them are aposematic. The existence of a Mullerian mimetic circle is particularly manifest for the group of several blue, white, and yellow Mediterranean and northeastern Atlantic species of Hypselodoris (Ros, 1976, 1977), but it is still not clear whether aposematism occurs in all members of this mimetic group. Virtually all Hypselodoris nudibranchs, as members of the family Chromodorididae, have special glands termed mantle dermal formations (MDFs) lying in the mantle rim, and storage of the food-derived defensive metabolites in the MDFs has been shown in several cases (e.g., Garcia-Gomez et al, 1990; Avila et al, 1991; Cimino et al, 1993; Fontana et al, 1993, 1994a; Avila and Durfort, 1996; Gosliner, 2001; Wagele and Klussmann-Kolb, 2005). This led to the general notion of MDFs as defensive glands (Gosliner, 2001; Wagele, 2004). Interestingly, a few exceptions diverge from the above generalization within the genus Hypselodoris, and this could suggest that they are not chemically defended. Gosliner and Johnson (1999) indicated Hypselodoris sagamiensis (Baba, 1949) and H. fontandraui (Pruvot-Fol, 1951) as the only known Hypselodoris species that entirely lack mantle glands (Garcia-Gomez et al., 1991; Baba, 1995), while Wagele et al. (2006) recently indicated their absence in five species (including the previous two) among 60 examined Hypselodoris species.

In the present work, we studied one of these species, Hypselodoris fontandraui, collected along Portuguese coasts (Fig. la). The lack of MDFs clearly distinguishes H. fontandraui from the co-occurring Mediterranean-Atlantic members of the blue chromatic Hypselodoris group--such as H. villafranca (Risso, 1818) (Fig. lb), H. cantabrica (Bouchet and Ortea, 1980) (Fig. lc), H. picta (Schultz, 1836) (Fig. Id), and H. tricolor (Cantraine, 1835) (Ortea et al, 1996; Gosliner and Johnson. 1999)--for which the presence of food-derived, feeding-deterrent terpenes in the MDFs underlying aposematism has been demonstrated (Cimino et al, 1982; Avila et al., 1991; Fontana et al, 1993, 1994b). With its blue body and white and yellow longitudinal stripes, H. fontandraui is very conspicuous and highly similar to the chemically defended species mentioned above (Fig. 1), but so far no experimental evidence exists that it also has a chemical defense. An earlier chemical study of H. fontandraui (cited in Avila, 1995) detected furanosesqui-terpenoids and suggested a dietary origin from the prey sponge Dysidea avara (Schmidt, 1862). However, the compounds were not identified, and no experimental tests of the pure compounds were performed to address their involvement in chemical defense. Therefore, we aim to determine whether H. fontandraui is truly aposematic and can be considered a member of the proposed Mullerian circle of the blue chromatic Mediterranean and Atlantic Hypselodoris species or, on the contrary, might represent a case of Batesian mimicry, as could be suspected by the absence of MDFs.

Materials and Methods

Sample collection

Eight specimens of the nudibranch Hypselodoris fontandraui (average size 20 mm) were collected by scuba diving during October 2007 along the western Portuguese coast, where other blue chromatic Hypselodoris nudibranchs also coexist (Cervera et al, 2006). All single specimens were identified by one of us (G. Calado). A voucher specimen (preserved in absolute ethanol) was deposited at "Institute Portugues de Malacologia," Portugal. An additional individual was found on a sponge of the genus Dysidea (Fig. le). In the latter case, the animal, its mucous secretion, and a fragment of the sponge were separately stored. All samples were frozen immediately after collection and stored at - 80[degrees]C until their chemical or histological analysis.

[FIGURE 1 OMITTED]

