Nematology 2003 Vol 5(2) 293-306

Comparative morpho-anatomical studies of the female gonoductwithin the Pratylenchidae (Nematoda Tylenchina)

Wim BERT curren Ruben VAN GANSBEKE Myriam CLAEYS Etienne GERAERT and Gaeumltan BORGONIE

Department of Biology Ghent University Ledeganckstraat35 9000 Gent Belgium

Received 5 November 2002 revised 10 February 2003Accepted for publication13 February 2003

Summary ndash The cellular morphology of the gonoduct of six Pratylenchus species three Pratylenchoides species Radopholus similisZygotylenchus guevarai Hirschmanniella loo and Nacobbus aberrans was revealed by dissection and light microscopy Except forNacobbus aberrans all studied species show an overall similarity in gonoduct construction ie an ovary often ending with a ring ofcells an oviduct formed from two rows of four cells and a 12-celled spermatheca followed by a tricolumella containing 16-24 cellsPratylenchoides magnicauda and Z guevarai did not diverge from the other Pratylenchidae in this respect although their gonoductdiffers from that of Amplimerlinius and Meloidogyne both formerly postulated as related genera The spermatheca structure observedin N aberrans has not been reported elsewhere in the Nematoda although the uterus is similar to that reported within the Heteroderinaeand Meloidogyninae and the uterus comprises more than 300 cells enlarging from a tricolumella to a polycolumella Transmissionelectron microscopy of Z guevarai revealed details of the cytoplasmatic contact between epithelial cells and the germ cells a nger-like ovarian wall cell extension was found penetrating the oocyte The oviduct lacks a preformed lumen and comprises eight cells withhighly plicated cell membranes The spermatheca is constructed from attened wall cells and is followed by columnar uterus cellswhere evidence of eggshell formation was demonstrated

Keywords ndash gonad Hirschmanniella morphology Nacobbus Pratylenchus Pratylenchoides TEM ultrastructureZygotylenchus

Light microscopic studies of the female reproductivesystem of some Pratylenchidae were reported by Sein-horst (1968) Roman and Hirschmann (1969) Geraert(1973) and Chizhov and Berezina (1988) The gonad de-velopment of Pratylenchuscrenatus Loof 1960 was stud-ied using light microscopy (LM) by Dickerson (1962)while on the ultra-structural level the female gonad ofPratylenchus penetrans (Cobb 1917) Filipjev amp Schuur-mans Stekhoven 1941 was examined in elaborate detailby Endo et al (1997 1999) with the transmission elec-tron microscope (TEM)

Identi cation of root-lesion nematodes is dif cult be-cause of little morphological variation and overlappingmorphometrical characters (Roman amp Hirschmann 1969)whilst many phylogenetic questions within the Praty-lenchidae remain unanswered the position of Pratylen-choides magnicauda(Thorne 1935)Baldwin Luc amp Bell1983 (Baldwin et al 1983 Ryss amp Sturhan 1994) andNacobbus (Baldwin amp Cap 1992) is questionable parsi-mony analyses of 26S rDNA of Pratylenchusspp showedthat the genus represented a paraphyletic assemblage (Al-

Corresponding author e-mail wimbertrugacbe

Banna et al 1997) Zygotylenchus has been suggestedas the evolutionary link to the Meloidogyninae (Geraert1997 Pourjam et al 2000) and in a recent phyloge-netic analysis of 18 SSU rDNA the Pratylenchidae didnot prove to be a monophyleticgroup (De Ley amp Blaxter2002)

In view of the importance of the female reproductivesystem in nematode systematics (Geraert 1981 1983)we examined the cellular gonoduct architecture of severalspecies and genera within the PratylenchidaeThe femalegonoduct of Pratylenchus spp Pratylenchoides spp Zy-gotylenchus guevarai (Tobar Jimeacutenez 1963) Braun ampLoof 1966 Radopholus similis (Cobb 1893) Thorne1949 Hirschmanniella loo Sher 1968 and Nacobbusaberrans (Thorne 1935) Thorne amp Allen 1944 was stud-ied by means of LM after dissection of the reproductivesystem Pratylenchuspenetransand Z guevaraiwere alsoexamined using TEM As the comprehensive studies onfemale P penetrans gonad ultra-structure by Endo et al(1997 1999) agreed with our ndings we only presentthe TEM results of Z guevarai in this paper

copy Koninklijke Brill NV Leiden 2003 293Also available online - wwwbrillnl

W Bert et al

Material and methods

NEMATODE MATERIAL

A list of the examined species is presented in Table 1The populations were extracted from soil samples or car-rot disk cultures Pratylenchus penetrans and Z guevaraiwere extracted from soil samples surface sterilised with2000 ppm streptomycin sulphate (Sigma St Louis MOUSA) for 12 h and rinsed three times with sterile distilledwater Five to ten individualsof each populationwere usedto establish monoxeniccultures on carrot disks (Moody etal 1973)

