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Zeitschrift/Journal: Bonn zoological Bulletin - früher Bonner Zoologische Beiträge.

Jahr/Year: 2007

Band/Volume: 55

Autor(en)/Author(s): Affeld Sven, Wägele Heike, Avila Conxita, Kehraus Stefan, König Gabriele M.

Artikel/Article: Distribution of homarine in some Opisthobranchia (: ) 181-190 © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

Bonner zoologische Beiträge Band 55 (2006) Heft 3/4 Seiten 181-190 Bonn, November 2007

Distribution of homarine in some Opisthobranciiia (Gastropoda: Mollusca)*

Sven Affeld"-2), Heike Wägele-*-''*, Conxita Avila^), Stefan Kehraus'* & Gabriele M. König"

' 'Institut fíir Pharmazeutische Biologie, Universität Bonn, Bonn, Germany -Institut ñir Evolutionsbiologie und Ökologie, Universität Bonn, Bonn, Gemiany -^Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Bonn, Germany ^Dept. of Biology (Invertebrates), Facultat de Biología, Universität de Barcelona, Barcelona, Spain

*Paper presented to the 2nd hitemational Workshop on Opisthobranchia, ZFMK, Bonn, Germany, September 20th to 22nd, 2006

Abstract. Homarine, a zwitterionic natural product, is only known from some marine invertebrates, including the opi-

sthobranch Phestilla luguhris (Klein 1986 in Karuso 1987). This natural product is described as having antipredatory and antifouling effects, hi the current study we present new results on the distribution of homarine in Opisthobranchia

and in a few of their food organisms. Our results indicate that homarine is mainly found in members of the Cladobran-

chia (with one exception) and is probably derived from cuidarían food sources. Other investigated opisthobranch ta.xa,

which feed on a variety of other marine organisms (algae, Porifera, Bryozoa etc.), lack homarine. An exception is the ce- phalaspid Aglaja tricolorata in which homarine was detected, and foraging of this predatory slug on homarine contai-

ning cladobranchs seems likely. It is shown in blainvillea that the characteristic glandular cells in the epider- mis of the Dendronotoidea are not adaptations to store homarine.

Keywords. Natural products, Cnidaria, food organisms, ,'Mí7n"oH/« hlaiiivillea. nistolOTy.

1. INTRODUCTION

A wide variety of natural products is known from Opistho- clinical trial (phase II) against hepatocellular carcinoma, branchia (Karuso 1987; Avila 1995; Cimino et al. 1999; non-small cell lung cancer and melanoma (Hamann

CiMiNO et al. 2001; Cimino & Gavagnin 2006; Wägele 2004). In some cases these sequestered chemicals are bio- et al. 2006). Some of the opisthobranchs are of particular transformed by the slugs. Thus Mediten'anean Oxynoe oli- interest due to their extreme toxicity and the high struc- vácea, Lobiger serraclifalci and Ascobulla fragilis^ all of tural diversity of their secondary metabolite content. which feeding on Caiilerpa prolifera, take up the algal

sesquiteipenoid caulerpenyne and modify it into oxytox-

One of these groups is the algae- or cyanobacteria-feed- in-1 and -2, which have an enhanced deterrent effect ing taxon Anaspidea, which store a high diversity of sec- against fish compared to caulerpenyne (Cimino et al. 1 990; ondary metabolites. Apai1 from terpenes, which are the Gavagnin etal. 1994a). According to Cimino & Ghiselin major structural class in Anaspidea, bioactive polyketides (1998), Cimino et al. (1999) and" Marin & Ros (2004) and peptides were also found (Carefoot 1987; Yamada highly derived sacoglossans are able to fonn their defen-

& KiGOSHi 1997; Cimino et al. 2001). One of these pep- sive compounds de novo. E.g. Gavagnin et al. (1994b) tides, i.e. dolastatin 10, showed potent cytotoxic activity. showed that the polypropionate elysione is produced by

In clinical studies (phase II), however it had no effect nei- Elysia viridis itself. ther against advanced colorectal cancer (Saad et al. 2002), advanced pancreaticobiliaiy cancers (Kindler et al. Within the Nudibianchia, the sponge-feeding Doridoidea

