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THE NAUTILUS 123(3):148-153, 2009 Page 148 The distribution of precursors and biosynthetic enzymes required for Tyrian purple genesis in the hypobranchial gland, gonoduet, and egg masses of Dicathais orhita (Gmelin, 1791) (Neogastropoda: Murieidae) Cliantel Westley Kirsten Benkendorff School of Biological Sciences Flinders Uni\ersit\', GPO Box 2100 Adelaide, South Anstralia, 5()01, AUSTRALIA [email protected] ABSTRACT Baker and Sutherland (1968) isolated the prochromo- 'rhe Biosyntheticorigin olTvrian purple in theadulthypoBran- gmeunr,ictivdr,inDdioxcyalthsanilsplioartbeil(aFi(gCumreel1i,n2,) f17r9o1m).thFerAoucshtrraolmioa-n chial gland and egg masses ot the Murieidae is unknown. geu hydrolysis by aiylsulphatase (Dubois, 1909; Baker Ilistociieinistn-' and mass spectrometn’ were employed to de- and Sutherland, 1968) and subsequent oxidation and termine the distriBntion ot Biosynthetic components essential lorT\rian purple precursors\nthesiswithin the InpoBranchial dimerization generates a suite ol bromiuated intermedi- gland, goiKKlnct, egg masses, and lan'ae ol Dicafhais orhita. ate dye precursors (Figure 1, 3-5) (Cooksey, 2001). Of Ilistochemical correlations suggest that dc novo sviithesis of these, tvriverdin (Figure 1, 4) is photol\4icallycleaved to the prochromogen, tvrindox'yl snlpliate, not only occurs within yield the pigment 6,6’-dibromoiudigo (McGovern and the hspoBranchial gland. But also within the gonodnet, cap- Michel, IfJOO; Cooksey, 2001). Depending on prochro- sule, intracapsnlar llnid, and encapsulated lan'ae. The coinci- mogen composition, 6,6’-dibromoindirubin, monobromi- ilence of tyrindoxyi sulphate and anIsnlpliatase in the capsule uated indoles and indirubins, indigo and indirubin may and alBnmen glands, alongwith the capsule w'all and intracap- also be formed (Wouters andVerhecken, 1991;Wouters, snlar llnid, suggest that the Biospithetic components required 1992; Koren, 1995; Cooksey, 2001; Cooksey and VVith- fmoartiTovnr.iaOnvepruarlplleitsaypnpteheasrisstahraetitnhtereogdugcmedasdsurniantgurcalappsruoldeufcotrs- uall, 2001; Karapanagiotis and De Villemeruil, 2006; Westley and Benkendorff, 2008). Despite the wealth of ot the Murieidae arise from a maternal source. information available on dye genesis from indoxyl sul- A(kIitio)uil kci/ivord'i: Bromopero.xidase, anlsnlphatase, tvrin- phate precursors, fewinvestigations have focused on the doxyi sulphate, capsule, intracapsnlar llnid, \itellns, natural biosvntlietic origin of procliromogens and the signili- products cance ol this biosvmthetic pathway. Secondaiv'metabolitesvnthesistxq^icallyoccursthrough tfie modification ofprimaiy metabolic pathways. Indoles are believed to arise from the essential amino acid tiypto- INTRODUCTION phan (Figure 1, 1) (Fox, 1983, Verhecken, 1989; Zinder- mau, 1990). Indeed, storage of tiyptophan has been Tyrian pui'ple is an ancient dye of religions and royal reportetl within muricid hypobranchial glands where Tyr- signilicance (Reinhold, 1970) obtained exclusively from ian purple genesis is known to occur (Bolognani-Fantin lixqrohranchial gland secretions ol inuricid inollnsks andOttaxiaui, 1981; Srilakshmi, 1991; NaegelandAguilar- (Cooksey, 2001). Altliough the east Mediterranean Tyr- Cniz, 2006). Among other euzymiatic conversions, tiypto- ian purple indnstiy ol the 13*'' Centuiy B.C. once nour- phan must then be bromiuated (Figure 1) to produce ished (McGovern and Michel, 