Odonatologica34(2):103-110 JuneI,2005 Theaccessory glandsof thefemalegenital tract inAeshna juncea(L.) andA. grandis(L.) (Anisoptera: Aeshnidae) A.Åbro Division ofAnatomy,DepartmentsofBiomedicine,UniversityofBergen,Jonas Liesvei91, N-5009Bergen,Norway ReceivedJune11,2004/RevisedandAcceptedDecember2,2004 Themicro-anatomyofthe $ accessory glandsinadult A. junceaand A. grandisis similarbutthesizeoftheA. grandisglandsisclearlylargerthan that ofA.juncea.The secretorycellsconstituteasimplecolumnarepitheliumsurroundingacuticle-lined lu- men.The glandularepitheliumisprovidedwith apeculiarsystemof deep,narrow, intercellular cryptsbordered with microvillar cell membranes. Lipidsreleased tothe cryptlumenarepresumablyforcedinto thecentralglandlumenby contractions ofthe muscularnetworkattachedtotheoutsideofthegland.Theefferentductofeach gland that openstothedistalpartofthevaginahasacomplicatedmuscularapparatus,prob- ablyservingasapump.The secretedsubstancesaccumulate in thecentralglandcavity mainlyduringthepre-reproductivephase,whichthedragonfliesspendawayfromwater. The secretioncontains substanceswithwax-likeproperties andbecomes darkened by osmication. Secretorycellsappeartopossessalimited lifespan;scatteredcellsinproc- essofdyingoccuralready duringtheearlyreproductivephase.Inthelatereproductive phasemostoftheglandularepitheliumpresentsadisintegratedappearance.Thereisno cell renewal intheglandinthecourseofadultlife. The patternofcell deathindicatesa decompositionbyapoptosis.Besidescontributingtoinvestmentoftheeggs,theglands presumablyintervene alsoinotheraspectsofthereproductiveprocesses. INTRODUCTION Inthedragonfly Aeshnajuncea(L.),apairofconspicuous accessory glandsmake upanintegral partofthefemalegenital tracttogether withsperm-storingorgans (ABRO, 2004a).However, theseglandsdonotseemtohavereceivedanyparticular attentionfromformerworkers concernedwith dragonflyanatomy(TILLYARD, 1917;WHEDON, 1919;WINKELMANN, 1973).Theaimofthepresentstudy hasbeentoprovide furtherinformationon thestructure andfunctionof thesene- glected glands. 104 A.Abro Preliminaryobservations havedisclosedthatthefemaleaccessory glandsofOdo- natamightvaryconsiderably betweendifferentspecies. Thetwo aeshnidspecies A. junceaandA. grandis arepresumed tobecloselyrelated. Theaccessory glandsof bothwereexaminedinordertorevealany dissimilaritiesintheirmicro-anatomy. MATERIALANDMETHODS Ovipositingfemales ofAeshnajuncea(L.)andA. grandis(L.)werecapturedatabreedingsitenear Bergen,westernNorway. Also,atclearingsin surroundingwoodland,afew specimenswerecollected duringtheirpre-reproductivephaseintheearlyflight season(lateJune- early July).Mostspecimens werefixed immediatelyinthefieldbyinjectingcoldfixativeintotheposteriorabdomenwith afinehy- podermicneedle. The femalegenitaltractwasnextdissected inalargervolumeofthesamefixative.As acontrol,freshtissuesofsomespecimens, anaesthetized incarbon dioxide,weredissected. Thefixative wasmadeup of3% glutaraldehydein0.2 M cacodylatebuffer (pH 7,3)with0.17 M sucroseadded;postfixationtookplaceina 1%solution ofosmiumtetroxide inthesamebuffer.Speci- mensweredehydratedthroughagradedseries ofethanolandclearedinpropyleneoxide,embedded in epoxyresin,and sectionedwithadiamondknife. Semi-thinplasticsectionsforlightmicroscopy,from material processed for electron microscopy, were stained in a 1%solution oftoluidine blue. Ultrathin sections for electron microscopywerecontrasted with uranyl acetate andleadcitrate. RESULTS Fromthegrossanatomyofthefemalegenital tractitisscarcely possible todistinguishbetween AeshnajunceaandA. grandis, exceptfortheac- cessory