GonadsandgametogenesisinChaetodactylusosmiae(Acariformes:Astigmata:Chaetodactylidae)aparasiteofsolitarybees

ElżbietaRożej-Pabijana,WojciechWitalińskib

aInstituteofBiology,PedagogicalUniversity,Podchorążych2,30-084Kraków,Poland.

bDepartmentofComparativeAnatomy,InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity,Gronostajowa9,30-387Kraków,Poland.

Received15December2017

Accepted28May2017

Published11September2018

CorrespondingauthorElżbietaRożej-Pabijan:elarozej@vp.pl

Academiceditor

MichelBertrand

DOI

10.24349/acarologia/20184269

Copyright

Rożej-PabijanE.andWitalińskiW.

Distributedunder

CreativeCommonsCC-BY4.0

ABSTRACT

Chaetodactylusosmiae(Dufour,1839)isamiteparasitizingthesolitarybee–OsmiarufaL.–usedasacommercialpollinator.Inthisstudywepresenttheanatomyoffemaleandmalereproductivesystemsofthisspeciesaswellasitsgonadalstructureandgametogenesisattheultrastructurallevel.ThereproductivesystemsaresimilartothoseofotherAstigmata.Theovariesarepairedandeachcontainsgerm-linecells–agiantnutritiveovariancellconnectedviafunnel-typeintercellularbridgestooogoniaandprevitellogenicoocytes.Germinalcellsareembeddedinseverallargesomaticstromacells.Numerousprotrusionsofthenutritiveovariancellpenetrateintothestromacellcytoplasm.ConspicuousERcisternsruncloseandparalleltothesurfaceofthegerminalcells.Oocytesenteringvitellogenesisdisassociatewiththenutritivecellandavitellineenvelopecomposedofheterogeneousmaterialappearsontheirsurface.Whenvitellogenesisiscompleted,theoocytesarefulloflipiddropletsandtwotypesofyolkspheres;thevitellineenvelopetransformsintoathinandhomogeneouschorion.

Pairedtestesarelocatedononesideofthebody,whereastheoppositesideisfilledbyamaleaccessorygland.Intestis,germinalcellsareembeddedinafewsomaticstromacells.Theearliestspermatogoniaformacompactgermarium,whereaslaterstagesaredispersedrandomlywithinthetestis.Spermatocytesarecharacterizedbyasuperficialspongylayer,formationofmitochondrialderivatives,lossofthenuclearenvelopeand

condensationofchromatininthreads.Asingleelectron-denselamellaappearsduringthespermatidstage,separatingchromatinthreadsfromalargespongybodysurroundedbyarcuate,double-membraneboundedcisterns.Inspermatids,thesuperficialspongylayerisabsent.Thetesticularcentralcellinthegermariumandstructuresrelatedtomeioticdivisionwerenotobservedinthetestes.

Spermatozoaaremultiformcells(approx.4×11µm)containingelectron-denselamella(ca.45nmthick)surroundedbymitochondrialderivativeswhichseparatechromatinthreads45-50nmthickfromremnantsofthespongybodyi.e.arcuatecisternprofiles.Spermatozoadepositedinfemalespermathecaaremoreelectrondense;theelectron-denselamellaisdeeplyfoldedseveraltimes,whereaschromatinthreadsarepresentinthecenterofthespermatozoonandareeitherflankedbylamellafoldsorlocatedmoreperipherallyundertheplasmalemma.Remnantsofthespongybodyarenotdiscernible.

Keywordsovariannutritivecell,intercellularbridges,ovary,oogenesis,vitellogenesis,testis,spermatogenesis,spermatozoa,gonadalsomaticcells,solitarybeeparasite,Acariformes

HowtocitethisarticleRożej-PabijanE.andWitalińskiW.(2018),GonadsandgametogenesisinChaetodactylusosmiae(Acariformes:Astigmata:Chaetodactylidae)aparasiteofsolitarybees.Acarologia58(4):801-824;DOI10.24349/acarologia/20184269

Introduction

Managedpollinatorssuchassolitarybeesandbumblebeesplayanimportantroleinfruitandvegetableproduction(VelthuisandVanDoorn2006).Recently,solitarybeeshavebeeninfocus,mainlythosebelongingtotheOsmiagenus.Theyaresuccessfullyusedforpollinatingearly-springfruitorchards.However,naturalpopulationsofOsmiaaredecliningasaresultofmultiplefactors(Krunićetal.1995;Krunićetal.2005).AstigmaticmitesfromtheChaetodactylidaefamily(suborderHemisarcoptoidea)areseriouspestsofmanagedpopulationsofOsmiacornutaandO.rufainEurope(Bosch1992;Krunićetal.2005).InthePalearctic,thebestknownandmostimportantisChaetodactylusosmiae(Dufour,1839)whileCh.krombeiniplaysasimilarroleintheNearctic(OConnorandKlimov2009;Klimovetal.2016).Recentworkindicatesthatthesetaxamaybefurtherdifferentiatedintotwopairsofsiblingspecies,Ch.osmiaeandCh.claudusinthePalearctic,andCh.krombeiniandCh.clavigerintheNearctic,probablyrelatedtorecentvicariancewithinthegenus(OConnorandKlimov2009).

Chaetodactylusosmiaehasseverallifestages(egg,protonymph,deutonymph,tritonymph,adultmale/female)(Michael1903;Fain1966;VanAsselt2000),butmostpeculiarforthegenusisthedeutonymphalstagewhichcanbeobservedintwomorphotypes,animmobileformwithintheprotonymphalexuviumandamobileform,theso-calledheteromorphicdeutonymph(hypopus)(forfurtherdetailssee:Stanisavljević1996;Krunićetal.2005).Theimmobiledeutonymphdevelopsbeforeautumnandallowsthesurvivalofmitesthroughthecolderpartoftheyear,whereasthemobiledeutonymphisadispersalstageandusesphoresytocolonizenewareasandhosts.Thedeutonymphalstagesdonotfeed.ManyexperimentalobservationsonCh.osmiaebiologywereprovidedbyChmielewski(1993)andStanisavljević(1996).