1985), traditional dye the prochromogeii tvriudoxyl sulphate (Westley et ak, jiroduction has now Been all but abandoned (Naegel 2006). Bromopero.xidase activip' has been detected in and Cooksey, 2002). Nevertheless, the historical inqtor- hypobranchial extracts oftlie muricid HexapJex fninculus tance ol Tyrian purple has prompted considerable inves- (lannaens, 1758) (Jannun and Coe, 1987),which provides tigation into the chemical composition and Formation ol e\4dence for precursor bromination and hence, dc novo this dye. pnichn)inogeu syntllesis In 1909, Eriedlander elucidated the dominant dye Early obsemitions bv Ari,stotle in ~350 B.C. (Peck, jrigment as b,b’-dibronioindigo (Figure 1, R). Much later. 1970) and Pliny the Elder in 1''* centuiy^ A.D. (Bailey, C. W'estley and K. Benkendorll, 2009 Page 149 COOH This investigation aims to provide new inlormation / on tlie concurrent dfstribntion ol the biosynthetic constituents essential lor Tyrian purple synthesis in the hvpobranchial gland, gonodnct, and egg masses ol Dicathais orbita. These compoimds and enzymes in- 1.Tiyptophan clude tn-ptophan, bromoperoxitlase, tvrindoxyl sulphate, Primarymetabolite and anTsulphatase. Overall, it is hoped these findings Bromoperoxidase+ will higlilight potential sites ol prochrornogen and Tyrian unidentifiedenzymes purple genesis and establish the importance of these OSO secondaiy metabolites in mmlcid reproduction and lar- val dey'elopment. 2.Tyrindoxy]sulphate MATERIALS AND METHODS Prochromogen A totalof27 femaleD. orbita specimens and 15 separate- ly spawned egg masses were sampled Irom the Flenrieii and Eyre Peninsulas of South Australia. Thepallial gono- dnct and hypobranchial gland ol 12 specimens, and the egg capsules and embi-yos from capsule glands yvere Iresh-lrozen ciwostat sectioned (15m). Transverse sec- tions were stained \y4th the acid-hydrolysis method for tvrindoxyl sulpliate adaptetl from Baker and Duke (4976), tlie bromo-phenol red method for bromoperox- idase modified from Krenn et ak (1989) and Wever et ak 1991 (Westley, 2008), andthe post-couplingmethod for ( ) aiyksnlphatase (Rutenbnrget ak, 1952). Gonodncts from 12 females, and capsules from 9 egg Tyrianpurplepigment masses were fixed in 10% neutral-buffered formalin and jiaraffin embedded. Transverse sections (5m) were Figure 1. Tlie proposed biosynthetic pathwayto Tyritin pur- .stained with the p-DAIAB-nitrite method (Adams, ple from tiyyitophan in the imiricid Dicathais orbita (adapteil 1957) to determine sites of tnptophan storage. Ciwostat from VVestleyetal. 2006), and paraffin sections were also stained with Haema- toxylin and Eosin (Thompson, 1966), Toluidine Blue (Kramer and Windrmn, 1954) and Periodic Acid Schiff 1929) indicated alinklietween Tvrian purplegenesis and (McManus, 1946) for morphological and biochemical reprodnction (Westley et ak, 2006; W^estley and comparisons. Benkendorll, 200S). This association was overlooked until Tvrindo.xyl sulphate distribution yvas determined by Tyrian puiple and intermediate precursors were recently liquid chromatography-mass spectrometiw (LC-MS). isolatecl from mnricid egg masses (Palma et ak, 1991; Ilyqyobranchial, albumen, and capsule glands were ex- Benkendori'f et ak, 2000, 2001, 2004). Subsequent cised from three females and capsules sampled from 12 obser\'ations reporteddeep redpigmentation in the gono- egg masses. Adult tissues and separate capsule constitu- dnct ol Dicathais orbita (Gmelin, 1791) (Benkendorll ents (capsule