glands which in A. grandis are clearly larger thanthose of A. juncea. However,their micro-anatomy issimilar.Thepaired accessory glandsoftheAeshnafemalearesituatedventral- ly in the ninthand tenthabdominalsegments. Infreshmaterialthelength ofeach gland from the ductproximally to thetip ofthetailmeas- ures, for A. grandisintherange2.9-4.0mm,for Fig. 1.Aeshnajuncea:distalregionof A.juncea 1.9-2.7mm. the adultfemale genitaltractshowing theposteriorlysituatedaccessoryglands Thepositioninsituoftheaccessory glandsof (Agl); lateral (left)and dorsal (right) A. junceahasbeendrawn in outlinefromsev- views. Based ondissections ofseveral eral dissectionsoftheadultfemalegenitaltract specimens,fixedandstoredin buffered (Fig. 1).Insomeindividualsthetwoglands are glutaraldehydesolution. Surrounding ofuneven size. musculature and nerves are omitted. G: position of theeighthabdominal Each gland isflattenedwithfoldsand clefts. 8 ganglion;od: oviduct;rs; seminal re- Whensectionedtheyappearlobulatedandmade ceptaclewith accessory sacs; v;vaginal up of microscopic subunitsrecognized as sac- canal;7-10;abdominal segments.[Scale bar2.0mm]. Accessory glandsinAeshnafemalegenitaltract 105 cularout-pocketings calledacinioralveoli(Fig. 2). Thegland unitsare formedby asinglelayer ofcolumnarepithelial cellsthatsurroundacentrallumen, linedwith athincuticlethatisnot incontact withthecellapices, givingrise to asubcuticular space(Figs3,4).Thecentralglandularcavityexhibitsnarrowbranchesandis con- nectedto ashortefferentductopening to thedistalpartofthevagina. Thecavity system, carrying theglandularsecretion, is initsentirety cuticle-lined.Thefemale accessory glandsofaeshniddragonfliesareofectodermalorigin,having developed by invagination of theintegument. Infresh material,thegland cavity containsagranulated substancedispersed in a liquid. Fixed glands exhibit an osmiophilic content. Components ofthe secre- tionindicatewax-like properties. Electronmicrographs show afinegranular pre- Figs2-4.Thefemaleaccessorygland:(2)Aeshnajuncea:theglandwiththeefferentductcutoffnear its outlettothe vagina(brokenline),tr,remainingtrachealtrees.Tissue fixedin bufferedglutaralde- hydesolution;unstained whole-mount incacodylatebuffer.Viewedintransmittedlight,brightfield. [Scalebar400pm]; —(3)A.juncea:view ofaglandlobule;pre-reproductivephase.Thelumen(L) containsexpelledsecretion.Notedeepintercellular cryptswithsecretedmaterial,(arrows), ac:glan- dular acinus;bl:basal lamina;c:liningcuticle;E:glandularepithelium.Toluidine blue.[Scalebar25 pm);- (4)A.grandis:partofgland(reproductivephase)showingtwomusclefibres(mu)connected with acommoninsertion tothe basallamina (bl)betweenadjacentacini, c:cuticle;E:glandularepi- thelium;L: lumen.Toluidine blue.[Scalebar25pm). 106 A.Abro cipitate onthecuticle(Fig.5). Thecolumnargland epithelium, mostly withovoidnuclei,rests onaprominent basallamina.Inelectronmicrographsthecellsexhibitanapical surfacewithsmall, intermittent, poorly-developed microvilli.Themicrovillarborderextendsintodeep intercellularcrypts, linedbymicrovillarcellmembranes(Fig. 5)reaching close to thebasalplasmamembrane.Inearly adultlife,theglandular epithelialcellscontain Fig,5.Aeshnajuncea:finestructureofthe glandularepithelium;pre-reproductivephase.Surveyviewof theepitheliumwith itsintercellularcrypts(cr)linedwithmicrovilli,fromthesamespecimenasinFig.3. Intraplasmiclipidispreservedassphericalglobulesstainedblack,presumablyowingtoconstituentsof unsaturated fattyacids.Inthecryptlumen,accumulations ofexocytosedlipids(whiteasterisks),as:ac- cumulated secretion;bl:basallamina;C:cuticle;L: glandularcavitywithsecretion;mu:musclefascicle; n:epithelialnucleus; si:subcuticular lumen.