Ultrastructural characterization of Hexisopus psammophilus (Arachnida: Solifugae: Hexisopodidae) spermatozoa in comparison to other solifuge spermatozoal traits

2011·7.2 MB·English

by  Anja E Klann

2011.TheJournalofArachnology39:280-286
Ultrastructural characterization ofHexisopmpsammophiius (Arachnida: Solifugae: Hexisopodidae)
spermatozoa in comparison to other solifuge spermatozoal traits
AnjaE.Klaim: InstituteofLegal Medicine, Ernst-Moritz-Arndt-UniversitatGreifswald, KuhstraBe 30, 17489
Greifswald, Germany. E-mail: [email protected]
TharinaL. Bird: Department ofArachnologyand Myriapodology, National MuseumofNamibia, Robert Mugabe
Avenue84, Windhoek, Namibia; DepartmentofZoology, DenverMuseum ofNatureand Science, 2001 Colorado
Boulevard, Denver, Colorado 80205, USA
GiovanniTalarko: DepartmentofEvolutionaryNeuroethology, Max Planck InstituteforChemicalEcology,
Hans-Knoll-StraBe 8, 07745Jena, Germany
Abstract. The family Hexisopodidae isendemic to southern Africa. Hexisopodids representavery peculiar groupof
Solifugae.Theydifferfromallothersolifugefamiliesthroughvariousautapomorpicadaptationstoasubterraneanmode
oflife,mostnotablythepresenceoffossoriallegs.ThephylogenyoftheSolifugaeiswidelyunresolved.Theultrastructure
ofspermatozoahassuccessfullybeenusedforphylogeneticanalysesinotheranimaltaxa.Therefore,thequestionarose
whetherthemorphologicalpeculiarityofthefamilyHexisopodidaemightalsobereflectedintheultrastructureoftheir
spermatozoa. Thiswas investigated for HexisopuspsammophiiusWharton 1981 (Hexisopodidae). Spermatozoa do not
seemtoaggregateinthetestes,norinthevasadeferentia.Spermcellsareaflagellate,roundish,andwithirregularlyshaped
chromatinbodies.Eachspermissurroundedbyasecretionsheath,thusrepresentingatypicalcleistosperm,thefirstrecord
ofthisformofspermtransferinsolifuges.Thespermcellsformfinger-likeprotuberancesandcontainputativegranulesof
glycogen,featuressharedwiththeAmmotrechidae,DaesiidaeandSolpugidae.Theacrosomalcomplexshowsadditional
similaritywiththeSolpugidae.Overall,thespermatozoaofH.psammophiiussharesomero.orphologicalfeatureswiththe
AmmotrechidaeandDaesiidae,butmostlyresemblethatofthefamilySolpugidae.
Keywords: Camel-spider,spermcell,finestructure,cleistospermia,phylogeny
Soiifuges,alsocalledcamel-spiders,occurworldwideonall charactersnot onlyatthe genuslevelbutatthespecies level
continents with the exception of Australia and Antarctica as well. For these reasons, various authors have critically
(Punzo 1998).Theyrepresentoneofthemesodiversearachnid challenged the current Roewerian system (e.g., Muma 1951,
orderscomprising 12familiesandabout 1087species(Harvey 1976; Harvey2002).