2005), nor advanced breast cancer (Perez et al. 2005). are probably the group among the Opisthobranchia best examined for the presence of secondaiy metabolites, most The Sacoglossa, which mainly feed on algae of the ta- of which are dietary derived teipenes (see e.g. the reviews xon Chlorophyta, obtain secondaiy metabolites from their Karuso etal. 1987;Avila 1995; Cimino et al. 1999; Gav- food. One of these compounds, the depsipeptide kahalalide agnin & Fontana 2000; Garson 2006; Wahidullah et

F that Elysia nifescens takes up from its food alga Biy- al. 2006; Miyamoto 2006). De novo biosynthesis, how- opsis spec, and algal associated bacteria is currently in a ever, has also been shown, e.g., for the sesquiterpenes © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

182 Sven Arfeld et al.: Homarine in Opisthobranchia

polygodial in Dendwdoris limbata (Cimino et al. 1983) its preferred prey, the soft coral Sinularia maxima (Slat- and (?/?r-pallescensin A in Dunopsilla areolata (Gavagnin tery et al. 1998). Only very few records for other com- et al. 2001). pounds are available. Ciavatta et al. (1996) discovered

two prenylchromanols ( 1 and 2) and a prenyl-p-hydroxy In contrast, the taxon Cladobianchia has been acid in the skin of Cratena peregrina. None of these com- poorly investigated chemically. Traditionially, Clado- pounds was detected in the prefeired food of C. peregri- branchia are divided into three taxa, the monophyletic Ae- na, namely the hydrozoan Eiidendriuin racemosiim. On olidoidea and Dendronotoidea, and the paraphyletic the other hand, other metabolites, usually not considered Anninoidea (see Wägele & Willan 2000). Members of as deterrent to predators have been isolated from Euden- the Cladobranchia mainly feed on octocorals and hydro- driiiin and its aeolid predators: carotenoids from Flabel- zoans (McDonald & Nybakken 1997). Within the Anni- lina iodinea (McBeth 1972), sterols from Cratena pere- noidea few terpenes have been detected. The briarane grina, Flabellina ajfinis and F. lineolata (Cimino et al. diterpenoids verecynarmin A to G and the cembranoid pre- 1980), carotenoids and alkaloids from Pbestilla verccynarmin are known from Annina macitlatu and its nielanobrachia (Okuda et al. 1982), sterols and alkaloids prey organism, the octocoral Veretilliim cynoiuoriuiii from P. liigubris (Targett 1983; Karuso 1987). Karu- (GUERRIERO et al. 1987, 1988, 1990). Letuimla millecra so (1987, after Klein 1986) reported also the presence of exhibits four different sesquiteipenoids, millecrone A and homarine in the latter .

B, as well as millecrol Aand B (Cimino et al. 2001 ). They all are sequestered while feeding on alcyonariids. Further Members of the Aeolidoidea are known for storing cnido- investigation revealed more natural compounds: isofura- cysts from their prey organisms in special structures lo- nodiene, (+)-8-hydroxycalamenenc, algoafuran, cated at the cerata tips (see Fig. lA). Here, a cnidosac is cubebenone, and a series of seven triprenylquinones and fonned as part of the digestive gland, and in the cells of hydroquinones. Alcyonium faitii and the gorgonian Lep- this sac, intact cnidocysts are incorporated and maintained togorgiti palma seem to be the source of at least some of functional. It is assumed that the slugs use these cnidosysts these compounds (McPhail et al. 2001 ). Origin of jano- as a major means of defense (e.g. Edmunds 1966; Green- lusimide, extracted from Janoliis cristatiis (Sodano & wood & Mariscal 1984a, b). This assumption is support- Spinella 1986) seems to be unknown, a de novo biosyn- ed by the studies of Frick (2003), showing that Flabelli- thesis can not be excluded (Cimino et al. 2001 ). na verrucosa responds to the presence of predators by the variation of nematocyst incorporation.