Chemical investigation

Three frozen specimens of H. fontandraui with their mucous secretion were placed in a mortar, immersed in acetone, and crumbled with a pestle. Subsequently, the mixture was treated with ultrasound vibration (1 min). The solvent was removed by filtration, and the extraction of the tissue was repeated four times exhaustively. After concentration, the extract was diluted with water and extracted with diethyl ether. Analysis of the extract by thin layer chromatography, carried out on pre-coated Merck silica gel F254 plates (n-hexane/diethyl ether in different ratios as eluent), showed the presence of a main UV-positive metabolite, which was also positive to Ehrlich's reagent. Subsequently, the extract was chromatographed on a silica gel column (Merck Kieselgel 60 powder) packed with n-hexane and eluted with a gradient of n-hexane/diethyl ether to give a pure colorless oil (10.1 mg), afterward identified by [.sup.1]H-and [.sup.13]C-NMR (proton and carbon nuclear magnetic resonance), and MS (mass spectrometry) analysis as the known furnosesquiterpene tavacpallescensin (Guella et al., 1985). The same procedure has been used to obtain the crude extract from both the putative prey sponge (Fig. le) and the mucus of the nudibranch found on it, which were directly analyzed by [.sup.1]H-NMR. [.sup.1]H-NMR spectra were acquired in CDC13 and C6D6 on a Bruker Avance 400 MHz and a Bruker DRX-600 MHz. [.sup.13]C-NMR spectra were recorded on a Bruker PDX-300 MHz and AMX-300 MHz. EIMS (electron impact mass spectra) were determined at 70 eV on a HP-GC 5890 series II mass spectrometer.

Anatomical distribution of tavacpallescensin

Three specimens of H. fontandraui were separately dissected into three parts: mantle border, residual external parts, and inner organs. The volume of each part was measured for each individual and extracted as described above. The chloroform-soluble part of all extracts was separately subject to [.sup.1]H-NMR quantification by adding a known amount of dimethylfumarate (Sigma Aldrich, Germany) as an internal standard. [.sup.1]H-NMR spectra were recorded on a Bruker Avance 400 MHz. The dimethylfumarate signals at [delta] 6.86 resulting from two protons and the signals of tavacpallescensin at [delta] 6.11 resulting from one proton were used for integration by using the Bruker software package XWIN-NMR 3.6 (Bruker, BioSpin GmbH, Germany). Natural volumetric concentrations were determined by dividing the calculated amount of tavacpallescensin by the respective tissue volume (mg/ml). Mean and standard error were calculated for each tissue part, and significance in distributional differences was calculated using a one-way ANOVA and the Tukey post hoc test as implemented in SPSS 15.0.1.

Feeding-deterrence assay

Tavacpallescensin was tested for its feeding-deterrence activity against the shrimp Palaemon elegans (Rathke, 1837). This generalist predator is widely distributed and occurs on the European coast of the Atlantic, the North and Baltic seas, as well as in the Mediterranean Sea (Janas et al, 2004). The shrimp occurs sympatrically with H. fontandraui in its distribution area (Ortea et al, 1996) and might there- fore be considered a potential natural predator of the nudibranch. Assays were performed as described in Mollo et al. (2008), by using food pellets treated with tavacpallescensin at concentrations of 0.5, 1.0, 1.5, and 3.5 mg/ml. Tavacpallescensin was dissolved with acetone to give twice the desired concentration, and 0.5 ml of each obtained solution was added to a mixture of 50 mg ground-lyophilized squid mantle, 30 mg alginate, 30 mg purified sea sand (granular size 0.1-0.3 mm), and mixed. After evaporation of the solvent, distilled water and a drop of red food color (El10 and E124) were added to give a final volume of 1.0 ml. The paste was mixed and exuded into a 0.25 mol [l.sup.-1] [CaCl.sub.2] solution with a syringe. After 2 min, the hardened strand was briefly rinsed in seawater and cut into 10-mm-long strips. Accordingly, controls were prepared with acetone only. Fifty individuals of P. elegans were available for the experiments. The shrimps were collected along the coast of Pozzuoli, Italy, and habituated to the control food in a 50-1 aquarium for a week before experiments. For each concentration and the control, 10 randomly chosen shrimps were assayed as a series of individual replicates (five series in total). Control and treatments were carried out in parallel. Shrimps were placed individually into 500-ml plastic beakers filled with 300 ml of seawater. Just one colored food strip was given to each shrimp, and shrimps were not re-used. The almost transparent exoskeleton of the shrimps enabled us to easily detect the occurrence of the red food in the gastric mill and the stomach of the shrimps. The presence of a red spot in the stomach after 30 min was considered as acceptance of food, while the absence of the spot was considered a rejection response. Statistical analysis between treatments and controls was performed using the two-tailed Fisher exact test with P < 0.05 regarded as a significant difference.

Histology

One frozen specimen was immersed in 4% paraformaldehyde (pH 7.4) and fixed overnight. Afterward it was dehydrated for 24 h using 70% ethanol followed by increasing grades of ethanol. Subsequently, the ethanol was eliminated by xylol and the tissue was imbedded in paraffin under vacuum. The imbedded tissue was cut to 6-[micro]m sections with a microtome. Sections were spread on glass slides. After removal of paraffin by xylol, the sections were hydrated using decreasing grades of ethanol, stained with toluidine blue, and then observed under a light microscope.