[Scalebar 10pm].Inset:Enlargementshowingmicrovillar borders. Eachvillusholdsatubuleofsmoothendoplasmicreticulum (arrows),[Scalebar5pm]. Accessory glandsinAeshnafemale genitaltract 107 Figs 6-7.Aeshnajuncea: (6)Electron micrographofstrongly vacuolated glandularepithelium;re- productivephase.Some intraplasmaticdarkened lipidsarestill present. Intheglandcavity thereis someresidual secretion(L).bl: basallamina;c:cuticle;n:epithelialnucleus;v:vacuole. [Scalebar20 pm]; —(7)Theefferentduct,transverselycut,displayingtissuecoatsaroundthefolded,cuticle-lined lumen (L).Theborder between theepitheloidcells(E)and themusculartunic (mu)isindicated by asterisks, bl: basallamina;C:cuticle;fc:fatcell;mf: musclefibres;ne:corticalnervoustissue.[Scale bar20pm],— Inset:Enlargementshowingcuticularintimanext tosomeresidual secretioninthe duct lumen(L).[Scalebar5pm]. 108 A.Abro organized arraysofgranularandsmoothendoplasmic reticulum,Golgicomplexes, numerous mitochondriaand membrane-boundlipidinclusions (Fig. 5). Tubular elementsofagranularendoplasmic reticulumcanbeseenextending intothemicro- villialmostto thetip(Fig. 5, inset). Minorlipidinclusionsgather intheglandular cytoplasm nearthecrypts; large lipid depositscanbe seeninthe nearbycrypt lu- men(Fig. 5).Secretedlipidinthecryptsandalsointhesubcuticularspaceislikely to traversethecuticleandaccumulateinthemaincavity. Surrounding theglandularunitsare muscularfascicles, forming anetworkwith insertionsonthebasallaminabetweenadjacent acini(Fig. 4).In addition,adven- Figs8-9. Aeshnajuncea:electron micrographs:(8)The wall ofthe efferentduct showingepitheloid cellswithcytoskeleton(cs)andglycogendeposits(g).Cellapicesfurnishedwithmicrovilli (mv)against cuticle(C); eachvilluscontainsatubuleofsmoothendoplasmicreticulum. Theborderbetween epi- theloid andmuscular (mu)layersisindicatedbyasterisks, bl:basal lamina;N:nucleus ofepitheloid cell;ne:corticalnervetissue.[Scalebar5pm]; —(9)Thewalloftheefferent ductshowingpartofa muscle fibrewithnerveterminals(openarrows). Withinthe axonterminals(ax)areseennumerous small (synaptic?) vesicles. F:muscle fibril;Z:muscularZ-band. [Scalebar 2pm]. Accessory glandsinAeshnafemalegenitaltract 109 titiouscellscarry tracheoles.Differences occur in thehistological appearanceof glands fromvariousindividuals, depending onwhetherthey are captured during pre-reproductive orreproductive phase. Secreted substances seem to accumulate inthecentralglandularlumenmainly duringthepre-reproductive phaseofadult life.Inearlyreproductive phase, glandularcellsoffunctionaldeclinecouldberec- ognized as localatrophy. Lateronthereoccurs cytoplasmic vacuolation(Fig. 6) and/orchromatincondensationofscatterednucleiin theglandularparenchyma, indicativeof dying by apoptosis. Thepresenceof apoptoticcells ischaracterized bynucleiandcytoplasm brokenupintoseveralcompactfragmentsandanumber of smallmembrane-boundglobules. Theefferentductofthegland isbuiltupofconcentrictissuecoatsaroundacuti- cle-lined,folded-uplumen.Thecuticleisratherthick,fibrousandwithanelectron- densecuticularintima(Fig. 7). Beneaththecuticleisasimplelayeroftallepitheloid cells, bordering amusculartunic(Fig. 8). Theepitheloid cellshaveinterdigitated lateralborders anda foldedapical membranewith microvilli, eachcontaining a tubuleofsmoothendoplasmic reticulum(Fig. 8).Inthecellsareintraplasmatic de- posits ofglycogenandlarge,branchedbundlesofcloselyspaced parallel microtu- bulesandinterspersed microfilamentsthatrunfromthecellbase upward through