Terpenes are also present in Dendronotoidea. Tiitonia haiuiw)-oriiin e.g. sequesters the feeding deterrent The incoiporation of cnidocysts is regarded as the main sesquiteipene julieannafuran from its food, which is the key character in the evolution of the Aeolidoidea (Wägele sea fan Gorgouiu ventalina (Cronin et al. 1995). Sever- 2004). Nevertheless, in some cases secondary metabolites al terpenes (mbifolide, pukalide, cuparane sesquiter- seem to be of importance for aeolids as well, e.g. in the penoides) and other compounds (tochuinyl acetate and its genus Pliyllodesmium where no incoiporation of cnido- dihydro-derivative, ptilosarcenone) are collected from cysts takes place (Slattery et al. 1998). The same holds Tochiiiiia tetraintetra. All these products seem to be food true for Pbestilla lugubris, which shows no cnidocysts in derived (Cimino et al. 2001 ). Further compounds in oth- their cnidosacs, but homarine was found (Karuso 1987, er members of Dendronotoidea are summarized by Cimi- after Klein 1986). no et al. (2001): An unknown Triioniu sp. revealed a prostanoid, also found in the octocoral Teleslo, and Tri- Homarine was first isolated in 1933 by Hoppe-Seyler. Its toniella belli sequestered a chimyl alcohol from the oc- biological function has been controversially discussed ever tocoral Clavularia frankliniana. De novo biosynthesis of since. This product seems to be restricted to marine or- teipenes is recorded from one member of the Dendrono- ganisms, and within the Metazoa it is only found in in- toidea: Barsby et al. (2002) showed through feeding ex- vertebrates (Carr et al. 1996). Therefore it was suspect- ,2-' periments with [ 1 -^Ci] acetate that Melibe leonina pro- ed that its purpose lies in osmoregulation. Beers (1967) duces at least one of its defensive metabolites (2,6-di- studied the distribution of homarine in Crustacea, Echi- methyl-5-heptenal) itself. nodcnnata and Tunicata and from these results infered that

the presence of homarine is "in basic accord with a role In the Aeolidoidea teipenes are only known from the genus in cellular osmotic phenomena". Dale (1971) though

Phyllodesmiuni. P. longicirniin accumulates three cem- could not find any contribution of homarine in osmotic branoid diteipenes, trocheliophorol, thunbergol and processes in decapod cmstaceans. Nevertheless, it is wide- epoxythunbergol, from the soft coral Sareophyton trocbe- ly accepted that homarine serves as an osmolyte in ma- liophonim (Coll et al. 1985). P. guamensis specifically rine algae. Initial studies have been caiTied out by Dick- sequesters the cembranoid 1 1-beta-acetoxypukalide from son & KiRST (1986) with the green alga Plat}-monas sub- © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

Bonner zoologische Beiträge 55 (2006) 183

cordiformis. Slattery et al. (1997) showed that the soft body of the dendronotoid Marionia blainvillea (Fig. 1 B) coral Gersemia antárctica releases a potent antimicrobial was investigated in detail. The epidermal tissue of the mixture of organic compounds into the suiTounding sea- mantle of M. blainvillea is densely packed with glands of water, and homarine is the compound responsible for most unknown function (Wägele et al. 2006) (Fig. IC). There- of this biological activity. In the tissues of the anthozoans fore a role of these glands in chemical defense was con- Actinia equina and Calliactis parasitica, homarine was de- sidered likely. tected at concentrations of 1.5 and 2.1 mg/g dry weight

(Mathias et al. 1960). Berking (1986, 1987) detected homarine in several hydrozoans, where it regulates the 2. MATERIAL AND METHODS colony morphology and prevents the metamoiphosis in larvae. Detection of homarine in gastropods is rare. Mc- Specimens of the opisthobranchs and of their food were

Clintock (1994) found the substance in the Antarctic collected worldwide during several field trips. Table 1

Marseniopsis mollis (Caenogastropoda. Marseniidae). It lists the investigated specimens, the collection details, and is probably derived from epizoic biyozoans and hydro- the means of extraction. The were fixated either zoans growing on the ascidian Cnemidocarpa verrucosa, in alcohol (ethanol or methanol) or by deep freezing. the main food of M mollis and serves as a feeding deter- rent against the seastar Odontaster validus. The supernatant alcohol was taken off and used for the preparation of a crude extract, or in the case of the deep Since homarine was found in aeolids without cnidocysts, frozen samples extraction with methanol was carried out. the question arouse, whether the occurence of homarine The solvent was evaporated under reduced pressure with with a possible defensive function is more widespread in a rotary evaporator in order to obtain a crude extract. opisthobranchs.The current study focuses on the distribu- tion of homarine in several opisthobranch taxa, with a The detection of homarine was carried out via 'H NMR main focus on Cladobranchia. since this group is hardly spectroscopy on a Bruker Avance 300 DPX spectrometer, investigated concerning its chemical defensive systems. using DtO as solvent. Furthermore the localisation of this compound within the