TERMINOLOGY

The terminologyof the reproductivesystem is based onGeraert (1983) who followed the interpretation of Chit-wood and Chitwood (1950) The genital system consistsof an ovary (Dgonad) and gonoduct The oviduct is aconstricted region between the ovary and the spermath-eca or uterus Here the term uterus is restricted to thecolumnar region of the gonoduct A uterine sac followsthe uterus The term tricolumella (Hirschmann amp Trianta-phyllou 1968) describe the spatial arrangement of theuterus cells The terms distal and proximal are used to de-scribe the position of whatever part in relation to the vulvaposition

LIGHT MICROSCOPY

The method used to study the cellular structure of thefemale reproductive system was based on Geraert (1973)Gonads were rst extruded four to six young femalesof every population being put in a drop of water on aglass slide and then cut with an eye-knife to expel thegut and gonad This preparation was either stained withacetic orcein (2 aqueous solution of orcein in aceticacid) or observed directly under LM Both methods wereapplied Using stains results in a stronger differentiationof the nuclei whereas general cell morphology is betterpreserved without staining Classical stains were unsatis-factory for staining the Nacobbus gonoduct cells To visu-alise the nuclei a drop of 46-diamidino-2-phenylindole(DAPI Sigma) at a concentrationof 1 sup1gml in PBS with01 Triton-X100 was added to the dissected gonads

The cellular structure of the ovary was only partly stud-ied with LM as it was dif cult to observe all structuresonly the ripeningzone of the ovary containeddistinct cellswhich could be visualised with the techniques used

TRANSMISSION ELECTRON MICROSCOPY

Adult females were picked out and placed on ice to re-lax They were killed and xed in ice-cooled Karnovskyrsquos(1965) xative composed of 2 paraformaldehyde25glutaraldehyde and 05 CaCl2 in 0134 M sodium ca-codylate buffer The nematodes were bisected to improvepermeabilityAfter approximately 15 h of xation at 4plusmnCthey were rinsed in 0134 M sodium cacodylate buffer for8 h Post- xation took place in reduced osmium a mixtureof 1 ml OsO4 (4) 3 ml Na cacodylaat (0134 M) and66 mg K3Fe(CN)6 for 36 h at pH 74 After rinsing withdouble distilled water the specimens were dehydrated in50 75 90 and absolute ethanol to which CuSO4 barswere added to remove any remaining water The speci-mens were subsequently in ltrated with a low-viscosityembedding medium (Spurr 1969) Ultrathin (50-90 nm)longitudinal sections were cut on a Reichert ultracuts ul-tramicrotome (Leica Vienna Austria) with a diamondknife (Diatome Ltd Biel Switzerland) and mounted onformvar coated single slot copper grids (Agar Scienti cStansed UK) The sections were stained (EM stain Le-ica) with uranyl acetate and lead citrate and viewed witha Jeol JEM-1010 (Jeol Ltd Tokyo Japan) transmissionelectron microscope operating at 60 kV

Results

A schematic overview of the results is presented inFig 3

PRATYLENCHUS FILIPJEV 1936 (FIG 1)

The ripening zone of the ovary comprises 16 cells Atthe transition to the oviduct in young females eight cellsform a sphincter-like structure although in older speci-mens those cells were more dispersed Eight randomlyarranged cells precede this circle The oviduct forms aclear constriction between ovary and spermatheca and iscomposed of eight attened cells in two rows In somespecimens of P scribneri Steiner 1943 an additional pairof smaller cells could be observed between the oviductand the ovary (Fig 1F) These cells looked like oviductcells but contained smaller and darker nuclei

In all species observed the spermatheca was formedfrom a total of 12 cells the arrangement of which isspecies speci c The spermatheca of P crenatus com-prises ten more or less rounded cells in variable posi-tions with two more elongated cells making the connec-tion to the uterus (Fig 1A) The spermatheca of P coffeae

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Table 1 Host origin and source of studied PratylenchidaepopulationsAll populations were extracted from carrot disk cultures exceptfor the eld populations and Nacobbus aberrans (from tomato)

Species Original host or Origin of Sourcesampling site population

Hirschmanniella Reed (Phragmites Bourgoyen- eld populationloo australis) Ossemeersen