TherelationshipsbetweenCh.osmiaeandbeesarecomplex.Althoughtheactivemitestagesdonotfeedonbees,theycanbedetrimentalbyeatingcollectedpollenandnectar,therebydecreasingfoodavailability(kleptoparasitism).Moreover,ifabundant,themitescancompetewithbeesforspaceinsidethenest,leadingtoconsiderablereductionofthecocoon(Krunićetal.2001;Krunićetal.2005).Theattachmentofnumerousphoreticdeutonymphstobeesseverelyimpedesflightandinhibitsforaging(Krunićetal.2005).ForthesereasonsCh.osmiaeisconsideredaparasiteofsolitarybees.

AnumberofsynapomorphictraitsofthereproductivesystemandgametogenesisofAstigmata(Witaliński2014)supporttheirmonophyly(OConnor1984,2009;Norton1998;Schatzetal.2011).Thefemalereproductivesystemconsistsofoogenetic(primary)andinseminatory(secondary)parts.Theoogeneticpartcomprisespairedovaries,pairedoviductsandanunpairedoviduct(sometimecalleda“uterus”)whichemptiesintothepreoviporalchamber.Thechamberopensbyaprimarygenitalorifice(oviporus).Theinseminatorypartconsistsofaninseminatoryopening,inseminatorycanal,spermathecaandtwosperm-conveyingcordsleadingtoovaries.Inthemale,pairedtestes,paireddeferentductsjoined

togetherinanunpairedejaculatoryductemptyingatthetipoftheaedeagus(penis),arepresent.Inaddition,oneortwoaccessoryglandsopeningatthebeginningoftheejaculatoryductmayoccur.Thevariabilityofreproductivesystems,gonadstructureandgametogenesisinAstigmatawasreviewedbyWitaliński(2014),whoprovidedsomepreliminaryobservationsonCh.osmiae.

Materialsandmethods

FemalesandmalesofChaetodactylusosmiae(Dufour,1839)collectedfromOsmiarufanestswereprocessedasfollows.AftersexidentificationunderaNikonSMZ1000stereomicroscope(NikonInstrumentsEurope,Amsterdam,Netherlands),thespecimenswereimmersedone

byoneintoadropletofKarnovsky’sfixative(Karnovsky1965)(mixturecontaining2%formaldehydeand2.5%glutaraldehydein0.1Mcacodylatebuffer,pH7.2)onaparafilm-coatedmicroscopicslide,theanteriorpartofthebodywascutoffwithafinerazorblade

andtheremainingrearpartwastransferredintofreshfixativefor24hat4°C.Fixedmaterialwasrinsed4x15minin0.1Mcacodylatebuffercontaining8%sucroseandpostfixedwith1.4%osmiumtetroxidein8%sucroseovernightat4°C.ThespecimenswerethenwashedanddehydratedinagradedethanolseriesfollowedbypropyleneoxideandembeddedinEponTM812substitute(Sigma-Aldrich).SemithinandthincrosssectionsweresectionedonaReichert-Jungultramicrotomefittedwithadiamondknife.SemithinsectionsonmicroscopicslideswerestainedwithanAzurIIandmethyleneblue(1:1)mixture,whereasthinsectionswerecollectedonformvarcoatedgrids,contrastedwithuranylacetateandleadcitrateaccordingtostandardprotocols(VenableandCoggeshall1965).SemithinsectionswereexaminedanddocumentedunderanOlympusBX51microscope(OlympusCorporation,Tokyo,Japan),whereasthinsectionswereexaminedunderatransmissionelectronmicroscope,JEOLJEM100SX(JEOLLtd.,Tokyo,Japan),at80kVintheDepartmentofCellBiologyandImaging,InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity.

Results

Thefemalereproductivesystem

Thefemalereproductivesystemiscomprisedofoogeneticandinseminatoryparts.Theoogeneticpartiscomposedof(1)pairedovarieslocatedsymmetricallyinthemostposteriorpartoftheidiosoma(Fig.1B),(2)twooviductspossessingposteriorly-directedandanteriorly-directedsections,(3)theunpairedoviduct,and(4)apreoviporalchamberendingin(5)anoviporus.Theinseminatorypartconsistsoffourelements.Itbeginswith(1)aninseminatoryopeninglocatedatthebottomofthetubularcuticularprotrusionattheposteriorendofthebody(Fig.1A-B),(2)ashortfunnel-shapedandcuticle-linedinseminatorycanal(lengthca.43µm,diam.fromca.7µmatinseminatoryopeningtoca.36µmatexittospermatheca)leadingto

(3)alargespermatheca(120x200µm,widthxlength)(Fig.1B-F),andtwoveryshort(4)conveyingcordsconnectingthebasalpartofthespermathecawithadheringovaries.

Theovary

Eachovary(Fig.1C-D)iscomposedofthreecellularelements:onenutritiveovariancell,previtellogenicoocytes,andseveralsomaticstromacells.Thenutritivecellandoocytesaregerm-linecells,whereasthestromacellsaresomaticandfillnarrowspacesbetweengerm-linecells(Fig.1C-D).

Nutritivecellandprevitellogenicoocytes

Theovariannutritivecell(Fig.1C)issubsphericalandverylarge(70–90µmindiam.),andadherestothepostero-dorsalmarginoftheovary.Itischaracterizedbyagiantmultilobularnucleuswithmanydistinctnucleoli(Fig.2).Thenuclearenvelopecontainsmanyporeareaswithadheringnuagematerial.Thecytoplasmisfullofribosomes/ribosomesubunits,crista-typemitochondriaforminglargeandcompactassemblages,multiformlysosomesandoccasionalGolgibodies(Fig.2).Mostconspicuousforthenutritivecellistheformationofnumerousprotrusionspenetratingdeeplyintothesurroundingstromacells(Fig.3).