Fig. 1. A. typical cnidosac {here Aeoliclia papillosa). B. Marimiia hlainvillea C. Histological section of dorsal mantle epitheli- um of Marionia blainvillea. Note the single vacuoles in the cells staining homogenously dark violet. © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

184 Sven Affeld et al.: Homarinc in Opisthobranchia

Table 1. Species investigated for homarine, with infomiation on location, date of collection, extraction type and result. Food or- ganism or substrate is indicated in the last column. These information are taken from Mc Donald & N>bakken (1997) if not stated otherwise. + homarine present; - no signal of homarine present in 'H NMR and total amount of crude extract >100mg; ? insuffi- cient amount of crude extract {<10mg); Column 5 (Extraction): tluid in brackets indicate that only the alcohol for fixation was used for the preparation of a crude extract

Higher ranking Location Number of Indiv. Fixation Extraction Homarine Food Taxon/Species Date of collection

Opisthobranchia Aeolidoidea

Crateini pilain [ilanes, Spain 04.11.1993 Deep frozen MeOH + Tubularia spp.. (Gould, 1X701 and other hydrozoans

Ciithona cíicnik'íi B;inyLils-sur-Mcr, France 05.2006 EtOH (EtOH) + diverse hydrozoan (Montiigu, 1X40) species

Ciilluma gymiiola Blancs, Spam 04. i 1 . 1 993 Deep frozen MeOH + Tuhularia spp., and (CoLithoiiy, IS3S) other hydrozoans

Flahellimi (i//iiiis Ikinyiils-sur-Mer, France 05.2006 EtOH (EtOH) Eudendrium spp. (Gmeliii. 17')1) Hermissciulii cnissiconns Blanes, Spain 5 Indiv. Deep frozen MeOH + Examined specimens (Eschscholtz, 1X31) 24.03.1994 found on Tubularia crocea, also on diverse hydrozoans

Plieslilla lui^iihris Data taken from Karuso 19X7 (after Klein 19X6) + Porites spp. (Bergh, 1X70) I'hvlliHlcsiiinini spec. Lizard Island, Australia 14.07.2006 MeOH (MeOH) Specialised on Xeni- idae. (pers. obs. HW)

Dendronotoidca

Marioiini l^ltiinvillcn Blanes, Spain 5 Indiv. Deep frozen MeOH e.g. Euiiicella siiigii-

(Ris.so, IXIX) (19.2004 laris, also on other gorgonians

Murionia lilaiiivillcn Blancs, Spain 10 Indiv. FlOH (EtOH) (Risso, 181X) 05.2006 Triloniu striata Blanes, Spain 15 Indiv. Deep frozen MeOH Paralcyonium ele- Haefclfinger, 1963 04.-07.2004 gaiis (see Schmekel & Portmann 1982)

Tritiiiiia manicata Blanes, Spam 11 Indiv. Deep frozen MeOH Clavularia sp., Deshayes, 1X53 03. -08.2004 Cormdaria spp. (Alcynonacea)

Doridoidca Dcmlrodaris graiiili/lora Tierra de Malgrat, Spain 05.11.2004 EtOH 96% (EtOH) Sponges e.g. Jrcinia

Rapp, I 827 fasciculata. Clatltria

toxystila, Spoiigia

officinalis

Platydiiris argd Giglio, Italy 05.2005 EtOH 70",, (EtOH) Demospongia (Linne, 1767) Adalaria prnxima Bretagne. France 07.1996 EtOH 93% (EtOH) Biyozoa (Alder & Hancock. 1X54) Arcliidoris psciidoargits Tierra de Malgrat, Spain 05,1 1.2004 EtOH 96"/o (EtOH) Demospongia (Rapp, 1827)

Cephalaspidea

Scapliaiidcr ligiiariiis Blancs, Spain 5 Indiv. EtOH 70% (EtOH) Predatoiy e.g.

(Linne, I 758) 11.11.2004 Polychaeta

Aglaja triciilorata Giglio, Italy 05.2005 EtOH 70% (EtOH) + Predatory, other Renier, 1807 Opisthobranchia

Anaspidea

Aplysia spec. La Coruña, Spain 08.03.2006 EtOH 96« 0 (EtOH) Algae Plivllaptlvsia spec. Banyuls-sur-Mer. France 09.1994 EtOH (EtOH) Algae 3 .

© Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

Bonner zoologische Beiträge 55 (2006) 185

Higher ranking Location Number of Indiv. Fixation Extraction Homarine Food Taxon/Species Date of collection

Pleurobranclioidea

Balhyberthella antárctica Antarctica 27.1 1.1 996 EtOH 70% (EtOH) - unknown Willan & Bartsch, 1987 61°43,30'S 59°12,40'W

Tylodinoidea í7/;;/wmi//;í/)Mím/)n«i//íí»; Meteor Bank ST 486 08.09.1998 EtOH 80% (EtOH) - Deinospongia (Lightfoot, 1786) (N-Atlantic) 29°45,7" N 28°22.9" W

Otiier gastropods „Prosobranchia" Littorina littorea Nordstrand. Germany 10 Indiv. EtOH 98% (EtOH) Linné, 1758 22.08.2006

Food of t lie examined Opistho branchia

Octocorailia Eiinicella singularis Banyuls-sur-Mer. France 05.2006 EtOH 98% (EtOH) + (Esper. 1791) Xenniidae Lizard Island, Australia 14.07.2006 MeOH (MeOH)

Hydrozoa Tiibidaria crocea Blanes, Spain 04. 11.1993 Deep frozen MeOH + Agassiz, 1862

3. RESULTS

From 1 g of the crude extract of one colony of the gor- The survey of several members of the Opisthobranchia re- gonian 4 mg of homarine were iso- vealed that the natural compound homarine is present in lated by RP- 1 8 VLC and HPLC. The VLC was canied out all exainined samples of Cladobranchia, apart from a new using a 2.5 cm glass column filled 10 cm with Silica gel species of Pliylloclesniium from Lizard Island. This com-

Polygoprep 60-50 CI 8. Elution was performed using a wa- pound was also not found in other members of the ter/methanol gradient starting with pure water, yielding 1 Opisthobranchia, except for Agla/a tricolorala. Results of

fractions. this survey are listed in Table 1

The HPLC was performed on a Shimadzu system The low field shifted 'H NMR signals of homarine ó 7.8 equipped with a Merk Hitachi Pump L-7100 and a Shi- - 8.9 ppm (see Fig. 2) are very characteristic for this com- madzu SPD-M6A Photodiode Array UV-VIS detector, us- pound, and can easily be detected in 'H NMR spectra of ing a Knauer C]^ Eurospher-100 (5 |irn, 250x80 mm) crude extracts. In all cases they did not overlap with any column. A constant flow of 1 mL/sec was applied using other resonance signals. The detection of these resonance

Water/Methanol (90:10 V/V) as eluent. signals is a clear indication for the presence of homarine,

however only if the concentration of the compound is suf-

The five deep frozen specimens of Maiioiiia blainvillea ficient, i.e. traces will not be detected. were dissected before extraction into mantle, foot, diges- tive gland and the rest of the viscera, in order to examine For specific localisation of the compound different body the distribution of homarine within the body of the ani- parts of M. blainvillea were extracted separately. 'H NMR mal. spectra of the extracts show that homarine is present throughout the body (see Fig. 3 A to D). For histological investigation, one specimen each of the dendronotoid Mariona blainvillea and the aeolid Cuthona Histological investigation revealed that the epidermis of caerulea have been preserved in formaldehyde/seawater. M. blainvillea contains many epithelial glandular cells After dehydration in alcohol, they were embedded in hy- with one large vacuole filling the whole cell. The vacuole droxyethylmethacrylate for serial sectioning and sections contains a substance that is stained homogeneously dark (2.5 i^m) were stained with toluidine blue. Investigation violett with toluidine blue (Fig. IC). These glandular cells was performed under a light microscope. are evenly distributed over the dorsal and lateral notum. © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

186 Sven Affeld et al.: Homarine in Opisthobranchia

PPM 8 6 8.4 8 2 8 0 7,8 7 6 7 4 7 2 7 0 6,8 6 6 6.4 6.2 6 0 5 8 5.6 5 4 5.2 5.0 4.8 4 6 4.4

Fig. 2. 'H NMR spectrum ofpuritled homarine in mcthanol-d4. 300 MHz. and ehemical structure of homarine.