Results

Tavacpallescensin identification

Nuclear magnetic resonance (NMR) and mass spectrometry (MS) data of the compound purified from crude diethyl ether extract (10.1 mg) obtained from three individuals of Hypeselodoris fontandraui were identical to those previously published for the furanosesesquiterpene tavacpallescensin (Fig. 2a) of both natural and synthetic origin (Guella et al., 1985; Ho and Lin, 1999). Furthermore, [.sup.1]H-NMR analyses on crude extracts from both the prey sponge (Fig. 2b) and the nudibranch mucus (Fig. 2c) clearly showed the presence of tavacpallescensin as the main metabolite.

[FIGURE 2 OMITTED]

Tissue distribution of tavacpallescensin in H. fontandraui

The [.sup.1]H-NMR quantification of tavacpallescensin in different mollusc parts showed that the compound is highly concentrated along the border of the mantle (25.98 mg/ml [+ or -] 1.41 S.E.), where mean volumetric concentration was about 4 times higher than in the remaining external parts (6.58 mg/ml [+ or -] 0.40 S.E.), including residual mantle and foot, and more than 20 times higher than in the inner organs (1.24 mg/ml [+ or -] 0.09 S.E.) (Fig. 3). One-way ANOVA analysis showed a very significant difference between the tissues (F = 78.715, df 2. P < 0.001) due to the difference between the mantle border and each of the other two tissues (each Tukey post hoc test, P < 0.001), but no significant difference was detected between the remaining external part and the inner organs, possibly due to the low sample size as a trend was visible (Tukey post hoc lest, P = 0.093).

[FIGURE 3 OMITTED]

Dose-response relationship in feeding deterrence

A minimum dose effect was observed at a concentration of 1.0 mg/ml of tavacpallescensin, which significantly deterred the shrimp Palaemon elegans from feeding, while a very highly significant rejection response was obtained when testing a concentration of 3.5 mg/ml of tavacpallescensin in the food (Fig. 4).

Mantle border anatomy

Even though light microscopy did not allow us to detect the presence of mantle dermal formations along the mantle border of the frozen individuals, it revealed the presence of an elongated, opaque structure along the mantle border at either side of the body. Transverse histological sections indicated an oval structure of about 0.8 mm in diameter containing granular material, brightly stained by toluidine blue, and enclosed by a thin membrane. Even though it is impossible to see the tissues properly in all sagittal serial sections, most probably because the only specimen used was fixed after it had been frozen, the above structures seem to appear discontinuously under the mantle rim.

Discussion

The furanosesesquiterpenoid tavacpallescensin, first isolated from a mixture of Mediterranean sponges (Guella et al, 1985), was detected in the chromododorid nudibranch Hypselodoris fontandraui (Fig. la). Although chemical defenses are typically multicomponent mixtures of diverse chemicals in other opisthobranchs, such as sea hares (Derby, 2007), in the case of H. fontandraui, tavacpallescensin represents the predominant lipophilic component in the extracts from the most exposed parts of the nudibranch body and in its mucous secretion (Fig. 2). The presence of tavacpallescensin both in the Dysidea sponge, on which one individual was found (Fig. le), and in the inner organs of the nudibranch, including the digestive apparatus, strongly suggests the dietary origin of the compound in the mollusc.

[FIGURE 4 OMITTED]

Different species of the genus Dysidea contain a variety of sesquiterpenoids, many of which are used defensively by quite a number of nudibranchs (Cimino and Ghiselin, 2009), and the available experimental evidence indicates that the Dysidea sponges biosynthesize the terpenoids. Variation patterns are of some interest. According to "Faulkner's rule," species that obtain their metabolites from food vary geographically, whereas those that biosynthesize them do not (Faulkner et al, 1990). Some "exceptions'' to the rule may be due to inadequate taxonomy. In a study like ours, failure to detect a particular metabolite in a sponge or a nudibranch would be mere negative evidence, and of minor interest where positive evidence is at hand. Although coincidence is a logical possibility, the co-occurrence of a particular furanosesquiterpenoid in both the sponge and a nudibranch upon which it has been found seems unlikely.