Previtellogenicoocytesaresituatedintheventralpartoftheovarybelowthenutritivecell(Fig.1C),aswellasinfrontofthenutritivecell(Figs1D,3A-B).Theyareirregularinshapeandeachoocyteisconnectedtothenutritivecellviaafunnel-shapedintercellularbridge(Fig.3A).Thenucleusofsmallprevitellogenicoocytesisirregularinshapeduetothepresenceofmanyconcavitiesontheenvelope,containingnuclearpores.Inadvancedprevitellogenicoocytes(Fig.4),thenucleusismorespherical,withanumberofshallow,pore-containingdepressionsoftheenvelopetowhichnuagematerialadheres(Fig.4A-B).Nuagematerialinthenutritivecellandsmallprevitellogenicoocytesissimilarlyfibrillar,butismorecompactinadvanced,

Figure1LightmicroscopicimagesofaChaetodactylusosmiaefemale.Femaleindorsalview(A)andtherearpartoftheidiosoma(B)withmarkedpositionofovaries(OV),nutritiveovariancells(NOC),spermatheca(receptaculumseminis)(RS)andinseminatorycanal(ic).C-F–Semithincrosssectionsofidiosomamarkedapprox.atlevelson(A).Notethesphericalshapeofnutritivecells(NOC)andirregular,wrinkledoutlineofprevitellogenicoocytes(po)(C,D).Earlyvitellogenicoocytes(vo’)locatedinthebackwardrunningoviductarespherical(D,E).Moredevelopedvitellogenicoocytes(vo)andeggs(eg)intheforwardrunningoviductsshowincreasingyolkfilling(D-F).Inthecentralpartofthespermatheca,sperm-containingmaterialdeliveredbyamaleispresent(D,E),whereastheposterior-mostandtheanteriorpartofthespermathecaarefreeofsuchmaterial(C,F).Sperm-containingmaterialalsoshowslarge,ovalstructuresofmediumdensity(D,E)..Scalebars:

0.1mm.

Otherabbreviations:I-IV–legsI–IV;blackarrow–inseminatoryopening;blackasterisk–basalpartofspermatheca;whiteasterisk–spacesintheovaryfilledwithstromacells;a–analslit;aa–analatrium;col–postcolon;eg–oviductaleggsurroundedbychorion;g–foodenvelopedbyperitrophicmembraneinalimentarytract;gn–gnathosoma;ic–inseminatorycanal;NOC–nutritiveovariancell;po–previtellogenicoocytes;RS–spermatheca(receptaculumseminis)filledwithmale-derivedmaterialcontainingspermatozoa;vo’–earlyvitellogenicoocyteinbackwardrunningoviduct;vo–advancedvitellogenicoocyteinforwardrunningoviduct.

Figure2Chaetodactylusosmiaefemale,twoaspects(A,B)ofnutritiveovariancell.TEM.Sectionsthroughmultilobularnucleus(n)containinglargenucleoli(nu).Cytoplasmfilledwithribosomesalsocontainslipiddroplets(L),manylysosomes(Ly),groupsofmitochondria(m)andsmallvesicularGolgibodies(Gb).Granularnuagematerial(asterisk)isusuallypresentinconcavitiesofthenuclearenvelope.Scalebars:A–10μm,B–5μm.

largerprevitellogenicoocytes(Figs2,3,4A-B).Insmallprevitellogenicoocytes,crista-typemitochondriatightlysurroundthenucleus,remaininginlargeroocytesclosetothenucleus(Fig.3).SmallGolgibodiesoccursporadically(Fig.4B).Intercellularbridgesarefunnel-shapedandfilledwithelectron-densegranulesandmicrotubulesinfan-likearrangements(Fig.4D).

Stromacells(Figs3,4A,C-D)fillallspacesbetweengerminalcells;theyarefewinnumberanddifficulttotally.Theirelectron-lucentcytoplasmcontainsribosomes,ERprofiles,mitochondriaandgroupsoflysosomes.StromacellshavecharacteristicsubplasmalemmalERcisterns(Figs3A-B,4A,C-D)runningalongthestromacell/nutritivecellborders.Suchcisternsarealsopresentalonglargerprotrusionsofthenutritivecell,aswellasalongthesurfaceofprevitellogenicoocytes.

Vitellogenicoocytesandvitellogenesis

Oocytesenteringvitellogenesisloseconnectionwiththenutritivecellviaintercellularbridgesandaresequesteredintotheoviductwhichstartsventrallyattheanteriorpoleofeachovary.Atfirst,theoviductsrunposteriorlyjustbelowtheovariesthenturnlaterallyandrunanteriorlytoeventuallyfuseintoanunpairedoviduct.Thus,pairedoviductshavebackward-runningandforward-runningparts.

Theoocytesintheoviductbecomemoresphericalastheygrow(Fig.1D-E).Sphericalnucleiare“empty”andhaveacentrallylocatednucleolus.Inoocytesenteringthesecond,forward-runningpartoftheoviduct(Figs1D-F,5A-B),twotypesofinclusionscanbediscernedinthecytoplasm:lipiddropletsusuallyassociatedwithglycogen,andproteinyolkspheresindifferentstagesofcondensation(Fig.5A).Atthebeginningofyolkformation,largeproteinyolkspheresfilledwithmoderatelydenseflocculentmaterialarepresent(Fig.5A).Later,theircentersgrowmoredensetofilltheyolkspherecompletely.Thisyolkmaterialisfurtherconcentrated,showinglesscondensedareasonadensebackground.Insomeyolksphereselectron-lightprofileswithsharpedgescanbeseen;thesearepossiblyremnantsofcrystallineinclusionsremovedduringsamplefixationforTEM(Fig.5B).

Vitellineenvelopeandchorion

Thevitellineenvelopeisformedonthesurfaceofearlyvitellogenicoocytes.Itiscomposedofmaterialdeliveredfromthecytoplasmviavesicles(Fig.5Ainset)andislayeredratherthanhomogeneous,visibleasalternatinglightanddarkstriae(Fig.5A).Asvitellogenesisproceeds,thevitellineenvelopereorganizesandformsamuchthinnerandhomogeneouslayer,thechorion(Fig.5B).

Themalereproductivesystem

ThemalereproductivesysteminCh.osmiaecontainstwotestes,paireddeferentducts,anunpairedejaculatoryductwhichopensatthetipoftheaedeagus,aswellasanunpairedaccessorygland.Testesarelocatedasymmetricallyontherightsideintheposteriorpartoftheidiosoma:therighttestisoccupiesalateralposition,whereastheleftoneisshifteddorsally(Fig.7A).Dependingonthespecimen,thelefttestismaybelongerandrunsunderthedorsalcuticleuptothelevelofthefourthcoxae,or,alternatively,bothtestescanterminateearlier,closetotheleveloftheaedeagus(compareFigs6and7B).

