Nematology,2002,V ol.4(8), 953-963 Thecellular structure of the female reproductive system within the Heteroderinae andMeloidogyninae (Nematoda) 1; 2 1 1 Wim BERT ¤, Gerrit KARSSEN , Rita VAN DRIESSCHE andEtienne G ERAERT 1 Departmentof Biology,Ghent University, Ledeganckstraat 35, 9000 Gent, Belgium 2 PlantProtection Service, Nematology Section, P .O.Box 9102, 6700 HC Wageningen,The Netherlands Received:18 March2002; revised: 5 August2002 Acceptedfor publication:5 August2002 Summary – Gonadsfrom living young females, representing 23 different species, were extracted to study the cellular structure of thefemale genital structure within the Meloidogyninae and Heteroderinae. All genera studied can be characterised by their cellular spermathecamorphology. Within Meloidogyne asphericalspermatheca is found with lobe-like protruding cells, most species having 16 to18 spermatheca cells with interlaced cell boundaries while M.microtyla and M.ichinohei havemore spermatheca cells with different cellboundaries. Heterodera and Globodera reveala comparablegonad structure. The spermatheca cells of Heterodera arecolumnar andarranged in a restrictednumber of rows, whereas in Globodera thespermatheca cells are squarish to rounded,depending on the species.The gonad morphology of Afenestratakoreana isclearlydifferent from what would be expectedbased on therelated genera Globodera and Heterodera.Theapparently simplest genital system was found in Meloidodera oridensis wherethe uterus has a limited numberof cells.In the other genera studied a largeand variable cell number was found. Keywords – Afenestrata ,femalegenital system, Globodera, Heterodera, Meloidodera , Meloidogyne ,morphology,SEM, variablecell number. Analysisof the cellular structure of thefemale repro- anduterine sac. The terms tricolumella (Hirschmann & ductivesystem for Secernenteawas mainlyexecuted by Triantaphyllou,1968), quadricolumella (Wu, 1958) and Geraert (1972,1976, 1978) and Geraert et al. (1980a, polycolumella(Chizhov & Swiliam,1986) describe the b)who also pointed out the importance of the female spatialarrangements of theuteruscells. Diversity and evo- reproductivesystem in nematode systematics (Geraert, lutionof Heteroderinae and Meloidogyninae are of spe- 1981,1983). More isolated studies of the reproductive cialinterest because world-wide they include some of the systemof Tylenchidawere undertakenby severalauthors mostdestructive plant-parasitic nematodes. In their com- (Wu,1958, 1967; Seinhorst, 1968; Bert & Geraert,2000). prehensivestudy, based on comparativedetailed morphol- Chizhovand Swiliam (1986) and Chizhov and Berez- ogy,of thephylogeny of theHeteroderinae, Baldwin and ina(1988a, b) studied several nematode species, espe- Schouest(1990) commented on the cellular structure of ciallyincluding Heteroderinae and Meloidogyninae .The thefemale reproductive tract as being potentially useful rst Meloidogyne spermathecadrawing was publishedby for interpretingphylogeny. However, they excluded ex- Nagakura(1930). Triantaphyllou and Hirschmann (1962) istinginformation from theirmatrix pending examination madea detailedillustration of the reproductive system ofmoreexcised reproductive tracts. More recently, Zunke of Heteroderaglycines andTriantaphyllou (1962, 1987) andEisenback (1998) remarked that additional investiga- focusedon some Meloidogyne species.The terminology tionsare needed, particularly on the nestructure of the ofthe reproductive system used here is basedon Geraert reproductivesystem, to improve our understanding of the (1983)who in turn followed the interpretation of Chit- phylogeneticrelationships among the numerous members woodand Chitwood (1950). The genital system consists ofthisgroup. In this paper, we examinethe cellular struc- of an ovary ( gonad)and gonoduct. The oviduct is the tureof several species and genera within the Heteroderi- D constrictedregion between the ovary and spermatheca naeand Meloidogynina einorder to shed new light on oruterus. The rest of the gonoduct is called the uterus suchquestions. * Correspondingauthor, e-mail: [email protected] © KoninklijkeBrill NV ,Leiden,2002 953 Alsoavailable online - www.brill.nl W. Bert et al. Materialand methods cellularstructure. Because of theelongation of theuterus innearlyall Heteroderinae and Meloidogyninae ,onlythe Alistof thespecies examined (and their authorities) is partfollowing the spermatheca was illustratedin those presentedin T able1. Tostudythe cellular structure of the caseswhere the uterus was demonstrablyhomogeneous femalereproductive system, gonads were rst extruded. instructure. Fourto six young females of every population were put ina dropof water on the glass slide and then cut with Results aneye-knife, leading to expulsion of gut and gonad. After removalof