wall, intracapsular Iluid and larvae) yvere et al, 2004) and mass spectroscopic analysis confirmed extracted in dimethyl formamide (DMF) and analyzed the presence ofTyrian purple and its precursors (Westley according to Westley and Benkendorff (2008) by high and Benkendorff, 2008). Although these findings imply a performance-liquid chromatography (W'aters Alliance) limdamental role lor these secondai'v metabolites in the coupled to a mass spectrometer (MS, Alicromass, Qua- reproduction and encapsulated development of the tro micro^^'). Tyrindo.xyl sulphate was identified by reg- Muricidae, the capacity' forbiosynthesis outside the hypo- istration of expected mass and isotopic clusters in mass branchial gland remains unknown. spectra (Westley and Benkendorff, 2008). It is currently assumed that the compounds in egg masses arise through maternal investment during cap- sule formation. However, lai-vae may possess the capaci- RESULTS ty to synthesize precursors de novo. Natural product biosynthesis has been suggested to commence at an ear- The distribution ofTyrian purple precursors and biosyai- ly lamil stage in some nndibranch species (Avila, 2006). thetic enzymes required lor natural product synthesis Non-\4able mnricid lanne are known to develop purple within the female hvpobranchial gland, gonodnct, and pigmentation (St. Amant, 1938; Gallardo, 1973; Spight, egg capsule constituents are summarized in Table 1. 1977; Pechenik, 1982; Boiler and Stickle, 1988; Naegek Tnptophan was detected by positive p-lDVIAB-nitrite 2004), which implies relevant biosynthetic competence. staining (Figures 2-3) yyfthin the hypobranchial gland. Page 150 THE NAUTILUS, Vol. 123, No. 3 Table 1. The (li.stribution of precursors and enzvmes required lor Tyrian purple syndiesis in the female Inq^obranehial gland, gonodnct and egg masses of D. orbifa. +. presence; — absence; IF, intracapsnlar fluid; NA, not attainable. Gonodnct Eggmass CoinponiKpenz\'ine Techniijne H\qrobranchial gland Albumen gland Capsnle gland Capsule IF Lan'al vitellns Tnptophan llistocheinistiy + + + + + + Broniopero.xitlase Histocheniistn' + NA + + + AnLsnlphatase Histocheinistn' T- + + - + Tyrindoxyl snlphate llistocheinistiy + - + + - + LC-MS + + + + + + gonoduct, egg cap.sule walls, intracapsnlar llnicl and lar- The concnrrence of aiylsnlphatase and tvrindoxyl \al vitellns (Table 1). As indicated by broniophenol-red snlphatewdthin the capsnle gland (Table 1) suggests that staining (Figures 4-5), bromopero.xidase displayed an intermediate precursor and dye genesis also occurs identical distribution (Table 1), altbongb the distribution within this gland. This is supported by detection of of broinoperoxidase in albumen gland tissue was not brominated indoles in Dicafhais orbifa capsnle gland acfjnired due to problematic posterior gonodnct section- extracts byWestleyand Benkendorff(2008). Aiylsnlpha- ing. Awlsnlphatase was localized within all adnit (Fig- tase was also found to coincide w4th tyrindoxyl snlphate ures 6-7) and lanal tissues (Table 1) examined and the in the albumen gland (Table 1), w'hich highlights this capsule wall (Figure 6), bnt not the intracapsnlar Ihiid gland as another prospective site for precursor synthe- (Table 1). Enzyme activiW was generally ot high activity’ sis. However, as tyrindoxyl snlphate w'as only detectable in the capsnle gland (Figure 6) and low activity in the by mass spectrometiy (Table 1), prochroinogen concen- albumen gland, capsnles (Figure 6), and