Thearrangementofdeferentductsiscomplicated.Theleftductstartsattheposteriorendofthelefttestisthenrunsforwardundertheaccessoryglandtoeventuallypasstotherightsideinfrontoftheanalatrium.Therightductappearsventrallyinthecentralpartoftherighttestisandrunsantero-ventrallytofusewiththeleftdeferentductintotheejaculatoryduct.

Theejaculatoryduct(Figs6B,7B)isatube(lumendiameternarrowingapprox.from6µmto2µm)withcuticlelining,enteringtheaedeagusandopeningatitstip(Fig.6B).

Apyriformmaleaccessorygland(diam.ca.90-100µm)(Fig.7A)islocatedontheleftsideoftheidiosomaandextendsfromtheposteriorendoftheidiosomauptothelevelofthecoxae

Figure3Chaetodactylusosmiaefemale,twoaspects(A,B)oftheperipheralpartofthenutritiveovariancell.TEM.(A)Nutritivecell(NOC)showsmitochondria(m)andlysosome(Ly),andisconnectedviaanintercellularbridge(blackasterisk)withanearlyprevitellogenicoocyte(po).Theotherprevitellogenicoocyteispartlyvisibleabove.Theoocytenucleus(n)isirregularinoutlineandenvelopeconcavitiescontainelectrondensenuagematerial(whiteasterisk).Mitochondria(m)intheoocytecloselysurroundthenuclearenvelope.Somaticstromacells(SC)showfewribosomesandarepenetratedbymanyprotrusionsofanutritivecell(whitearrows).NoteERcisterns(blackarrows)runninginstromacellparalleltoitscontactswithgerm-linecells(i.e.nutritivecellandoocytes).(B)Anotheraspectofthenutritivecellperipherysimilarto(A).Notealobeofthenucleus(n)withanucleolus(nu)inthenutritivecell;concavitiesofthenuclearenvelopearefilledwithnuagematerial(whiteasterisk)whereasmitochondria(m)areclusteredclosetothenucleusinboththenutritivecellandoocyte(po).NutritivecellprotrusionsandERcisternaeinstromacellsareindicatedbywhiteandblackarrows,respectively.

Otherabbreviations:Ly–lysosomeinstromacell.Scalebars:A–2μm,B–5μm.

Figure4Chaetodactylusosmiaefemale.TEM.(A)Advancedprevitellogenicoocyteshowingnucleus(n)withnucleolus(nu).Thenuclearenvelopehasconcavitiesinsitesrichinpores,withadheringnuagematerial(asterisk).Mitochondria(m)aregroupedaroundthenucleus.Anotheroocytesectionedtangentially(po)isalsopresent.Inthestromacell(SC),anarrowmarksaconspicuousERcisterna.(B)Enlargedfragmentoftheoocytemarkedin(A):notenucleus(n),aGolgibody(Gb),nuage(asterisks)andmitochondria(m).Notethedifferentappearanceofnuagematerialwhichisnotgranularasinthenutritivecellandearlyoocytes(comparewith2Band3A-B)..(C)Agapbetweenanutritivecell(NOC)andprevitellogenicoocyte(po)filledwithastromacell(SC)inwhichseveralnutritivecellprotrusions(whitearrows)areembedded.ERcisternsinthestromacellarealsopresent(blackarrows).(D)Intercellularbridge(IB)connectinganutritivecell(NOC)andprevitellogenicoocyte(po),filledwithelectron-densegranularmaterialinfan-likearrangement.Golgibody(Gb)innutritivecellandERcistern(arrow)instromacell(SC)aremarked.Scalebars:A–2μm,B-D–1μm.

Figure5Chaetodactylusosmiaefemale.TEM.(A)Vitellogenicoocyteinupper(forward-running)partofoviduct(Od),containingyolkspheresinsubsequentstagesofdevelopment(Y’,Y’’andY’’’)andlipiddroplets(L)surroundedbyglycogen.Lamellarvitellineenvelope(VE)coversthesurfaceoftheoocyte.Inset:Highermagnificationofvitellineenvelopeadheringtooviduct(Od).VesicledeliveringvitellineenvelopematerialfromGolgibodyisindicatedbyanarrow.(B)Moreadvancedvitellogenicoocytecoveredwiththinandhomogeneouschorion(Ch)intheupperoviduct(Od).Yolk(Y)spheresoftwotypes,largerandlessdenseandsmallerandmoredense,containingcristallineelectron-lucidinclusions.Inset:Yolkspherewithelectron-lucidinclusionsandmeanderingstructure(arrow).Scalebars:A,B–5μm,Ainset,Binset–2μm.

Figure6LightmicroscopicimagesofChaetodactylusosmiaemale.Maleinventralview(A)andtherearpartofidiosoma(B).Scalebars:

0.1mm.Abbreviations:I-IV–legsI-IV;linesAandB–levelsofcross-sectionsinFigure7;a–analslit;ae–aedeagus;DE–ejaculatoryduct;gn–gnathosoma.

ofthefourthleg.Thewalloftheglandisvariableinthicknessandformsseveralconcavitiesmainlyinitsrearpartandalongitsdorsalsurface.Thelumenisfilledwithahomogeneouslucentmaterial(Fig.7A).Theglandemptiesventrallyintotheleftdeferentductapproximatelyattheleveloftheanteriormarginoftheanalatrium.

Thetestis

Testesareirregularinshapeandcontainmalegermcellsembeddedintesticularstromacells.Ineachtestisearlyspermatogoniaformagermarium(Fig.7A-B)inwhichcellsadheretightlytoeachother(Fig.8A-B).Cellsthatlosecontactwiththegermariumproceedwithspermatogenesis(Fig.8B).Suchcellsdispersesingly,thusthesequentialdistributionofstagesofspermatogenesisisnotmaintained.

Spermatogenesisandspermstructure

Theearliestspermatogoniathatformthegermarium(Fig.8)aresmallpolygonalcells(ca.4.5x6.5µm)tightlyadheringtoeachother,withaspherical,relativelylargenucleuscontainingacentralnucleolus.Thecytoplasmcontainsfreeribosomes,crista-typemitochondriaandoccasionallyvisibleGolgibodies.Nuagematerialadheringtoporousareasofthenuclearenvelopealsooccurs.