2002, 2003). Also known for their great sprint speeds and The ultrastructure of spermatozoa has been shown to
voracious feeding behavior, knowledge about, for example, contributevaluablecharacterstoelucidateandtosubstantiate
their systematics, phylogeny, behavior and life cycle is still phylogenetichypothesesinmanyanimalgroups(e.g.,Alberti
superficialandfragmentary.Amongthe 12familiesknownto 1984, 1991; Baccetti 1970; Baccetti & Dallai 1978; Dallai &
date, one family, Hexisopodidae (mole- or teddybear soli- Afzelius 1995; Jamieson et al. 1999; Dallai et al. 2003;
fuges) strikingly differs morphologically from all other DowningMeisneretal.2005;Liana&Litvaitis2007).Within
solifuge families through the presence of various autapo- arachnids three different types ofsperm cells are known: 1)
morphiccharacters, most notablytheirfossorial, ratherthan filiformflagellate-presentonlyinscorpions(daCruz-Landim
cursorial, legs. Highly modified adaptations such as these & Ferreira 1972, 1973; Alberti 1983; Vignoli et al. 2008;
often obscure real relationships with other taxa, and the Michalik & Mercati 2010), 2) coiled flagellate - present in
putative hexisopodid relationships are no exception. The Amblypygi,Uropygi,Araneae,PseudoscorpionesandRicinu-
family Hexisopodidae Pocock 1897 only occurs in southern lei(Alberti 1985, 1990,2000;Alberti&Palacios-Vargas 1984;
Africa (mainly in South Africa and Namibia, but also in Callaini&Dallai 1993;Michaliketal.2004a,2004b;Michalik
Angola, Zimbabwe, Zambia, and Botswana). Twenty-three &Huber2006;Talaricoetal.2008;Talarico&Michalik2010)
described species are distributed among the two genera and3)aflagellate-presentinAcari, Solifugae,Opilionesand
Hexisopus Karsch 1879 and Chelypus Purcell 1902 (Harvey Palpigradi (Alberti 1980a, b, c, 2000, 2005a; Alberti et al.
2003). Themainmorphological differencebetweenthesetwo 2009; Klannetal. 2005,2009).
genera is the presence of well developed spines on the The morphological variety of spermatozoa within these
pedipalpsofChelypus,whereassuch structuresarelackingin groups differs strongly. Spiders and mites exhibit a large
Hexisopus(Roewer1934).Duetotheirprimarilysubterranean diversity in, e.g., morphology and size oftheir spermatozoa
mode of life, the species of Hexisopodidae are extremely (Alberti 1980a, b, 1990, 2000), whereas othergroups suchas
difficulttostudyinanyaspect. scorpions, ricinuleids, and solifuges apparently show only
The current classification of solifuges does not recognize slight variability in their characteristic ground patterns
any subordinal or superfamilial arrangement (Harvey 2003). (Alberti 1983; da Cruz-Landim & Ferreira 1972, 1973;
This classification, based mainly on the taxon delineation Talarico et al. 2008; Klann et al. 2009; Michalik & Mercati
established by Roewer (1934), relies on highly variable 2010). Nevertheless, within arachnids these results are only
KLANNETAL.—SPERMCELLSOFHEXISOPUSPSAMMOPHILUS 281
includedinmodernphylogeneticanalysestoalimiteddegree producedbythesecretorycellsintheglandularpart(Figs.4,
(Weygoldt&Paulus1979a,b;Shultz2007;Dabertetal.2010; 5). Medium electron-dense secretion fills the testis lumen
Pepatoetal. 2010). Because manytraditional morphological homogenously (see Figs. 3, 7). Additionally, very electron-
characters of solifuges have been shown to fail substantial dense secretions are released as droplets into the lumen
systematic and phylogenetic requirements as already men- (Fig. 5).Thespermcellsarelocatedinthegerminalpartofthe
tionedabove,theultrastructureofsolifugespermatozoamight testis(Fig. 6).Theydonotaggregatetogroups.Oncereleased
beanimportantcontributiontowardsproposingphylogenetic individually into the lumen of the testis (Fig. 7), they are
hypotheses,notonlywithinsolifuges,butalsoforhigherlevel transportedtothevasadeferentia.