Our results seem to conflict with a previous report by Avila (1995) of unknown furanosesquiterpenes in H. fontandraui and its proposed sponge prey Dysidea avara because of the absence of avarol, the "chemical marker" of D. avara (Muller et al., 1986), in the extracts of both the nudibranch and the sponge here analyzed. It remains uncertain whether H. fontandraui is a monophagous species or can prey on a variety of sponges, presumably of the genus Dysidea (a known source of furanosesquiterpenoids), which could lead to the prey-dependent uptake of different furanosesquiterpenoids, as has been demonstrated for the congeneric H. picta (as H. webbi (d'Orbigny, 1839) in Fontana et al., 1994a).

In the present study, the sponge-derived compound tavacpallescensin is accumulated mainly in the outer part of the nudibranch, and especially in the mantle border, where the concentration is more than 20 times higher than in the internal part and almost 4 times higher than in the rest of the external part of the nudibranch's body. Analysis by light microscopy confirmed the previously noted absence of mantle dermal formations, but led to the observation of an elongated, opaque structure on both sides of the nudibranch body. Because the preparation was poor, preliminary histological study of the mantle border did not allow us to unambiguously ascertain the existence of reservoirs of the compound in the mantle border (Fig. 5). Nevertheless, the study led us to detect structures, oval in transversal section (Fig. 5C), containing granular parts that are included in the body wall and located immediately under the mantle border. They appeared discontinuously in the sections under the mantle rim. The high concentration of tavacpallescensin in the section of the wall including the mantle border would be explained if these structures represent accumulation reservoirs of the terpene, but further histological studies and electronic microscopy, preferably on freshly fixed samples to avoid tissue disruption, are needed to confirm this hypothesis.

[FIGURE 5 OMITTED]

Feeding experiments conducted on a generalist shrimp led us to propose that the extremely high concentration of the compound in the mantle border of H. fontandraui, which is the body part most exposed to predator attacks, is related to a mechanism of chemical defense. As in other cases among the family Chromodorididae (e.g., Mollo et al., 2005), the presence of the feeding-deterrent compound in the mucous secretion of H. fontandraui could be related to a first line of chemical defense that might act by giving the animal an unpleasant taste, while the extremely high concentration of tavacpallescensin in the mantle border should work as the last chance if the most exposed animal tissues were wounded by the pincers, claws, or teeth of a predator. In fact, even when artificial food was significantly unpalatable, starting from the minimum effective dose of 1.0 mg/ml of tavacpallescensin, it is noteworthy that in the border of the mantle the concentration of the compound (25.98 mg/ml [+ or -] 1.41 S.E.) considerably exceeds the threshold value of 3.5 mg/ml that showed very high significant differences in rejection rate compared to control food in the bioassays. This is in concordance with previous data on chromodorid nudibranchs, including Mediterranean-Atlantic Hypselodoris species, for which an accumulation of food-derived feeding-deterrent furanosesesquiterpenes in the mantle dermal formations has been suggested (Avila et al., 1991; Fontana et al., 1993, 1994a, b; Cimino and Ghiselin, 1999; Mollo et al., 2005). In particular, data on six compounds from three northeastern Atlantic blue chromatic Hypselodoris species suggest the feeding deterrence of the compounds to be in the same range found for the freshwater goldfish Carassius auratus (Linnaeus, 1758), but one compound was 10-fold more active. The same study showed that only two of five tested compounds were toxic to the freshwater mosquitofish Gambusia affinis (Baird and Girard, 1853) (Fontana et al., 1993). The differences in feeding deterrence reported in the literature for various metabolites from nudibranchs are in accordance with the concept that Batesian and Mullerian mimicry are two extreme ends of a continuum and that the species within a Mullerian mimetic circle might vary in their deterrent potential (Mallet, 1999; Balogh et al., 2008; and references therein). Even though the ecological relevance of the reported chemoecological data is questionable, since the tests were performed neither against co-occurring predators (Wagele et al., 2006) nor with statistical evaluation of the results or reliable quantification of the compounds in the animal, the cited studies are widely accepted as evidence of chemical defense and discussed in evolutionary terms (Cimino and Ghiselin, 1998, 1999, 2001). In this report, we used quantitative NMR on crude extracts to avoid degradation and loss of compounds, which often occurs during chromatography and leads to unreliable quantifications.