Ghent BelgiumNacobbus Tomato not known G Karssen

aberrans Plant Protection ServiceWageningen The Netherlands

Pratylenchus Apple orchard Kerkom Agricultural Research Centrepenetrans Belgium Department Crop Protection

Ghent Belgium (CLO-Gent)Not known Azores PortugalApple orchard Eksaarde Belgium eld populationGrass land Bourgoyen-Ossemeersen eld population

Ghent BelgiumGrass land with plum tree Ingelmunster Belgium eld population

P coffeae Banana not known CLO-GentP crenatus Maize- eld Eksaarde Belgium eld populationP akkensis Grassland (dominated by Eksaarde eld population

Arrhenatherus elatius Belgiumand Holcus lanatus)

P scribneri Maize Vero Beach FL USA CLO-GentP thornei Chick-pea Santaella CLO-Gent

Cordoacuteba SpainP vulnus Unknown Unknown CLO-GentP zeae Maize South Africa CLO-GentPratylenchoides River bank (dominated by Redu Ardennes A Ryss Russian Academy of

magnicauda Trifolium sp Quercus sp Belgium Sciences St Petersburg Russiaand Salix)

Pratylenchoides Wheat Apulia Italy A Trocolli Instituto di Nematologiaritteri Agraria Bari Italy

Pratylenchoides Lawn in the vicinity Botanical garden eld populationcrenicauda of a willow tree Ghent University

(Salix matsudana Koidz) BelgiumRadopholus Banana Sennar Sudan G Elbadri Crop Protection

similis Wad Medani SudanZygotylenchus Pistachio Kerman Iran E Pourjam Tarbiat

guevarai Modares University Iran

(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 is not clearly differentiated from the uterus Twelvecells were counted but an unambiguouscellular architec-ture could not be described (Fig 1B) The spermatheca ofP thornei Sher amp Allen 1953 is partly offset the offsetportion comprising on average four cells the spermathecacells being more or less round and equally sized (Fig 1C)The spermatheca of P penetrans is asymmetrical and theoviduct is not connected to the spermatheca entirely axi-

ally The slightly protruding corner of the spermatheca onthe oviduct side comprises small cells of various shapetwo large rounded cells making the connection to theuterus (Fig 1D) The oviduct of P zeae Graham 1951 ispartly enveloped by the spermatheca which has six cellsvariably arranged in the region close to the oviduct fol-lowed by four slightly larger cells with two cells makingthe connectionto the uterus (Fig 1E) The spermatheca ofP scribneri is asymmetrical and slightly offset due to the

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protruding and larger spermatheca cells at one side of thespermatheca Four spermatheca cells make the connectionto the uterus (Fig 1F) The spermatheca of P akkensisSeinhorst 1968 and P vulnus Allen amp Jensen 1951 alsoseems to comprise 12 cells but the number of successfuldissections was too low to obtain reliable results

The uterus of all studied Pratylenchus species consistsof three rows of cells (Da tricolumella) each row beingfour cells long The uterine sac follows the columnaruterus

ZYGOTYLENCHUS GUEVARAI

Light microscopy

The ovary connects to the oviduct via a ring of eightcells (Fig 2A) Two rows of four cells comprise theoviduct The spermatheca has 12 cells the two cellsconnecting it to the uterus being clearly larger The spermcells are variously shaped and often swollen They are alsofound at the beginningof the uterus which is formed fromthree rows of four cells

Transmission electron microscopy

The description below is based on the posterior branchof the didelphic reproductive system (Fig 4A) with theexception of one picture of the spermatheca taken fromthe anterior branch (Fig 6)

The ovarian wall cells form a thin layer around the germcells and have extensions between some of the germ cellsThese cytoplasmatic nger-like extensionscan even enterthe oocytes (Fig 4B) In the germinal zone the oocytesarranged in a single le have a clear cell membraneand the large variably shaped nucleus contains a darkspherical nucleolus As the oocytes become older theyincrease in size and contain more and more lipid dropletsand yolk

The oviduct (Fig 5) consists of irregularly shapedcells having highly plicated cell membranes In onelongitudinal section four adjacent oviduct nuclei could beseen two nuclei at the same level being observed close tothe spermatheca No preformed lumen was observed Theoviduct cells contain unknown secretions and nuclei withirregularly shaped nuclear membranes lined with densechromatin

The spherical spermatheca (Fig 6) occupies almost thewhole body cavity as observed in a transverse sectionThe spermatheca wall cells are highly attened theirnuclei being oval with dispersed dense chromatin Thevariably shaped sperm cells have a thick head portioncontaining the nucleus and a thinner more elongated

tail The nucleus lacks a membrane and is seen as aprominent mass of chromatin surrounded by clusters ofmitochondria