arachnid phylogeny. The present study aims to characterize Thevasa deferentia are surrounded by an extremely thick
the fine structure ofHexisopuspsammophilus Wharton 1981 muscle layer (Fig. 8). In contrast to the epithelium of the
(Hexisopodidae)spermatozoainordertorevealwhetherthere testis, theepithelium ofthevasa deferentiaisapparently not
are any spermatozoal traits that distinguish Hexisopodidae composed oftwo parts. The epithelium possesses apically a
fromothersolifugespermcells. microvilli border (Fig. 8) and consists ofirregularly shaped
METHODS cells with prominent nuclei. The cell borders strongly
interdigitate(Figs. 8,9). Mitochondriaandglycogengranules
Amale ofH. psammophiluswascollected at the Gobabeb arealsopresent(Fig. 9).Numerousindividualspermcellsand
Research Station, Namibia (23°34'40.5"S, 16°02'31.3"E) secretionsofdifferentelectron-densitiesarevisibleinthewide
(Voucher specimen deposited at the National Museum of lumen (Fig. 10). Dependingon the orientation ofthe section
Namibia,SMN 14218).Themalegenitalsystemwasremoved plane,thespermcellsappearovalorcircular(Fig. 10).Their
and preserved in 2.5% glutaraldehyde in phosphate buffer average diameter is about 2-2.5 pm. Very rarely, few sperm
(0.1 M, pH 7.2, 1.8% sucrose) at4° C overnight. Thereafter, cells are attached to each other and form small stacks
thespecimenwasrinsedinphosphatebufferandpost-fixedin (Fig. 11).
2%aqueousOSO4at4°Cfor2h.Afterbeingrinsedinbuffer, Eachindividualspermcellissurroundedbyathinsecretion
the male genital system was dehydrated in graded ethanol sheath (Fig. 12). The sperm cell forms finger-like protuber-
series(60%-absolute).ThespecimenwasembeddedinSpurr’s ances around its entire surface (Figs. 11-15). The chromatin
medium (Spurr 1969). Semi-thin sections were made for body is very irregularly shaped and exhibits a granulate
general orientation and stained according to Richardson et structure (Figs. 11-15). No nuclear envelope could be
al. (1960). Ultrathin sections (50-70 nm) were made using a observed. Multilamellar bodies and areas with conspicuous
Diatome diamond knife. These sections were placed on electron-densegranules,mostlikelyglycogen,areenclosedby
uncoated copper grids and stained with saturated uranyl- the chromatin body (Fig. 12). The acrosomal complex is
acetate in 70% methanol and lead-citrate according to the deeplyembeddedinthechromatinbody. Itisrelativelysmall,
methodafterReynolds(1963). Transmissionelectronmicros- consisting of the acrosomal vacuole and the acrosomal
copywasperformedwithaJEOL-JEM-1011. filament, the latter originating underneath the small, conical
Abbreviations: AF = acrosomal filament,AV = acrosomal acrosomal vacuole (Fig. 13). The acrosomal filament runs
vacuole, bL = basallamina, C = centriole, CB = chromatin through the chromatin body and apparently coils preferen-
body, Epi = epithelium, Gly = glycogen, Hd = hemidesmo- tiallyaroundtheareawheretheglycogenparticlesarelocated.
some, Lu = lumen. Mi = mitochondrium, MIB = multi- Itdoesnotseemtocoilaroundthechromatinbody(Figs. 14,
lamellarbody. Mu = muscle, Mv = microvilli,N = nucleus, 15).
Nu = nucleolus, Pt = protuberances, rER = rough
endoplasmic reticulum, Sec = secretion. Sec1-3 = secretions DISCUSSION
withincreasingelectron-densities,Sp= spermatozoon,SSh = Ithasbeenshowninformerstudiesthatthespermatozoaof
secretionsheath,Tr = trachea. solifugesexhibitcertainseeminglyfamily-specificmorpholog-
RESULTS icalcharacters(Klannetal. 2009), butsomesimilaritieswere
also found between families. The finger-like protuberances
The male genital system ofH. psammophilus corresponds andtheputativepresenceofglycogenfoundhereinthesperm
morphologicallytothemalegenitalsystemsofothersolifuge cells ofH. psammophilus are similar to those found in the
species. Two pairs oflong testes (four in total) are present. spermatozoaofAmmotrechidae,somemembersofDaesiidae
Eachpairoftestes merges into onevas deferens, whereas in and Solpugidae. Multilamellar bodies, previously only ob-
turnthevasadeferentiaenterthecommongenitalchamber. served in Gluviadorsalis(Latreille 1817) (Daesiidae) (Klann,
Thetestesaresurroundedbysmalltracheaeandmusclecells unpubl. data), were also found in the hexisopodid species
(Fig. 1). The muscles are embedded in the layered basal studiedhere.