The epithelium of Cuthoiui caenilca showed very few which is in accord with its role as a moiphogen. suppos- glandular cells, these were usually filled with dark stain- edly in all hydrozoans (Berking 1986, 1987). The exam- ing granular substances. ined gorgonian E. singiilaris also contained homarine as

described for other gorgonians where it serves as an an-

tifouling agent (Targett et al. 1983). Uptake of homa- 4. DISCUSSION rine can also occur by predation on opisthobranchs con-

taining homarine. According to Rudman ( 1 972), members Secondary metabolites clearly seem to play a role in the of the genus Aglaja are active predators, feeding on vagile biotic interactions with potential predators throughout all prey. Whereas some aglajid species, like .4. cylindrica, spe- systematic groups of Opisthobranchia. These chemicals cialize on polychaetes and nemeilines, others (e.g. A. aii- are often taken up from the food while de novo synthesis reopimctata) mainly focus on opisthobranchs. It seems is rare (review see Cimino et al. 2004). likely that Aglaja tricolorata obtained the homarine by feeding on other opisthobranchs. Only a few field studies on the deteiTcnt effect of the ob- served compounds have been cairied out (Thompson Several of the investigated species belong to the Aeoli-

1960; Avila & Paul 1997; Slattery et al. 1998; John- doidea with functional cnidocysts incoiporated, which are son & Willows 1999; Marín et al. 1999; Avila et al. a good means of defense against predatoiy fishes (Frick

2000; Becerro et al. 2001; Iken et al. 2002; Rogers et 2003) but certainly useless against cmstaceans and prob- al. 2002; Penney 2004). While even a few studies focus ably without effect against seastars. Both latter groups are on parasites on opisthobranchs (Arnaud 1978; Ho 1981; potential predators on opisthobranchs, though only few Carefoot 1987; Jensen 1987; Huys 2001; Schrödl thorough investigations on predation have been earned out

2002, 2003), the effect of secondai7 metabolites on (review see W.ägele et al. 2006). The efficiency of homa- pathogens is hardly investigated. Teeyapant et al. (1993a, rine as a feeding deteiTent is shown e.g. in Marseniopsis b) studied the uptake of secondai^y metabolites in Tylo- mollis (McClintock 1994). But similar studies have nev- diiui perversa (Tylodinoidea) from its food sponge er been perfonned for opisthobranchs and ftirther inves- Aplysiiui {Veivngia) aerop/ioba and the antimicrobial ac- tigations are needed to clarify the role of this compound tivity of the sponge metabolites. However they only found in cladobranchs. The presence of a further defensive sys- antimicrobial activity in compounds that were not pres- tem, like homarine, would provide additional protection, ent in Tylodiiui perversa. e.g. for the cnidocyst containing Cuthoua caerulea. In

species without cnidocysts, e.g. Mariouia hlainvillea.

Incorporation of homarine is shown here for slugs feed- homarine might even represent the sole defensive strate- ing on Hydrozoa as well as on the gorgonian octocoral E. gy- singularis. The examined Hydrozoa contained homarine. © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

Bonner zoologische Beiträge 55 (2006) 187

Fig. 3. 'H NMR spectra of crude extracts of dissected Marionia blaiinillea in methanol-d4. 300 MHz. A. Mantle, B. Foot, C. Digestive gland. D. Viscera without digestive gland. © Biodiversity Heritage Library, http://www.biodiversitylibrary.org/; www.zoologicalbulletin.de; www.biologiezentrum.at