Figure7LightmicrographsofcrosssectionsthroughidiosomaofChaetodactylusosmiaemaleatlevelsasindicatedinFigure6.Outlinesoftestesaremarked.Scalebars:0.1mm.

Abbreviations:IV–legIV;asterisk–germarium;a–analslit;AG–maleaccessorygland;col–postcolon;DE–ejaculatoryduct;OG–opisthonotalgland;LVD–leftdeferentduct.

Figure8Chaetodactylusosmiaemale,twoaspects(A,B)ofgermarium.TEM.(A)Earlyspermatogoniaassembledingermariumadheretightlytoeachother.Theypossessalargenucleus(n)withprominent,usuallycentralnucleolus(nu)andmitochondria(m)dispersedinthecytoplasm.Nuagematerial(asterisks)ispresentinseveralsitesaroundthenuclei.(B)Earlyspermatogoniaingermariumshowfeaturesasin(A),whereasdevelopingspermatogoniumseparatedfromgermarium(rightuppercorner)islarger,roundishandhasalargernucleus(n’)withperipheralnucleolus(nu).Spacesbetweendevelopingspermatogoniumandgermariumarefilledwithasomaticstromacell(SC).Scalebars:A–2μm,B–5μm.

Figure9Chaetodactylusosmiaemale.TEM.(A)Closerviewofdevelopingspermatogoniumsurroundedbyastromacell(SC).Nucleus(n)isroundishandcontainsaprominentnucleolus(nu).Thereareanumberofribosomesinthecytoplasm,aswellasdispersedmitochondria(m).Nuagematerial(asterisks)adherestonuclearenvelopeinsitesrichinpores.Notethatthesurfaceofthecellissmoothandnotcoveredwithaspongylayer.(B)Earlyspermatocyteshowingspongylayer(SpL)atcellsurfacewhichiswrinkledandadherestostromacell(SC).Nuagematerial(asterisk)adherestolargenucleus(n).Numerousmitochondria(m)arelocatedratherperipherallyinthecell.Scalebars:2μm.

Figure10Chaetodactylusosmiaemale.TEM.(A)SpermatocytemoreadvancedthanthoseinFigure9B,embeddedinstromacell(SC).Notethickerspongylayer(SpL)coveringthecellsurface,largenucleus(n)andnuagematerial(asterisks).Peripheralcytoplasmcontainsmanymitochondrialderivatives(md):theyareroundishandpossessonecircularcrista.AGolgibody(Gb)ispresentunderthespongylayerofaneighboringspermatocyte(rightbottom).(B)Peripheralpartsoftwospermatocytesseparatedbyastromacell(SC).Comparethethicknessofthespongylayer(SpL)intheupper,lessadvancedspermatocyteandthatofthelower,moreadvancedcell.BothcellshavesimilarGolgibodies(Gb)andmitochondrialderivatives(md)butthemoreadvanced,lowerspermatocyteshowsemptyspacesinitsperipheralcytoplasm.Scalebars:2μm.

Figure11Chaetodactylusosmiaemale.TEM.(A)Advancedspermatocyteseparatedwithstromacell(SC)fromneighboringspermatocytes.Itshowsaspongylayer(SpL)andemptyspacesinperipheralcytoplasminwhichmanymitochondrialderivatives(md)arepresent.Nuagematerial(asterisks)surroundsthenucleus(n)containingasmall,compactnucleolus(nu).(B)Spermatidinanearlystageofspermiogenesisembeddedinstromacell(SC).Thenucleusisnolongervisibleandchromatin(Ch)formsfinethreadssurroundedbyfociofelectron-densematerial(whitearrows).Mitochondrialderivativesaremultiform,eitherlargewithmeanderingcristae(md)orsmallandvesicular(blackarrow).(C)Sectionthroughspermatidatasitewhereanelectron-denselamella(La)isformed(thearrowindicatesadiscontinuityinthelamellasuggestingtheadditionofdensematerialtoitsmargin).Spongybody(SB)derivedfromthesuperficialspongylayerofthespermatocyteisalsovisible.Moreorlessdensemitochondrialderivatives(md)canflankthelamella.Spongybody(SB)derivedfromthesuperficialspongylayerofthespermatocyteisalsovisible.Scalebars:A,B–2μm,C–1μm.

Figure12Chaetodactylusosmiaemale.TEM.Twoconsecutivestepsinspermiogenesisshowingspermatidjustafterlamella(La)formation

(A)andsubsequently(B)whenmultiformdensemitochondrialderivatives(md)appearalongthelamella.In(A)thespermatidissectionedthroughthelamella(La),separatingchromatinthreads(Ch)andorganizingthespongybody(SB).In(B)thespermatidsectionshowsthelamella(La)flankedbydenseandmultiformmitochondrialderivatives(md).Severalsmallvesicularmitochondrialderivativescanbeseen(arrows)closetotheleftendofthelamella.Scalebars:2μm.

Otherabbreviations:SC–stromacell.

Figure13Chaetodactylusosmiaemale.TEM.(A)Latespermatidshowingchromatinthreads(Ch),denselamella(La)andelaboratespongybody(SB).Smallvesiclesofmitochondrialderivatives(md)arepresentaroundthespongybodyandalongthedenselamella;however,largerandmoreelectron-densemitochondrialderivativesarealsopresent(arrows).Anastomosingtubulesofthespongybodyareencircledbycharacteristicarcuatecisterns(cSB)formedbydoublemembranessimilartoannulatelamellae.(Inset)Arcuatecisternsandtubulesofspongybodyunderhighermagnification.(B)Twoverylatespermatidsnotyetcondensed(1)andfragmentsoffinallycondensedspermatids(2,3).Eachspermatidcontainsonemeanderingdenselamella(La)whichseparateschromatinthreads(Ch)fromdoublemembranevesicles(vSB)putativelyderivedfromarcuatecisterns(cSBinA)surroundingthespongybody.Multiformmitochondrialderivatives(md)arefrequentlylocatedalongdenselamella.Scalebars:A,B–2μm,Ainset–1μm.