lamina(Fig. 2).Theepithelialcellsinturnareattachedtothe Hexisopodid spermatozoa have a unique feature in that
basallaminaviahemidesmosomes(Fig. 2).Theepitheliumof everyindividualspermcellissurroundedbyitsownsecretion
thetestisconsistsoftwodifferentparts,namelytheglandular sheath,thusformingatypicalcleistospermium.Theformation
and thegerminal parts. Cells formingthe glandular part are of sperm stacks in the lumen of the vasa deferentia could
characterized by an extensive amount ofrough endoplasmic possibly be caused by occasional adhesion of the secretion
reticulum. Centrioles arranged in a tandem position can be sheathsofdifferent spermcells. Thiscan also beobserved in
seenintheapicalpartofthiscelltype(Fig. 3).Thenucleiare othersolifugidspeciesinwhichsinglespermcellsarecommon.
ovalorroundishinshape.Apically,thecellsareprovidedwith Cleistospermia have not been observed in other solifuge
amicrovilli border (Fig. 3). Different kinds ofsecretions are species so far. In contrast to cleistospermia, coenospermia
282 THEJOURNALOFARACHNOLOGY
—
Figures 1-7. Testis. 1.Overviewoftheepitheliumofthetestissurroundedbymusclesandtracheae.Scalebar10pm.2.Theepitheliumofthe
testisisattachedtothebasallaminaviahemidesmosomes.Musclesareembeddedinthelayeredbasallamina.Scalebar2pm.3.Intheapicalpartof
epithelialcellsconstitutingtheglandularpartofthetestiscentriolesarearranged intandemposition. Largecisternaeofroughendoplasmic
reticulumarevisible.Thenucleusisroundish.Scalebar5pm.4.Differentsecretionsareproducedbyepithelialcellsofthetestis.Scalebar2pm.5.
Secretionsarereleasedintothelumenofthetestisandmostlikelytransportedtothevasdeferens.Scalebar2pm.6.Individuallydistributed
spermatozoaarelocatedwithinthegerminalpartofthetesticularepithelium.Scalebar2pm.7.Singlespermcellsarereleasedintothelumenofthe
testis.Inthissectionthetestislumenseparatescellsofthegerminalpart(lowerleft)fromcellsoftheglandularpart(upperright).Scalebar2pm.
KLANNETAL.—SPERMCELLSOFHEXISOPUSPSAMMOPHILUS 283
—
Figures 8, 9. Vas deferens. 8. The epithelium ofthe vas deferens is surrounded by a very thick muscle layer. The epithelium itselfis
comparativelyflat.Thelumenofthevasdeferensisfilledwithsecretionsofdifferentelectron-densities.Spermatozoaanddropletsofhigh-dense
secretion(Sec3)canbefoundinthehomogenousmedium-densesecretion(Sec2).Scalebar5pm.9.Theepithelialcellisprovidedwithanoval
nucleuswithaprominentnucleolus.Thebasallaminaislayered.Notethehemidesmosomes.Scalebar2pm.
consistofseveralspermcellssurroundedbyasecretionsheath convergences, reversals and parallelisms in morphological
(Bertkau 1877). This type of transfer form occurs in characters, spermmorphologyappeartobeveryconstant, at
Galeodidae and certain members of the family Daesiidae, leastwithinsomefamiliesoftheorderSolifugae(Klannetal.