mostof thenon-reproductive tissue, the Asummaryof theresults with emphasis on the diver- preparation,covered by a coverslip,was studiedat once gentcharacters is presentedin T able2. underthe light microscope. In order to have an ideaabout thevariability of thestudied structure, this procedure was repeateduntil at least 20 preparations could be observed MELOIDOGYNE GÖLDI, 1892 for eachpopulation. The dissection process must have Theoviduct-spermatheca region of allthe species stud- anin uence on the size and shape of the cells due iedis characteristicand typical of thegenus Meloidogyne . toa changingosmolarity. However, repeated dissections Theoviduct comprises two rows offourthick cells. The demonstratedthat such in uences are relatively constant spermathecais spherical and formed by avariablenumber withina species.When preparations are stained with oflobe-likecells. The uterus consists of threelong rows aceticorcein (2% aqueous solution of orcein in acetic of cells ( atricolumella).In M.incognita 3 65 to 3 95 D £ £ acid)or acidfuchsin (aqueous solution), the nuclei of the cellshave been counted. cellsare more clearly visible. This can be very helpful Thespermathecae of M. arenaria, M.ardenensis,M. for smallernematodes, but for membersof Heteroderinae artiellia, M.chitwoodi , M. exigua, M.graminicola , M. andMeloidogyninae no additionalinformation is obtained hapla, M.hispanica , M.incognita , M.javanica,M. from suchstaining. morocciensis and M.trifoliophila aresimilar, comprising Observationson the internal structures can best be 16to 18 lobe-like cells with interlaced cell boundaries madeon freshly dissected specimens, which usually plas- (Fig.1A). Thespermatheca of M. fallax is slightly molyseand decompose within 1 h.With the purpose of differentin that in 35% of the specimens four or ve permanentlyrecording the morphology of the reproduc- spermathecacells are clusteredtogether to form separate tivesystem, we havealso produced some short video clips lobes(Fig. 1B). Meloidogynemicrotyla hasa spermatheca thatmimic multifocal observation through the lightmicro- with18 to26cellsand thecellboundaries are only slightly scope.This was donewith the aid of aVideoCapture and interlaced(Fig. 1C). The spermatheca of M.ichinohei is Editing(VCE) method(De Ley& Bert,in press) andthe clearlydifferent from theremaining Meloidogyne species resultscan be viewedon http://www.vce.be.tf . andhas 18 to30cells forming irregular lobes (Fig. 1D). For scanningelectron microscopic (SEM) observation, Sperm was onlyobserved in the spermatheca of M. excisedreproductive systems from 50specimens were artiellia and M. fallax.SEMof the reproductive system transferredand xedin freshly prepared 4% formalde- of M.incognita (Fig.2) con rms thelight microscopic hyde(from paraformaldehyde)in phosphate buffered observations.The two rows offourlarge oviduct cells can saline(PBS) pH7 atroom temperature. The reproduc- beobserved as protruding cells. The membranes of the tivesystems were xedovernight and subsequently dehy- lasttwo oviduct cells seem to befusedin somespecimens dratedin 25, 50,75 and95% ethanolat 2-hourlyintervals, andappear more like spermatheca cells (Fig. 2B). The followedby anovernight dehydration in absoluteethanol lobe-likecells of thespermatheca are pronounced, but the and nalremoval of theremaining water using silica gel. interlacedcell boundaries, as seen by light microscopy After critical-pointdrying using CO 2 (Wergin,1981) as (Fig.1), cannot be seenwith SEM. SEM also fails to show thedrying liquid and sputtercoating with 20 nmgold,they theexternal cell boundaries of theuterus. were examinedwith a JEOL JSM-840at 15 kV. Thecellular structure of theovary is excludedfrom this GLOBODERA SKARBILOVICH , 1959 study,because it proved dif cult to observethe cellular ar- chitectureusing only lightmicroscopy. Furthermore, there Theoviduct is made up of two rows offour cells. isno reported evidence for anytaxonomic value of this Thespermatheca of an excised gonad forms thecorner 954 Nematology Cellularstructure of the female reproductive system Table 1. Listof speciesstudied, identi cation method, examination method(s) and source. Species Identication Examination Source method method Meloidogynearenaria (Neal, Light LM & Video GerritKarssen 1889)Chitwood, 1949 microscopy Capture and PlantProtection Service, (LM) & Editing(VCE) Wageningen,The Netherlands isozymes M.ardenensis Santos,1968 Idem LM Idem M.artiellia Franklin,1961 Idem LM & VCE Idem M.chitwoodi Golden,O’ Bannon, Idem LM & VCE Idem Santo& Finley,1980 M. exigua Göldi, 1892 Idem LM Idem M. fallax Karssen,1996 Idem LM & VCE Idem M.graminicola Golden & Idem LM Idem Bircheld, 1965 M. hapla Chitwood,1949, race A Idem LM & VCE Idem M.hispanica Hirschmann,1986 Idem LM & VCE Idem M.ichinohei