The anatomy and histology of the Fijian Anadare mollusc are described. Its structure is similar to other lamellibranchs of the family Arcidae. The gender determination of live specimens was deemed impossible, due to the inaccessibility of the gonads. Peculiar white patches were found on the mantle of all specimens, suggesting a reaction to disease or pollution which warrants further investigation

De Ramon N'Yeurt, Antoine

University of the South Pacific Electronic Research Repository

ïI8I418-AQUACULTUREPRACTICAL PBOJECTI',IÀlr. ,rI1IItIt,têAN INVESTIGATION INTO THE BIOLOGY AND SPAT{NING MATURITY OFANêDARA SPP. (BIVALVIA : ARCIDAE) IN FIJI.ABSTRACT : The anatomy and histol-ogy of the FijianAnêdare molluscr are described. Its structure issimilar to other lamellibranchs of the familyArcidae. The §ender determination of livespecimens was deemed impossible, due to t'heinaccessibility of the gonads. Peculiar whitepatches ï{ere found on the mantle of all specimens,suggesting a reaction to disease or poll-utionwhich warrants further investigation.TNTRODU-CTION :The bivalves of the genus Anadara are a marine group,and belong to the family Arcidae, subfamily Anadarinae.They arean important source of protei-n in many tropical, subtropical andwârm temperate âreas. In Fiji, the most common species is A.antlquata (Lin. ), followed by the second most-common species A.subcrenata (Lischke) and lastly A. inequivalvis (frugiere).They are epibenthic bivalves, showing some primitivemorphological features {eg. external chevron ligament, straighttaxodont hinge, unfused mantle margins, and byssus ) . The familyis also peculiar in possessing the red blood pigment haemoglobin'The fossil record shows that the f irst fe** species appeared inthe Ol igocene , but by the Mi-ocene many , widely di-stributedAnadara spp were present. Today, there are about 60 livingspecies, mostly tropical and sub-troplcal. They inhabit inter-tidal mudflats and sub-tidal areas, seaward of mangrove forests.Nothing much is known about recruitment of Anadara spp.in Fiji, but Maybin (1989) showed that apparently some part ofthe population is mature throughout the year, with a definitepeak in rnaturity in December (the hot and rainy season).It has been suggested (Fiji MAF, 1982) that Anadara sppcould be a potential orglanism for aquaculture in the FijiIslands. fn this respect, a knowledge of the spawningperiodicity, species composition and distribution and growthrates is essential in order to a,ssess the viability of themariculture of these molluscs. The subject of this presentinvestigation is to throw some light on the reproductive biologyand popr.llation structure of Anadara in Fiii, as well as to find aT.râÿr if possible, of determining the sex of live a.nimal-s withminimum damage, which would be of obvious advantage ifmariculture af these organisms is envisaged. Also, the nature ofcertain peculiar patches that have recently appeared in certainAnadara populations is looked into, especially so as to determineany possible disease and/or pathological reactions in presentbivalve populations.MATERTALS AND METHODS :At.the outset of this proiect, it was proposed to makean aim of identifying the sex of live Anada-ra specimens with anovel approach, which would consist of of attempting to drill ahole of suitable size at the right location through the shel1 ofthe animal, so as to enable extraction of gonadial samples via anhypodermic needle for further investigation of its content andmaturity stage.For this purpose, it i,-as envisaged to procure asuitable drill (eg. an electronic printed-circuit board dri11, indefault of a much more expensive dentist's drill-) and to carryout dissections on a few specimens so as to determine the exactlocation of the glonads in respect to the shell' It is impossi"bleto determine the gender of these molluscs from externalcharacteristics, as sexual dimorphism is only apparent in thecontent and color of the gonads, being pink for ovarian tissueand vrhitish for testes (Maybin' 1989 ) .About 50 Anadara antiquata specimens r,ùere bought fromthe Suva market, and kept in an aquarium supplied with mudflatsilt. Preliminary investigati-ons into the general morphology ofthe molluscs were carried out, and dissections revealed that theinternal anatomy of Anadara closely ressembles that of otherlamellibranchs of the family Arcidae. Results and drawings of thedissections are to be found in Appendix A'?hese dissections revealed that the gonads l{7ere farless accessibte than previously thought, being intimatelyÉtssoc iated with the f oot and digest ive gland , anrl locateddorsally