thelatterexhibitingthelargestspermatozoalvariabilitywithin 2009). Members ofthe family Galeodidae, for example, are
theSolifugae(Klannetal. 2009). united byvarious morphological characters such as shape of
In spiders, coenospermia are considered to be ancestral, a the flagellum, opisthosomal “Stigmenkamme” and setose
hypothesisderivingfromthefactthatthistransferformoccurs tarsal claws (Roewer 1934). This is also reflected in the
inprimitivespidertaxa,suchasMesothelaeandMygalomor- morphology oftheir sperm cells; the sperm cells ofdifferent
phae(Alberti 1985, 1990;Michaliketal.2003;Michaliketal. galeodid species are extremely similar and difficult to
2004b). Since onlyfine structural data on spermatozoa from differentiatefromeach other. Nonetheless, it is important to
seven solifuge families (including now Hexisopodidae) are notethatspermcellscanundergowiderangesofmorpholog-
knowntodate(Klannetal.2009),theevolutionarypathways icalmodifications(Pitnicketal.2009),andsimilaritiesdueto,
ofthistraitinsolifugescannotyetbejudged.Nonetheless,our forexample,convergencescanthereforenotbeexcluded.
findings suggest that the sperm cells of the investigated Insummary,althoughasetofuniqueexternalmorpholog-
hexisopodidmostcloselyresemblethefinestructureofsperm ical characters (e.g., fossorial legs) clearly separates the
cells ofthe family Solpugidae. This resemblance is not only Hexisopodidae from other solifuges, the onlyautapomorphy
foundinthepresenceoffinger-likemembraneprotuberances found in their sperm morphology was the presence of
and glycogen, but also in the organization and form ofthe cleistospermia. Their spermatozoa thus do not reflect their
acrosomalcomplex. BothinSolpugidaeandintheinvestigat- peculiar outer morphology, but rather indicate a closer
edhexisopodid,theacrosomalvacuoleisconicalinshapeand relationshipwiththefamilySolpugidae.
locatedwithinthechromatinbody. Interestingly,whereasthe Similar challenges based on spermatozoan ultrastructure
currentclassification places the Hexisopodidae as a separate have been made in higher level arachnid phylogeny studies.
family within the order, Hewitt (1919) regarded the Hexiso- Variousauthors(Alberti 1980c;Alberti&Peretti2002;Klann
podidae as being a potential subfamily of Solpugidae. It et al. 2005) questioned the commonly accepted clade
remainsto beseen whetherthe hypothesis ofHexisopodidae Haplocnemata (Solifugae + Pseudoscorpiones) based on the
as a subfamily of Solpugidae will persist in modern coiled flagellate sperm cells of pseudoscorpions versus the
phylogeneticanalyses. However, Hewitt’s hypothesis, seen in aflagellate sperm characteristic of solifuges. These findings
thelightofsimilaritiesinthefinestructureofSolpugidaeand havebeencorroboratedbyrecentmolecularstudies(Dabertet
Hexisopodidaespermatozoa,could indicateacloserrelation- al. 2010; Pepato et al. 2010)which, independently, recovered
ship between thesetwo families than iscurrently recognized. the clade Actinotrichida (Acari)-i-Solifugae, thereby implicat-
Although morphological similarities do not automatically ing the diphyly ofthe Acari (Anactinotrichida and Actino-
indicate a close relationship between taxa due to potential trichida as distinct groups, previously suggested by van der
284 THEJOURNALOFARACHNOLOGY
—
Figures 10-15. Spermatozoa. 10. Spermatozoa aredistributed individually in thelumen ofthe vasdeferens. Dropletsofelectron-dense
secretionsarealsovisible.Scalebar5|im. 11.Spermcellsseldomclingtogetherandformsmallgroups.Scalebar1pm.12.Eachindividualsperm
cellissurroundedbyasecretionsheath.Finger-likeprotuberancesapparentlysurroundtheentirecell.Multilamellarbodiesoccurinsidethecells.