188 Sven Affeld et ai.: Homarine in Opisthobranchia

Barsby, T, Linington, R.G. & Andersen. R.J. 2002. De No- Homarine could also serve as an antibacterial agent in the vo terpenoid biosynthesis by the dentronotid nudibraneh Me- mucus layer of the slugs, independent of its supposed func- lihe leonina. Chemoecology 12 (4): 199-202. tion as a feeding deteiTcnt, since any kind of mucus is a Bi c ERRO, M.A., Goetz. G., Paul, V.J. & Scheuer, RJ. 2001. perfect environment for bacteria or fungi. Therefore, or- Chemical defences of the sacoglossan mollusk Elysia rufesceus and its host alga BiTopsis sp. Journal of Chemical ganisms with a mucus layer can adopt two ways of keep- Ecology 27: 2287-2299. ing their body surface clean. They can cither produce big Beers, J.R. 1967. The species distribution of some naturally-oc- amounts of mucus that wash off continuously or add a sub- curring Quaternary Ammonium Compounds. Comparative stance that inhibits growth of microorganisms. The for- Biochemistry and Physiology 21: 11-21 mer strategy is known, e.g. from certain soft corals (Duck- Berrincí, S. 1986. Is homarine a morphogen in the marine hy- low & Mitchell 1979; Duerden 1986), the latter could droid Hvdractinia? Roux's Archive of Developmental Biol- 33-38. be the case in the Aeolidoidea and Dendronotoidea. ogy 195: Berkinc, S. 1987. Homarine (N-methylpicolinic acid) and trigonelline (N-methylnicotinic acid) appear to be involved in WÄGELE et al. (2006) noted the presence of homogenous- pattern control in a marine hvdroid. Development 99: ly staining violet glands as a characteristic of Dendrono- 211-220. toidea. In their survey on glandular structures in Opistho- Carefoot, T.H. 1987. Aplysia: its biology and ecology. branchia, they observed these glands for nine (out of 17 Oceanography and Marine Biology: An Annual Review 25: investigated) members of the Dendronotoidea. These 167-284. Carr. W.E.S., Netherton. J.C, Gleeson, R.A. & Derby, CD. glands are absent in any members of the Aeolidoidea and 1 996. Stimulants of feeding behavior in fish: Analyses of Tis- "Arminoidea". We continued the presence of these par- sues of Diverse Marine Organisms. Biological Bulletin 190: ticular glands in Marionia hlainvi/lea and their absence 149-160 in Ciithoiia caerulea. The uniform distribution of homa- Ciavatta, M. L., Trivellone, E.. Villanl G. & Cimino, G. rine in the different body parts of M hlainvillea suggests 1 996. Prenylphenols from the skin of the aeolid mollusc Cra- that no bioaccumulation in the glandular epithelium takes tena peregrina. Gazzetta Chimica Italiana 12: 707-710. CiMiNO,G., DE Rosa, C, de Stefano, C, Sodano, G. 1980. place in this case. Homarine is a very small and very po- Cholcst-4-en-4, 16ß, 18. 22R-tetrol-one 16,18-diacetate, a lar molecule, therefore it is likely that it is taken up pas- novel polyhydroxylated steroid from the hydroid Eiidendri- sively in the digestive gland or even in the whole diges- um sp. Tetrahedron Letters 21: 3303-3304. tive system. Quantitative analyses still should be carried Cimino, G., de Rosa, C. de Stefano, C, Sodano, G. & Vil- out to support this supposition. Since homarine is also lani, G. 1983. Dorid nudibraneh elaborates its own chemical present in C. caerulea, we can conclude that these parti- defense. Science 219: 1237-1238. Cimino, G., Crisping, A.. Di Gavagnin, M. Ros, cular epidemial glandular structures of the Dendronotoidea Marzo. V. & J.D. 1990. Oxytoxins. bioactive molecules produced by the are not a morphological adaptation to store homarine. marine opisthobranch mollusc Oxynoe olivácea from a diet- Their function remains unclear. derived precursor Cellular and Molecular Life Sciences 46(7): 767-770. Cimino, G. & Ghiselin, T.G. 1998. Chemical defense and evo- Acknowledgments. We thank Sabrina Blcidissel (Wuppertal). lution in the Sacoglossa (Mollusca: Gastropoda: Opisthobran- Katharina Händeicr (Bonn), Yvonne Grzymbovvski (Bonn) and chia). Chemoecology 8(2): 51-60. Wolfgang Wägcle (Bonn) for help in collecting material. This Cimino, G.. Fontana, A. & Gavagnin, M. 1999. Marine opistho- study was partly funded by the Gemían Science Foundation to branch molluscs: chemistry and ecology in sacoglossans and - HW (SPP 1127, "Adaptive radiation origin of biological di- dorids. (Review) Cunent Organic Chemistry 3: 327-372. versity": Wa 61 8/8). Cimino, G.. Ciavatta, M. L., Fontana, A. & G.wagnin, M. 2001. Metabolites of marine opisthobranchs: chemistry and

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