and closely apposed to the shell hinge. Hence, it wasdeemed not practically feasible to sample gonadial tissue viadrilled holes, since there wâs too much risk of damaging adjacentvital organs in the process. Consequently, this phase of theinvestigation was abandoned, and the project took a different(](fltrse.During the initial dissections, it was noticed thatnearly al1 of the specimens investigated had peculiar whitepatches on the mantle edges, particularly at the posterior end.These superficially looked like calcareous incrustations of somekind, and ItIere apparently not present in Anadara populationslooked aL a few years ago (Maybin, personal com. ). The mysteriousaspect of this phenomenon induced some research into the natureand possible origin of these patches, as well as any implicationsfor the potential culture and consumption of these molluscs.The t.hird phase of this investigation, which ranconcurrently with the second, consisted in carryin$ outhistological analysis of the gonads, in order to determine thereproductive maturity of the animals.The method used was to fix selected gonad tissue int<:Bouin's fluid for a set time, followed try progr.=uit" dehydrationin grade{ ethanols. Xylene clearing foll-owed, after which thetissues were embedded in wax. Sections 1lfn thick were obtainedof, the blocks using a rotary microtome, eind the resulting slides\^iere stained using eosin and haemotoxylin. A detailed f ixing andstaining schedule is to be found in Appendix B.During the côurse of this investigation it was a'lso{ecided to try a different staining technique, in order tolighlight any stellate cells and âssociated granules in thetissuesr âs the above dyes do not stain these features (Sullivan,1960 ). The distribution of stellate cells follows that of whitishmottling on the mantle of A. trapeaia (Sullivan, 1960) and it wasconsidered worthwhile to invest igate whether this was also thecase in A. antiquata.To this effect, waxed tissue sections of the mantle andgonad were brought to water and stained with Gomori's aldehyde-fuchsin dye f or elastic f ibres ( Drur-v et al ' 1967 ) , However, thedesired effect was not obtained, and the slides remained blank.Possible reasons for the failure of this exercise are given inthe discussion. A detailed account of the staining schedule is tobe found in Appendix C. Photomicrographs of relevant slides eiereobta.ined usingf an Olympus photo-miclroscope.RESULTS :f. Mantle-tissue investigation zA drawing of a section of so-called "patchy" mantle isshown in Fig. l- r âs well as the accompânying photornicrographs.Portions where the patches r^rere present appear darker-staining,due to the presence of numerous, small cel1s. Teased-out "patch"material, viewed through a phase-contrast microscope, revealedwhat looked tike long "strands" of non-descript material, withrrumerous black "granules" embedded in it. It could not beascertained whether these r^iere part of the mantle epithelium orforeign bodies r but their structure r^ras evidently more complexthan initially thought -Anadara antiquata specimens collected from the mudflatsgpposite the Suva Pony Club turned out to also possess thepeculiar white patches, albeit to a much lesser extent. Here themantle was soft and smooth, in contrast to the Market specimens,where the incrustations r^iere profuse on the posterior mantleedges, ctenidial suspensory arches and adjacent tissue, impartinga tough and apparently "calcareous" texture to these organs.Hr:wever, the fact that these incrustations survived long periodsof decalcification in Bouin's fluid suggest that they are notentirely calcareous in nature.II. Gonad Histology :A number of acceptable slides were obtainecl of bothovarian and testicular tj-ssue, and these were looked-at under themicroscope. Drawings of these can be found in Figs. 2 and 3, a,ndthe accompanying photomicrographs.The ovarian tissue contained â large number of oocytestand was very similar to the ovarian tissue of mature Anadaragrarrosa specimens investigated by Broom ( 1983 ). The testiculartissue contained many spermatozoa, apparently at various meioticstages. In addition, some ciiiated stomach epithelium and anumber of concave erythrocytes are visible. It was found that avariation of the treatment shown in Appendix B gave better-contrasted slldes; this consisted in a brief immersion \4-5 sec)in Eosi-n following blueing in l-ithium carbonate, and prior toimmersion in haemotoxylin. These slides are 1abelled "rrar".DISCUSSTON :The peculiar aspect of the "patches" on the mantle istheir apparent recent emergence, and thi-s suggests that they arenot