Otherabbreviations:SC–stromacell.

Spermatogoniathatseparatefromthegermariumstarttogrowandbecomespherical,withalargesphericalnucleuscontainingaprominentnucleoluseitherinitscenteror,morefrequently,closetothenuclearenvelope(Figs8B,9A).Theenvelopepossessesmanyporesandnuagematerialinitsvicinity.Thecytoplasmcontainsanumberofribosomes,ERprofiles,crista-typemitochondriaandfewGolgibodies.

Duringspermatogenesis,mitochondria,ERprofilesandGolgibodiesdislocatetotheperipheralcytoplasm.ActiveGolgibodiesprovidevesicleswhichfusetogetherformingaspongylayerofvariablethicknesswiththeplasmalemma(Fig.9B).Sincenosignsofmeioticdivisionwereobserved,wearbitrarilynamedgermcellssurroundedbyaspongy

layerasspermatocytes(seeDiscussion).Atthistime,mitochondriatransformgraduallyintomitochondrialderivatives,atfirstbecomingsphericalandwithonecircularcrista.Thenucleusandnucleolusarestillpresent,butmeanderingemptyspaces(i.e.unoccupiedbyribosomes)appearintheperipheralcytoplasm(Figs10A-B,11A).

Atthebeginningofspermiogenesisthenuclearenvelopedisappearsandnuclearmaterialisvisibleasthinthreadssurroundedbyelectron-densegranules(Fig.11B).Thesuperficialspongylayerflowstoonepoleofthecellandtheninternalisestoformaspongybodythatisvisibleasagroupofmeanderingtubularprofiles(Figs11C,12A,13A)surroundedbypeculiararcuatecisternsformedbyannulateddoublemembranes(Fig.13A,inset).Somemitochondrialderivativeswithdistinctcristaearestillpresent,butmanydiminishtoformsimplifiedvesicularstructures.Thespermatidcytoplasmcontainsanelectron-denselamella(Fig.11C)locatedbetweentwocompartments,thechromatinthreadsandthespongybody(Figs12A,13A).Insomespermatids,bothsidesofthelamellaareflankedbyelectron-densebodiesorgranulesthatmaybemitochondrialderivatives(Figs12B,13B).

Spermcellsfoundinthemalearemultiform,approximately4x11µm,andcanrepresentdifferentstagesofcondensation(Fig.13B),increasingwithmaturity.Theycontainmeanderingdenselamellaseparatingthechromatinthreadsfromarcuatecisternprofiles,beingremnantsofspermatidspongybody.Chromatinthreadsare45-50nmthick,whereasthethicknessofdenselamellaisca.45nm.

Spermatozoadepositedintospermatheca(Fig.14AB)tightlyadheretoeachotherandshowconsiderable,uniformelectrondensity.Thedenselamellaisdeeplyfoldedandflankedbylesscondensedcytoplasmcontainingmitochondrialderivativesofvariableshapeanddensity.Chromatinthreadsarefrequentlylocateddeeplywithinthelamellainfoldinginasubcentralpositioninthecell(Fig.14B),butsometimesmoremarginally(Fig.14A).Aschematicrepresentationofamaturespermcell(fromthespermatheca)isgiveninFig.14C.

Besidesspermatozoa,thespermathecacontainslarge,ovalstructuresofmoderatedensitiesofunknownorigin(Fig.1DE).

Thestromacells

Thenumberofsomaticstromacellsinthemalegonadislowastheirnucleiareonlyoccasionallyobserved.Thecytoplasmofthestromacellislessdensethanthatofgermcellsandsimilartotheovarianstromacell,thuscontainingmanyvesicularortubularprofiles.Stromacellsfillverynarrowspacesbetweenmalegermcellswiththeexceptionofthegermariumandgroupsofspermatozoa.

Discussion

Arecentreviewonreproductivesystemsandgametogenesisinastigmaticmites(Witaliński2014)showedthatthefemalereproductivesystemsaremuchlessvariablethanthoseofmales.Accordingly,thefemalereproductivesysteminCh.osmiaeisverysimilartothoseinotherspeciesstudied,i.e.itpossessestwoparts,aprimary(theoogenetic)andsecondary(theinseminatory)part.Bothsectionsmeetatsiteswheresperm-conveyingcords–emergingfromthebasalpartofthespermatheca–reachtheovarysurface.Thelengthofsperm-conveying

Figure14Chaetodactylusosmiaefemale.TEM.Spermatozoadepositedinspermatheca.(A)Twospermatozoa,eachwithhighlyfoldedlamella(La),chromatin(Ch)andmitochondrialderivatives(md).(B)Spermatozoasimilarto(A)inhighermagnification.Notelocationofchromatin(Ch)withindeepinfoldingofdenselamella(La).Variablestructureofmitochondrialderivatives(md)isalsoevident.(C)schematicrepresentationofspermcellfromfemalespermatheca.Scalebars:A,B–2μm,C–5μm.

Otherabbreviations:Ch–threadsofchromatin;La–electron-denselamella;md–mitochondrialderivatives.

cordsvariesinAstigmata,butisroughlycorrelatedwiththelengthoftheinseminatorycanal.Thisisbecausethepositionofthespermathecaalsovariesrelativetotheposteriorendoftheidiosomawheretheovaries,thetargetforspermcells,areusuallylocated.AnextremeexampleisFalculiferrostratus,whoseinseminatorycanalisupto300µmlong(Witaliński2014)andsperm-conveyingcordsareaccordinglyveryelongated.InCh.osmiaetheinseminatorycanalisveryshortandwide,andthebasalpartofthespermathecaislocatedjustovertheovaries,resultinginminutesperm-conveyingcords.