laiwae. LC-MS tration must be comparatively low'. This is consistent revealed the presence of tyrindo.xyl snlphate within the with the intracapsnlar fluid of D. orbifa capsnles (Ta- hypobranchial, allmmen and capsnle glands of D. orbifa Ide 1), which is thought to originate in the albumen .specimens, and all capsnle constituents including lamre gland in some Mnricidae species (D’Asaro, 1988). Low (Table 1). Prochroinogen concentration wms below' the prochroinogen concentrations coupled with low aiylsnl- detectable limit byhistochemical techni(|iies in the albn- pliatase activity may also explain w'hy bioactive inter- )nen gland and intracapsnlar tlnid (Table 1). mediates were not previously reported in antolyzed albumen tissues (Westley and Ilenkendorff, 2008). Overall, the limited biosynthetic capacity of the albn- DISCUSSION men gland suggests it is unlikelyto contribute significant concentrations of brominated indoles to D. orI)ifa egg Coincidence ol tnptophan, broinoperoxidase, and txrin- masses. dox)'l snlphate in the hypobranchial gland of Dicaihais In comparison to the intracapsnlar fluid, laiwal vitellns orbifa (Table 1) confirms prochromogen sy'iithesis from W'as found to contain all the bios)'nthetic components the priman' metabolite, tnptophan. These findings ex- recpiired for Tvrian pnqrle genesis (Table 1). This is pand on the know'll occurrence ofbroinoperoxidase ac- consistent with previous reports of intermediate precur- tix'itv in hxpobranchial gland homogenates of Hcxaplex sors and Tvrian purple in mnricid egg capsnle extracts fnincuhis (fannnn and Coe, 1987) and provide further (Palma et ak, 1991; Benkendorff et ak, 2000, 2001). evidence forthede novo sy'iithesis of brominated indoles Mnricid embiyos are largelycomposed ofnutritivevitel- in the Mnricidae. Detection ofthese biosy-nthetic consti- his (Roller and Stickle, 1988; Naegel, 2004). These yolk tuents within the capsnle gland (Table 1) indicates that grannies are synthesized by ovarian follicle cells and prochroinogen .synthesis is also pos.sible w'ithin the mnr- ooc)4es (Martel et ak, 1986; Amor et ak, 2004), and icid gonodnct. The capsnle gland hmctions in the depos- consumed over the course of development (Gonzalez ition ol capsnle laminae (Fretter, 1941; D’Asaro, 19SS) and Gallardo, 1999). As tix'ptophan must be derived and correlations between capsnle gland and capsnle from the diet (Graw'ford, 1989; Bentley, 1990; Hermann biocheniistiY (Table 1) confirm the introduction ol tvr- et ak, 1992), it is likely that the ovaiwcontributes tn'i^to- indoxyl snlphate and the bio.synthetic components for phan to yolk grannies during vitellogenesis. In the case prochroinogen .synthesis during capsnle formation. The of broinoperoxidase, aiwlsnlphatase and possibly tv'rin- presence of both h'rindoxyl snlphate and aiwlsnlphatase do.xyl snlphate, it is unclearwhethertheseoriginate from in capsnle walls is supported by previous reports ol pur- follicle cells or the oocvte. ple pigmentation in capsnles oi Mnricidae (Benkendorff The findings ofthis investigation stronglyindicate that et ak, 2004). Overall, these findings provide a means bioactive intermediate precursors in the egg masses of ol incorporating natural products into capsnles and D. ori)ifa are synthesized w'ithin the capsnle w'all, lanal eliminate the need to transfer precursors from the h\'|ro- vitellns and, to a lesser extent, the intracapsnlar Ihiid, brancliial gland as previously suggested (Westley et ak, from biosynthetic components ol maternal origin. As 2006). the caenogastropod pallial gonodnct evolved from an C. Westlev and K. Benkeiulorli, 2009 Pa.t^e 151 Figure 2-7. Dicatliaisorhita. Transwrse liistological sections. 