an innate physical trait. The fact that these patches aremore prominent on the posterior, light-exposed part of the mantlehas some parallels with observations made by Sullivan ( 1960 ) onA. trapezia, where "peculiar translucent granules" ïvere found inthe connectlve tissue of the mantle border, the ctenidial arches,etc. , being more concentrated on the posterior end of the animal.Their composition was not determined, and they t/ere believed tobe associated with the processes of the "stellate ceIIs",peculiar branched cells peculiar to the molluscan phylum, andwhich remain colourless with haemotoxylin stain.Sullivan (1960) also states that in fresh specimens, awhitish mottling of the mantle border is found, whosedistribution corresponds to that of the translucent granules inthe tissues. Atkins (1936, in Sullivan, 1960) considered thatthese patches are present on tissues that are exposed to light.Whether the latter observations are relevant to thewhite patches observed in present Anadara populations is notcertain, however it may be significant that their distribution isalso in areas of the mantle most likely to be exposed to l-ight.The actual significance of this observation is yet unknownThe presence of numerousin "patchy" âreas of the mantlesome extent of t'reactiôn tissue"hence brings up the question ofresult" of disease, pathogens orindeed pathologieal in origin,epidemic proportion, consideringthe samples had profuse distribut, small and dark-staining celIsepithelium are reminescent tofound in other organisms, andwhether these features are theirritant agents. If these rÿereit would certainly be of anthe fact that virtually 100% ofions of patches on them" Alsot5the restriction to areas likely to be reached by tight is notentirely explained by a pathologiical cause. A further alternativernight be that they are caused by pollution of some sort, whichtriggers this peculiar (defensive?) reaction from the moiluscs.sullivan ( 1960 ) states that in A. trapezia, thedistribution of white patches on the mantle is concurent to thedistribution of stellate cells and peculiar translucent granulesin the mantle epithelium. The latter remain colourless with eosinand haemotoxylin, and stain red with Azart and purple withGomori's aldehyde-fuchsin. However staining the A. antiquatatissues with this stain proved fruitless, and a possible reasoncould be that. the basic fuchsin employed was not pure or not ofthe kind especially prepared for use with this and Schiff'sreagent (Drury et a1r 1967). Also, the fixation techniqueempioyed may have been inadequate (excessive decalcification inBouin's) for this kind of material to survive. Had this effortproved successful, it would certainly have thrown some Iight intothe nature of these white patches r and whether they areassociated with the stellate ce11s.Hence, it is proposed that further investigati-on isrequired into the nature of these patches, particularly toascertain whether they are associated with the "granules" ofstellate cell-s. To achieve this, sections of mantle tissues wouldneed to be stained with Azan or Gomori's aldehyde-fuchsin' asrecômmended by Stillivan (1960), Lrut using the appropri-ate basicfuchsin, If the latter association proves true, then perhapsresearch into the actual function of these granules and stellatecel1s could throw some light into the nature of these whitepatches.In addition, a survey could be carried out of Anadaraspp. in various sites around Fiii, to establish the occurence anddistribution of these patches. An investigation into the leveIand types of any pollutants in the water and mollusc tissuescould also be undertaken, so as to determine any possiblecorrelation between pollution levels and patch occurence.II. Gonad seetions :The close ressemblance of the gonad tissue to that ofA. granosa (Broom, 1983) indicates that the reproductive patternof both species is similar. It seems that the specimen inquestion was fairly mature, which was not immediately evidentsince the gonad was not particularly large. It was found upondissection that gonad growth proceeds a fair bit into the footand around the digestive glandr which makes it less obviousexternally, and also complicates any attempt to sample the tissueôn l ive spec imens , as fitentioned earl ier.Out of about I sPecimens dissected,males, suggesting that the male to femaleonly two wereratio in thispopulation may not be6L:1, as reported for A. granosa (Broom,1983 ) . This fact shsuld add further impetus for research intosex-determinatj-on of these molluscsr &s the latter is essentialif breeding of cockles in an aquaculture context