SincePrasse(1968),whofirstobservedtheovariannutritivecellinAstigmata,thiscellwasfoundinallspecies.However,itslocationvarieswithintheovary.Theovariesinthestudiedspecieshavealargeovariannutritivecelllocatedintheupperpartofthegonadandprevitellogenicoocytesaresequesteredtoitsventralpart.Thisissimilartopsoroptidianovaries,

e.g.inSarcoptesscabieithenutritivecellintheovaryislocatedeitherantero-dorsally(Desch2001)ordorsallyinNotoedrescati(Witaliński2014).InGlycyphagusdomesticus,thelocationofthenutritiveovariancellissimilarbutmorevariable,eitherdorsalorsubcentral(Witaliński2014).Thefunctionofthenutritiveovariancellismanifestedbyanenlargedmultilobularnucleuscontainingmanyprominentnucleoli,andacytoplasmrichinribosomesandotherorganelles.Allearlyprevitellogenicoocytesareconnectedtothenutritivecellviafunnel-typeintercellularbridgesallowingthedeliveryofsubstancesfromthenutritivecelltooocytes.ThistypeofintercellularbridgeischaracteristicforAstigmataexceptforHistiostomatoidea(Histiostoma,Bonomoia)whereadifferenttype,thediaphragm-crossedbridge,wasfound(FlorekandWitaliński2010b,Witalińskietal.2014).Interestingly,Ch.osmiaehasaveryhighovipositionrate,producingfrom135upto515eggs(from1.2to10.3eggsdaily)duringtheovipositionperiod(21to46days)(Chmielewski1993).However,ourstudydidnotshowanynoteworthyspecializationsinbridgestructurethatcouldberelatedtoahighrateofnutritivecell-oocytesupplementation.

OvariesinCh.osmiaeexhibittwodistinctivefeatures:thenutritiveovariancellpossessesnumerousprotrusionspenetratingthesurroundingstromacells,andstromacellsshowconspicu-ousERcisternsrunningparalleltocontactsofstromacellandgerm-linecells(nutritivecellandoocytes).ProtrusionsformedbythenutritivecellwerealsofoundinSarcoptesscabiei(Desch,2001)andGlycyphagusdomesticus(Witaliński2014),butERcisternsunderthestromacellsurfaceareuniqueforthestudiedspeciesandtheirroleisunknown.Wecanonlyspeculatethatgerminal-somaticcellcontactsareimportantand–atleastinthisspecies–requireenlargementandarepossiblyequippedwithsomespecializations.

Giantnutritivecellsareconnectedwithalargepoolofoocytes.Wedidnotobservesynaptonemalcomplexesinnucleioftheearliestgerminalcells,suggestingthatoogoniaenteringthemeioticprophaseoccurintheearlierinstar,andinadultfemalesnutritivecell-connectedgerminalcellsareprevitellogenicoocytes.ThismatchesobservationsongonaddevelopmentinHistiostomainwhichmeioticdivisionswerecompletedinthetritonymph,thusbeforethefinalmolttoadultfemale(Witalińskietal.2014).However,insomespeciessuchasSancassaniaberlesei,themeioticdivisioniscompletedmuchlater,invitellogenicoocytespassingthroughthemiddlepartoftheoviduct(Walzletal.2004).

Adetailedaccountofvitellogenesiswasnotanaimofthisstudy,thereforeweonlyprovideaninitialassessmentofthisprocessinCh.osmiae.Vitellogenesisentailsautosynthesisandoccursinoviductaloocytesonthebasisofmaterialsdeliveredfromthenutritivecellviaintercellularbridgesandstoredinooplasm,asequenceofeventsgenerallyacceptedforallAstigmata.Typically,vitellogenesisleadstotheformationoftwotypesofyolkmaterial,lipidyolkandproteinyolk,tightlyfillingtheoocyte.Theformerconsistsoflipiddropletsassociatedwithglycogengranules,whereasproteinyolkformslargesphereswithheterogeneouscontent;somecontainelectron-lucentparacrystallineinclusions.

DuringvitellogenesistheoocyteiscoveredwithavitellineenvelopewhichinthisandotherspeciessuchasSancassaniaanomala(=Caloglyphusanomalus)(Reger1977)andAcarussiro(Witaliński1993)islamellar,butlaterconvertsintoathinnerandhomogeneouslayer,thechorion(forfurtherdiscussiononvitellineenvelopeandchorionorigin,aswellaschorional

glandsinAstigmata,see:Witaliński2014).ItisbelievedthattheentranceofspermintotheoocyteoccursbeforethedevelopmentofthevitellineenvelopeinallAstigmata(Prasse1968,Witalińskietal.1986,Witaliński1988)andiscorrelatedwiththelackofanacrosomalcomplexinastigmatanspermatozoa(LianaandWitaliński2005,FlorekandWitaliński2010a,Witaliński2014).

TheAstigmatareproductivesystemsinmalesaremorevariablethaninfemales(Witaliński2014).Differencesmainlyconcernthenumberoftestes(unpairedorpaired)andtheirdistribution,presenceorabsenceofatesticularcentralcellinthetestisgermarium,aswell

asthenumberandstructureofmaleaccessoryglands.Inthestudiedspecies,thetestesarelocatedone-sidedlyduetotheoccupationoftheothersideoftheidiosomabyalargeaccessoryglandasinCaloglyphusberlesei(WitalińskiandWalzl1995).InotherAstigmata,however,aseparateaccessoryglandiseitherabsentanddoesnotinfluencethesymmetricdistributionofthetestes,ortwoglandsofdifferentstructurearepresentasinLardoglyphuskonoi,leadingtothepositionofonetestisinfrontoftheother(VijayambikaandJohn1975).

Theearlieststagesofspermatogenesisarespermatogoniaassembledinthegermarium.Withinthegermarium,aconspicuous,so-calledtesticularcentralcellmaybepresent.Thecentralcellbelongstothegerminallineandindevelopinggonadsisconnectedtotheearliestspermatogoniaviatubularintercellularbridges,whichlaterdisappear(Witalińskietal.2014).Thus,thetesticularcentralcellseemstobefunctionalindevelopingtestis,butlater,itcaneitherremainwithinthegermariumormaydeteriorate.ThecentralcellwasnotobservedinthetestisofthestudiedspeciesasinmanyotherAstigmata(Walzl1992;WitalińskiandWalzl1995;FlorekandWitaliński2010a),althoughitispresentinsomespecies(Prasse1968;BakerandKrantz1985;Witalińskietal.1990;FlorekandWitaliński2010a;Witalińskietal.2014).Aroleforthegermariumandtesticularcentralcellinsuppressingspermatogoniadevelopmenthasbeenpostulated(forfurtherdetailssee:Witaliński2014).