Areascontainingglycogen are present within thechromatin body. Scale bar 1 pm. 13. Theacrosomalcomplex isdeeply embedded in the
chromatinbody.Theacrosomalvacuoleisconicalshaped.Scalebar0.5pm. 14.Inthissectiontheacrosomalfilamentpenetratestheglycogen
aggregation. Scale bar 1 pm. 15. This section indicates that the acrosomal filament additionally coils several times around the glycogen
aggregation,butitdoesnotcoilaroundtheentirechromatinbody.Scalebar 1 pm.
KLANNETAL.—SPERMCELLSOFHEXISOPUSPSAMMOPHILUS 285
Hammen (1989) and Alberti (2005b)) and refuting the Alberti,G.&A.V.Peretti.2002.Finestructureofmalegenitalsystemand
Pseudoscorpiones-Solifugaesistergrouprelationship. sperminSolifugaedoesnotsupportasister-grouprelationshipwith
mnoeleItedciusltcaolre,abreasthbawateslhelidgahseornalesqvuueilatlSiootflyibfuaogtnahdeepcxhatyrelerofnguaelllnyeatnicdconaisnniatdleeyrrsneeadsl BacIPBcnaesctectTueidht,otesiBc,.Sope1rd9pe.7i)r0.om.nAeTCcshecle(alAdsrepaascemhrinamainadNtaIao)nzz.dioJeoooxnnuaroolnffealAAdrerotitfhhLArrironoapcpcoeohid,dnao.RlPIohoXygm.lyeoPg3pe0.n:e21s66i89s--.2178(41B...
morphological characters, ifrobust phylogenetic hypotheses Baccetti, B. & R. Dallai. 1978. The evolution of myriapod
forthisgrouparetobeproposed. spermatozoa.AbhandlungenundVerhandlungendesnaturwissen-
ACKNOWLEDGMENTS schaftlichenVereinsinHamburg21/22:203-217.
Bertkau, P. 1877. UberdieUbertragungsorgane unddieSpermato-
The authors greatly acknowledge G. Alberti for valuable zoenderSpinnen. SitzungsberichtederNiederrheinischen Gesell-
comments on the manuscript and want to thank G. Uhl for schaftfurNatur-undHeilkundeBonn34:28-32.
use of her TEM-facilities. Additionally, we would like to Callaini,G.&R.Dallai.1993.ThespermatozoonofPseudoscorpions
thank R. Wharton for the help to collect and identify the (Arachnida). Bollettinodell’AccademiaGioeniadiScienzeNatur-
mfiantaenrciiaalllyinvseusptpiogratteedd ibnytthheepGreesremnatnstRuedsye.arTchhisFosutunddyatwiaosn DaalCir2u6z:-3L5a-n5d2.im,C.&A.Ferreira.1972.Spermdifferentiationinthe
(ScDiFenGceAF11o3u8n/d1a2t-i1onaw(aDrBdIed03t4o63G7.8Aalwbaerrtdie)datnodP.thEe.NCautsihoinnagl DasCcorrupzi-oLnanTdiitymu,sCb.ah&ienAs.LsFe(rPreeritrya)..1C9a7r3y.oTlohgeiasp2e5r:m1a2t5o-z13o5a.ofTityus
arencdeivDeEsBap0o2st2-8d6o9c9toarawlarsdteipdentdofLr.omPrtehnediMnaix).-PGl.aTn.ckcuSrorceinettyl.y DabbDeaarhbtie,erntsM.i.s,2(0P1We0t..ty)WMiostlcaelocirnupslikaoinr,-pABh.uytlKhoaigzdeamneiy.erCosyfktio,alcoaZgr.iiafOol3rs8m:z1am8ni7ot-we1ss9k4i(.Ac&ariJ,.
Finally, we thank two anonymous reviewers for suggestions Arachnida):Strongconflictbetweenphylogeneticsignalandlong-
forimprovingthemanuscript. branchattractionartifacts.MolecularPhylogeneticsandEvolution
56:222-241.
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