The columnar uterus cells (Fig 7A B) have plicatedmembranes The cytoplasm is lled with mitochondrianumerous ribosomes and some inclusionswith both heav-ily and slightly stained zones The nucleus contains aprominent nucleolus and some chromatin adjoins the nu-clear membrane A preformed uterus lumen was not ob-served the columnar cells being pressed against eachother and forming lsquomembrane-junctionsrsquo (Fig 7B) as de-scribed by Endo et al (1997 1999)

The columnar uterus is followed by a uterine sac(Fig 8) lled with a nely granular substance Thecuticula of the vagina is continuouswith the ventral liningof the uterus sac while a attened cellular wall lines therest of the uterine sac

PRATYLENCHOIDES WINSLOW 1958 (FIG 2B C D)

The ripening zone of the ovary consists of 16 cells witha ring formed from a variable number of cells connect-ing the ovary to the oviduct The oviduct of P magni-cauda (Fig 2C) and P ritteri Sher 1970 (Fig 2D) com-prises two rows of four cells In P crenicauda Winslow1958 (Fig 2B) only three cells were observed in each rowfor the majority of the observed specimens although anoviduct with two rows of four cells has also been ob-served The spermatheca and oviduct of an excised go-nad forms the corner of an angle between ovary anduterus The spermatheca of the studied Pratylenchoidesspecies comprises 12 variously shaped cells the sper-matheca cells of P ritteri being more rounded and pro-truding The uterus is constructed from three rows of sixto seven columnar cells

RADOPHOLUS SIMILIS (FIG 2E)

The ripening zone of the ovary has four groups offour cells The oviduct comprises two rows of four attened cells The spermatheca always contains 12variably positionedcells and is followed by a tricolumellacomprising 12 cells

HIRSCHMANNIELLA LOOFI (FIG 2F)

The ripening zone could not be distinguished fromthe remainder of the ovary The oviduct comprises tworows of four cells The spermatheca cells are indistinctlydelimited and their cell borders are twisted The number

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Fig 1 Partial female reproductive systems of Pratylenchus spp A Pratylenchuscrenatus B P coffeae C P thornei D P penetransE P zeae F P scribneri End of ovary (ova) oviduct (ovi) spermatheca (sp) and uterus (ut) (Scale bars D 10 sup1m)

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Fig 2 Partial female reproductive systems of Pratylenchidae A Zygotylenchus guevarai B Pratylenchoides crenicauda C Pmagnicauda D P ritteri E Radopholus similis F Hirschmanniella loo G Nacobbus aberrans spermatheca region H Nacobbusaberrans uterus closer to the vulva End of ovary (ova) oviduct (ovi) spermatheca (sp) uterus (ut) and uterus closer to the vulva(utv) (Scale bars D 10 sup1m)

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Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

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Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

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Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

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Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

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Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

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Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

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Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

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AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

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Table 1 Host origin and source of studied PratylenchidaepopulationsAll populations were extracted from carrot disk cultures exceptfor the eld populations and Nacobbus aberrans (from tomato)

Species Original host or Origin of Sourcesampling site population

Hirschmanniella Reed (Phragmites Bourgoyen- eld populationloo australis) Ossemeersen

Ghent BelgiumNacobbus Tomato not known G Karssen

aberrans Plant Protection ServiceWageningen The Netherlands

Pratylenchus Apple orchard Kerkom Agricultural Research Centrepenetrans Belgium Department Crop Protection

Ghent Belgium (CLO-Gent)Not known Azores PortugalApple orchard Eksaarde Belgium eld populationGrass land Bourgoyen-Ossemeersen eld population

Ghent BelgiumGrass land with plum tree Ingelmunster Belgium eld population

P coffeae Banana not known CLO-GentP crenatus Maize- eld Eksaarde Belgium eld populationP akkensis Grassland (dominated by Eksaarde eld population

Arrhenatherus elatius Belgiumand Holcus lanatus)

P scribneri Maize Vero Beach FL USA CLO-GentP thornei Chick-pea Santaella CLO-Gent

Cordoacuteba SpainP vulnus Unknown Unknown CLO-GentP zeae Maize South Africa CLO-GentPratylenchoides River bank (dominated by Redu Ardennes A Ryss Russian Academy of

magnicauda Trifolium sp Quercus sp Belgium Sciences St Petersburg Russiaand Salix)

Pratylenchoides Wheat Apulia Italy A Trocolli Instituto di Nematologiaritteri Agraria Bari Italy