SpermatogenesisinAstigmatacanproceedinoneoftwoways,withorwithoutmeioticdivision,ineffectdeterminingtheploidyofmales.Inhaplo-diploidspeciesinwhichfemalesarediploidandmaleshaploid,arrenotokyoccursandmeiosisisabsent.Meiosisoccurs,however,indiplo-diploidspeciesandtwoofitsconsequencescanbeobserved:synaptonemalcomplexeswithinprophasenucleiofspermatogoniaenteringtheprimaryspermatocytestage,aswellastwo-orfour-cellclonalgroupsresultingfrommeioticdivisions.InCh.osmiaeneithersynaptonemalcomplexesnorclonalgroupsofgerminalcellswerefound.ThisconfirmsthesuggestionbyStanisavljević(1996)thatthespeciesishaplo-diploid(withhaploidmales),whichisincontrasttoChmielewski’sopinion(Chmielewski1993)thatthespeciesisdiplo-diploid.

Theabsenceofmeiosisentailsadifficultyindefiningthespermatocytestage.ForCh.osmiaeweusedadifferentcriteriontodefinethespermatocytestage,asobservedindiploidmalesofAstigmataandGamasida,inwhichGolgibodiescontributetotheformationofadistinctivesuperficialspongylayerenvelopingthespermatocyte.

Atthebeginningofspermiogenesis,meanderingmembranesofthespongylayerflowtoonepoleofthecellwheretheyareinternalised,formingtheso-calledspongybody.This

conspicuousstructurehasbeenfoundinCarpoglyphuslactis(FlorekandWitaliński2010a)andwasalsoreportedinDiplaegidiacolumbae(Buchholz)andGlycyphagusdomesticus(DeGeer)(Witaliński2014).Insomecases(e.g.Ch.osmiae,G.domesticus),thespongybodycandevelopintoaquitecomplexstructureofunknownfunction.Inspermcells,however,onlysimplifiedremnantsofspongybodycanbediscerned.

Asspermatogenesisproceeds,germcellmitochondriasubstantiallymodifytheirstructuretoformroundishbodiescontainingonecircularcristainspermatocytes.Subsequently,however,someundergostructuraldegradationleadingtotheformationofsmallerelectron-densebodiesinwhichmembranesarenolongervisible.Despitetheoccurrenceofthesemitochondrialderivatives,mitochondria-derivedbodiesofmoreconventionalstructure(i.e.withvisiblemembranes)canstillbepresenteveninspermdepositedinthefemale.

DistinctivecharactersofAstigmataspermcellsincludechromatinthreadsandelectron-denselamellae.Denselamellaecanbesingular,asinHistiostomaferoniarum(Lianaand

Witaliński2005),Carpoglyphuslactis(FlorekandWitaliński2010a)andCh.osmiae,butareusuallymultipleandvariableinlengthanddistribution(LianaandWitaliński2005,Witaliński2014).TheiroriginisrelatedtothetransformationofERcisterns.InthecaseofCh.osmiae,theoriginofsingulardenselamellawasnotobservedindetail,butsomeimages(Fig.11C)suggesttheformationoffocaldensitieswithinlamellamargins.Suchdensitiescanlatercoalescetoformthedensefillingoflamella.Theroleofdenselamellaeisstillunderspeculation,butsincespermchromatinisfrequentlysurroundedbythelamella(e),themechanicprotectionofgeneticmaterialmaybeanexplanation.InCh.osmiae,thedenselamellaofinseminatingspermatozoaisfoldedmanytimesover,andchromatinthreadsfrequentlyoccupyacentralpositioninsuchinfoldings.SomeprotectionofchromatinmaterialseemstobebeneficialsinceinmostAstigmatathespermisdeliveredintothefemalespermathecaunderconsiderablepressure,firstthroughanarrowmaleejaculatoryductandthenthroughthefemaleinseminatoryduct.Furthermore,spermatozoacrawlviasperm-conveyingcordsfromthespermathecatoovarieswhichcanleadtoahighincidenceofdeformation.ThereforesomeunusualcharactersofAstigmataspermincluding1)multiformshapeofspermatozoa,2)chromatininthreadsinsteadofinarigidnucleus,and3)thecentrallocationofthechromatinshelteredbyelectron-denselamellae,canbeexplainedasanadaptiveresponsetomechanicalstressduringandafterinsemination(Witaliński2014).IntheCh.osmiaefemale,theinseminatoryductisshortandwideandsperm-conveyingcordsareminute.Therefore,themainsourceofmechanicalstresstospermatozoamaybethepumpingofspermbythemalethroughthenarrow(diameterca.2

µm)ejaculatoryduct.

Conclusions

FemaleandmalereproductivesystemsinChaetodactylusosmiaematchthegeneralfeaturesofotherastigmatanspecies.Thefemalereproductivesystemandovarystructureistypicalanddiffersonlyindetailsfromotherspeciesstudiedtodate.Thefunctionalstructureofintercellularbridgescouplingtheovariannutritivecellandprevitellogenicoocytes,andalsothechemicaldetailsofvitellineenvelopetransformationintothechorionremaintobeestablished.Furthermore,additionalcharacteristicsofthechorionareneededtodeveloppotentialmethodsofmitecontrol.

Inthemale,thecourseofspermatogenesisconfirmsarrhenotokyinthisspecies,whereasspermstructureissimilartothatofotherAstigmata.Inthefuture,detailedstudieson

theformationoftheheterogeneous,male-derivedmaterialdepositedinthespermatheca,containingspermatozoabutalsolargeglobularstructures,areplannedtobetterdescribethefunctioningofthespermatheca.

Acknowledgments

TheauthorswishtothankProf.Dr.MichałWoyciechowski(InstituteofEnvironmentalSciences,JagiellonianUniversity)forsupplyingChaetodactylusosmiaeforstudies,aswellastoDr.DagmaraPodkowaforperfecttechnicalassistanceinTEM.TEMobservationsweredoneintheDepartmentofCellBiologyandImaging,InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity.TheEnglishoftheMSwaskindlycorrectedbyDr.MaciejPabijan,InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity.TheworkwassupportedbyDSGrantK/ZDS/006307andK/ZDS/007345(fundsforstatutoryactivitiesoftheJagiellonianUniversity).

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