Terpenoids are known to be effective in deterring a wide range of grazers and predators. Here we evaluated the activity of tavacpallescensin as a feeding deterrent, using the co-occurring marine decapod Palaemon elegans This shrimp ate fresh squid strips when offered them in the field (E.M., unpubl. obs.), and other co-occurring palaemonids are known to feed on molluscs, including gastropods (Guerao, 1993; Guerao and Ribera, 1996). The generally high visual sensitivity of marine crustaceans to blue-green wavelengths (Johnson et al., 2002) strongly supports the ability of this model predator to recognize the blue color of H. fontandraui and similarly colored species. Furthermore, taking into account that aposematic coloration works only against visual predators with a learning ability, such as fishes (Gimcnez-Casalduero et al., 1999), it is noteworthy that crustaceans are also known to be able to detect and process signals by both chemosensory and visual systems (Wight et al., 1990; Johnson et al., 2002; Barr et al., 2008; Derby and Sorensen, 2008). In particular, decapods exhibit complex learning ability, and there is good evidence for memory lasting several days (Tomsic et al., 1996; Feld et al., 2005; Gherardi and Atema, 2005). Our feeding experiments unambiguously proved that pure tavacpallescensin, identified by NMR and mass spectroscopy, significantly deters, in a dose-dependent manner, the shrimps from feeding. Consequently, this secondary metabolite is one of many diet-derived furanosesquiterpenoids that have been implicated in nudibranch chemical defense (Cimino and Ghiselin, 2009). Derby and Sorensen (2008) emphasized the importance of the identification of the chemical cues and signals for a better understanding of how crustaceans process anti-feeding compounds. In this respect, our results would represent the starting point for future investigation on neural processing and perception of a defined feeding-deterrent stimulus in crustaceans.

In conclusion, our results imply that H. fontandraui is chemically defended in much the same way as its aposematic co-occurring and blue-colored congeners of a proposed Mullerian mimetic circle and that it does not rely on Batesian mimicry. In view of the monophyletic origin of this group of sympatric Hypselodoris nudibranchs, the similar color pattern and their aposematism may have been inherited from their common ancestor and therefore not the result of convergent evolution. The retention of the ancestral state would then seem to be maintained by stabilizing selection due to the advantages of Mullerian mimicry (Gosliner and Johnson, 1999; Gosliner, 2001) and the selective advantage of maintaining a broad search image for recognition by visual predators. As previously suggested, the selective pressure exerted by predators that do not use visual sense--for example, echinoderms or such molluscs as predatory opisthobranchs--might explain the apparent absence of Batesian mimicry in nudibranchs (Tullrot and Sundberg, 1991; Avila, 1995; Wagele, 2004).

Acknowledgments

We thank the staff of the INETI and ICB MS and NMR services. We also thank R. Coelho and G. Villani for providing the photos of Hypselodoris picta and H. fontandraui, respectively. M. H. acknowledges financial support by the EU RTN Marie-Curie action FP6, BIOCAPITAL, contract no. MRTN-CT- 2004-512301. The work has been partially funded by the Italian-Portuguese bilateral project (FCT/ CNR) and the Portuguese research project PDCT/MAR/65854/2006.

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Received 23 October 2009; accepted 25 January 2010.

* To whom correspondence should be addressed. E-mail: emollo@iemib.na.cnr.it

Abbreviations: MDF, mantle dermal formation.

MARKUS HABER (1), (2), SONIA CERFEDA (1), MARIANNA CARBONE (1), GONCALO CALADO (3,4), HELENA GASPAR (5), RICARDO NEVES (4), VEERAMANI MAHARAJAN (6), GUIDO CIMINO (1), MARGHERITA GAVAGNIN (1), MICHAEL T. GHISELIN (7), ERNESTO MOLLO (1), *

(1) Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy; (2) Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel; (3) Centro de Modelacao Ecologica IMAR, FCT/UNL, Quinta da Torre, 2825-114 Monte da Caparica, Portugal; (4) Universidade Lusofona de Humanidades e Tecnologias, A. Do Campo Grande, 376 1749-024 Lisboa, Portugal; (5) Institute Nacional de Engenharia, Tecnologia e Inovacao (INETI), Estrada do Paco do Lumiar, Edificio F, 1649-038 Lisboa, Portugal; (6) Istituto di Cibernetica "Eduardo Caianiello", CNR, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy; and (7) Department of Invertebrate Zoology, California Academy of Sciences, 55 Concourse. Drive, San Francisco, California 94118
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Author:Haber, Markus; Cerfeda, Sonia; Carbone, Marianna; Calado, Goncalo; Gaspar, Helena; Neves, Ricardo; M
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:7ISRA
Date:Apr 1, 2010
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