is envisaged.No attempt was made to induce spawning in the A.antiquata specimens, although the latter has been triedpreviously without success, rnainly due to failure of i-ndentifyingthe sex of the brood-stock {Maybin, pers. comm. ). Broom (1985)recommends artificial induction of spawning! in Anadara spp. bythermal shock techniques, or immersion of ripe females in asperm-seawa.ter suspens ion .CONCLUSIONS AND FURTHER RESEARCH :From this investigation, it is clear that thedetermination of the glender of live 4". antiquata specimens isvery difficult ' due to the absence of external sexual dimorphismand the relative inaccessibility of the gonads. since the sex-determination of the brood stock in an aquaculture venture is ofcruci.al importance, further research into the sex-determinationof these molluscs is warranted. The possible effects of diseaseor pollution, especially with respect to the peculiar whitepatches on the mantle of present Anadara population, needslooking into.REFERENCES iBROOM, M.J. (1983). Gonad development and spawning ingranosa ( L. ) . Aquaculture, 30 : 2tl-219 . AnadaraBROOM,DRURY,M.J. (1985). Themolluscs of thebiolopfy and culture of marine bivalvegenus Anadara. ICLARM reviews, Mani-la.R.A.B. ; WALLINGTON, E.A. ; CAMERON, S.R. ( 1967 )."Carleton's histological technique". Oxford UniversityPress.FIJI MTNISTRY OF AGRTCULTURE, FTSHERIES AND FORESTS ( 1982 ).Fisheries Division annual- report for 1981. FisheriesDivision, Suva.MAYBÏN, J.A. (1989). Ecological and taxonomic aspects of Anadarain Ffj i and some neighbouring island groups. MScthesis, USP, Suva.SULLIVAN, G.E. (1960). Functional morphology, micro-anatomy, andhistology of the "Sydney Cockle", Anadara trapezia(Deshayes). Aust. j. zool-., 9 : 2L9-256.td?t i:g{I-Q *llt*r aGËC.r'}j,vjr"d\|giË-:tt'['üii.!+§pË§^Bg*.t' . : i:*?d\ ^§t;&ô ;y,'* , ':i; .,.:3r#§ÿil:*i'+Cxu-*i§*.''ffiiïr:"e, i.Mffi ï"ll:$§,:)f:'': i'**+\.-\ ' .Bqt$,* '..1 -t,*ïnl .'t"5nffi;. ", r', ,,_l--.ô *, - t-18..i,t^À,r-Lt t"CD- 1i -t fAr-c ri " (< q O) fAacÀ.lx4oc)æ' coÿt)Êë-îinE TtgçùE aÇ y\xÿ7LL-.LX ?Jiÿ) æ R?o Biza'? czttj' (ç,rc7çw Ft rTEK-,;x rtüt)APPENDIX AAnadara antiquala : General ÂnatomyV"47s&taRA11V1ro1a-Yivt cL I fiutelzt'ar:-haoL,cron hÿ§êLE,<.ctc:ttfîî At..._- vfg.efirznà99(Fèt4/tLr: oÊANAD)- foo"fèALcàaêêO§tEfoshs?A.4t)iLe/{\?r4ts1f ÿËA-LhN5TLoVTfi,,+-" - Avrzic.ll!.i- **ncullÿePrR+êLâAN'|EBt'aæ t+î?ûeî?x<1^ûcècai- Ç4Ûff3)L' iv'rLL'cL:LfÇr. 4a17,'or',,.,ÿtÿE<.a.*efEËlr?t'.4l--..---Jl. cr*r-ftÿ 3' Atn*âuc. ,r -bqr,ro'*rn ,lf,,l't"»er-o\.'.*:reSez- VisW orz Vt'ÿcerîAL ;üaS-ç.8APPENDIX BFixinA and staininq schedule for the srrecimens :I. Tissue Fixation :SOLUTION TIME IMMERSEDBouin's Ftuid10% ethanol20% "50% "75% "95% "ABS ,,Xylene48h12h,,,,tt4hth12}]II. I{axinÉ :The tissues ?ÿere immersed in paraffin wax, and placed in avacuum oven at 65C for 12h to remove the xylene.After this liquid rvax hras poured into plastic moulds, andthe tissues l,irere placed in theseOnce set, the r^rax blocks hrere 1abe1l-ed and attached towooden chucks for placement onto the mierotome stage.Sections were cut at LL,U*, and floated on a warm water bath.Albumen-coated Élass slides were used to pick up selectedsections.III. Staining :SOLUTION TIME IMMERSEDXylene (removes wax)ABS95% ethanol75% "45% "2A% "Distilled waterLithium Carbonate (removes picrictap water washHaemotoxylin (Chr. )tap water washLithiun Carbona.te ( "blueing" )tap water washEosin2 min1"acid),,,,,t,,indefinitedip1 mindip30 secThe sl ides r^Ierein xylene, and mountedthen dehydrated in graded ethanols, clearedwith Canada Balsam.APPENDIX C_Q-<:Uû-r-1*'s Aldehvde Fuchsin stain fr:r elastic f i-bres It. PgepAr,ation :Basic fuchsin ( Schiff-compatible ) 0.5C7 o% ethanol 1oonilConc. Hydrochloric acid 1mlParaldehyde l-mlIT. Method :The dye is dissolved into the alcr:hol, and the acid andparaldehyde added. The mixture is teft at room temperature fort2-72 h, or until a deep purple colouration is assumed.III. Technique IThe sections are taken to water, and treated as per thefollowing schedule :SOLUT]ON TIME IMMERSEDLugol's iodinetap water wash2,5% sodium thiosulphatetap water **ash1A% ethanol r,rashAl dehr--vde f uchs 1n95% ethanol wash10 min2 min1C rni-nThe slides can be counterstained as desired.Mounting should be done using a synthetic resin medium.

An investigation into the biology and spawning maturity of Anadara Spp. (Bivalvia : Arcidae) in Fiji

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An investigation into the biology and spawning maturity of Anadara Spp. (Bivalvia : Arcidae) in Fiji

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