2. Encapsulated lanae, slicrn'ingtnptopliaii distiihiitioii e\iilenceil byblue p-DMAB-uitiite stainingwitliin the\'itellns (Vit) and intracapsulai’ llnid (IF). 3. Tnptoplian distribution i'\idenced b\- blue p-DMAB-iiitrite stainingwitliin the capsule (Cgl) and h\pobranchial gland (Hg). 4. Capsule gland containingapartiaiK lonned egg capsule. Bronioperoxidase acti\it\’ (arrows) indicated In broinophenol-bhie staining ol capsule material (Cm). 5. 11\pobranchial gland,showingbroinoperoxidaseacti\it\'(arrows)exidencedIn broniophenol-blnestaining.6.Capsulegland,shon'inganIsnlphatase actixity(arrows) displax-edbyred(= loxx'lexels) stainingot capsule material. 7. IIxpobranchial gland, shoxx'ingaiAIsulphatase actixitx (arrows) displax'ed by purple (= high k'X'els) staining. Abbrexiations: Bm, basement niembiane; Cgl, cajisnie gland; Cm, capsule material; Hg, hxpobranchial gland; IF intracapsulai' fluid; Lu. lumen;Vit, larxal xitellus;Vs,xascnhirsinus. Scale bars = 100 pin. Page 152 THE NAUTILUS, Vol. 123, No. 3 ancestral right li)pohranchial gland (Fretteret ah, 1998), Benkendorll, K., J. Breinner, and A. Davis. 2001. Indole ileri- it appears tliat the capacih’ for Tyrian purple s)aithesis vativ'es from the egg masses of mnricid molluscs. Mole- has been retained in various reproductive glands of the cules 6: 70-78. Mnricidae over the course ofevolution. The presence of Benkemlorff, K., C. Westley, and C. Gallardo. 2004. Observa- Tyrian purple precursors (Benkendorff et ah, 2001, tions ofpnqile pigments in the egg capsules, h)qiobran- chial and reproductiv'e glands from seven species of the 2004) in the egg masses of species from the monophylet- AInricidae (Gastropoda: Alollusca), Invertebrate Repro- ic snhlamilies Rapaninae, Mnricinae, Ocenebrinae, and duction anil Development 46: 93-102. Ergalataxinae (Claremont et al. 2008) suggests this phe- Bentley, R. 1990. The shikimate pathway - A metabolic tree nomenon is widespread in the Mnricidae. ILwevei", the with many branches. Critical Review's in Biochemistiy absence ofTvrian purple precursors from some Oceneb- and Alolecnlar Biology25: 307-384. rinae species (Benkendorff et ak, 2001, 2004) indicates Bolognani-Fantin A.AI. and E. Ottaviani. 1981. The lypobi'an- that gonodnct bio.syiithesis ol hypobranchial gland meta- chialglandofsome Prosobranchia(Alollusca: Gastropoda) bolites may be ciade-specific. Nevertheless, this chemo- living in different habitats: a eomparativ'e histochemical taxonomic dbide coiipled with male Tyrian purple stnily. AlonitoreZoologico Italiano 15: 63-76. genesis (Eisner and Spanier, 1985; Verhecken, 1989; Claremont, Al., D.G. Reid,andS.T.Williams. 2008. A molecular phylogeny ofthe Rapaninae and Ergalataxinae (Neogastro- Michel et ak, 1992; Benkendorff et ak, 2004; Westley poda: AInricidae). jonrnalofMollnscanStudies74:215-221. and Benkendorlf, 2008), indicates that maternal provi- Cooksey, C. 2001. Tyrian Purple: 6,6’-dibromoindigo and sioning to support lanal development is not the sole related compounds. Alolecules 6: 736-769. hmction of these natural products. Cooksey, C. and R, Withnall. 2001. Chemicalstudieson Nticella lapilltis. Dv'es in IlistoiyandArchaeology 16-17: 91-96. Crawford, l.P, 1998. 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