thecell body to end atthe apex, apparentlyserving as cytoskeleton (Fig. 8).The layer ofmusclefibresappearsthickestaroundtheproximalpartof theduct. Mus- clefibresandepitheloid cellsarecloselyattachedwithoutabasallaminainbetween butthereis aborderoftightly-woven texture withsomeshortbranching fingers ontheepitheloid side oftheborder(Fig. 7). Musclefibresare seen concentrated nearest to the epitheloid layer. A corticalsheathofnerve tissue canbe seen close to thebasallamina:axons accompanied along theircourse by neuroglial cells and alsoneuromuscularjunctions(Figs 7-9).Outsidethemusculartunicis anirregular investmentoffatcellsandtracheolarcells(Fig. 7). DISCUSSION Theaccessory glandsofthefemalegenitaltractaretypicalexocrineglandsandare presumed tohavemultiple functionsinthereproductive processes(ABRO, 2004a). Theglands are thought to secrete the bulkoftheir production early inadult life andstore itinthecentralcavity;laterthey secrete continuously atalowrate. Un- dercertainconditionstheymight bestimulatedto secrete larger volumesoftheir products. However, thereare no morphological criteriaby whichitis possible to determinewhetheragivengland isstimulated.Thebranching cavitysystem ofthe gland anditsefferentductcarry thesecretionto thedistal vaginalcanal. Thelip- idsarepredominantly releasedto theintercellularcrypts byexocytosis. Fromthere they arelikely tobesqueezed outtothesubcuticularspaceandthenthrough pores in thecuticleto thecommongland lumenby contractions ofthemuscularreticu- lumsurrounding theglandularlobules/aciniandthemuscularapparatusoftheef- 110 A.Abro ferentduct.Blackening ofthesecretionby osmiumfixativesindicatesthepresence ofunsaturatedlipids. TheAeshnaglandsexhibitseveral morphological featuresin commonwith theegg-waxing glands ofixodid ticks (BOOTH, 1989).Thus they possess similarcrypt systems in their glandularepithelia, thecells ofwhichbear microvillicontaining tubularelementsofsmoothendoplasmic reticulum.Smooth reticulumisknown tobeinvolvedinlipid metabolism(FAWCETT, 1981).Theef- ferentductsofA.juncea andA. grandisglands exhibitstructuralsimilarityto that ofthe damselflyPyrrhosoma nymphula(ABRO,2004b)butareofgreater size, so thateachinvesting tissuecontainmorecellularelements.Themanynerveterminals to themusclefibres, apparentlywithpolyneural innervation, indicateanintricate nervous control.Theductprobably functionsasapump(ABRO, 2004b). Likethe glandular epithelialcells, theepitheloid cellsoftheefferentductare provided with microvillicontaining tubulesof smooth endoplasmic reticulum, whichindicates thatbothcelltypesareof commonorigin. Theaveragelongevity oftheadultAeshnafemaleisnotknownbutwillcertainly not extendbeyond afewweeks. As to the activity ofthegland, thepresentstudy indicatesthatmostoftheproduced substancesaccumulateintheglandularlumen duringthepre-reproductive phase,whichthe dragonfliesspend away fromthewa- ter.Secretory gland cells seemtopossessalimitedlife spanofafewdays, followed byafunctionalandstructuraldecline.Thusdecompositionofscatteredsinglecells in the gland parenchyma startsearly in thereproductive phase. Thisbecomes an accelerating trendinthecourse ofthereproductive phase. Inthelatereproductive phasemostofthegland exhibitsdisintegratingtissues.Renewalofsecretorycellsin theadultgland appearsnotto occur.Theshortadultlifetimepresumably doesnot need proliferation ofundifferentiatedrenewalcells. Degradation withintheglan- dularepithelium appearstoaffectindividualcells ratherthantractsofcontiguous cells dying spontaneously by apoptosis. 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