Pratylenchoides Lawn in the vicinity Botanical garden eld populationcrenicauda of a willow tree Ghent University

(Salix matsudana Koidz) BelgiumRadopholus Banana Sennar Sudan G Elbadri Crop Protection

similis Wad Medani SudanZygotylenchus Pistachio Kerman Iran E Pourjam Tarbiat

guevarai Modares University Iran

(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 is not clearly differentiated from the uterus Twelvecells were counted but an unambiguouscellular architec-ture could not be described (Fig 1B) The spermatheca ofP thornei Sher amp Allen 1953 is partly offset the offsetportion comprising on average four cells the spermathecacells being more or less round and equally sized (Fig 1C)The spermatheca of P penetrans is asymmetrical and theoviduct is not connected to the spermatheca entirely axi-

ally The slightly protruding corner of the spermatheca onthe oviduct side comprises small cells of various shapetwo large rounded cells making the connection to theuterus (Fig 1D) The oviduct of P zeae Graham 1951 ispartly enveloped by the spermatheca which has six cellsvariably arranged in the region close to the oviduct fol-lowed by four slightly larger cells with two cells makingthe connectionto the uterus (Fig 1E) The spermatheca ofP scribneri is asymmetrical and slightly offset due to the

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protruding and larger spermatheca cells at one side of thespermatheca Four spermatheca cells make the connectionto the uterus (Fig 1F) The spermatheca of P akkensisSeinhorst 1968 and P vulnus Allen amp Jensen 1951 alsoseems to comprise 12 cells but the number of successfuldissections was too low to obtain reliable results

The uterus of all studied Pratylenchus species consistsof three rows of cells (Da tricolumella) each row beingfour cells long The uterine sac follows the columnaruterus

ZYGOTYLENCHUS GUEVARAI

Light microscopy

The ovary connects to the oviduct via a ring of eightcells (Fig 2A) Two rows of four cells comprise theoviduct The spermatheca has 12 cells the two cellsconnecting it to the uterus being clearly larger The spermcells are variously shaped and often swollen They are alsofound at the beginningof the uterus which is formed fromthree rows of four cells

Transmission electron microscopy

The description below is based on the posterior branchof the didelphic reproductive system (Fig 4A) with theexception of one picture of the spermatheca taken fromthe anterior branch (Fig 6)

The ovarian wall cells form a thin layer around the germcells and have extensions between some of the germ cellsThese cytoplasmatic nger-like extensionscan even enterthe oocytes (Fig 4B) In the germinal zone the oocytesarranged in a single le have a clear cell membraneand the large variably shaped nucleus contains a darkspherical nucleolus As the oocytes become older theyincrease in size and contain more and more lipid dropletsand yolk

The oviduct (Fig 5) consists of irregularly shapedcells having highly plicated cell membranes In onelongitudinal section four adjacent oviduct nuclei could beseen two nuclei at the same level being observed close tothe spermatheca No preformed lumen was observed Theoviduct cells contain unknown secretions and nuclei withirregularly shaped nuclear membranes lined with densechromatin

The spherical spermatheca (Fig 6) occupies almost thewhole body cavity as observed in a transverse sectionThe spermatheca wall cells are highly attened theirnuclei being oval with dispersed dense chromatin Thevariably shaped sperm cells have a thick head portioncontaining the nucleus and a thinner more elongated

tail The nucleus lacks a membrane and is seen as aprominent mass of chromatin surrounded by clusters ofmitochondria

The columnar uterus cells (Fig 7A B) have plicatedmembranes The cytoplasm is lled with mitochondrianumerous ribosomes and some inclusionswith both heav-ily and slightly stained zones The nucleus contains aprominent nucleolus and some chromatin adjoins the nu-clear membrane A preformed uterus lumen was not ob-served the columnar cells being pressed against eachother and forming lsquomembrane-junctionsrsquo (Fig 7B) as de-scribed by Endo et al (1997 1999)

The columnar uterus is followed by a uterine sac(Fig 8) lled with a nely granular substance Thecuticula of the vagina is continuouswith the ventral liningof the uterus sac while a attened cellular wall lines therest of the uterine sac

PRATYLENCHOIDES WINSLOW 1958 (FIG 2B C D)

The ripening zone of the ovary consists of 16 cells witha ring formed from a variable number of cells connect-ing the ovary to the oviduct The oviduct of P magni-cauda (Fig 2C) and P ritteri Sher 1970 (Fig 2D) com-prises two rows of four cells In P crenicauda Winslow1958 (Fig 2B) only three cells were observed in each rowfor the majority of the observed specimens although anoviduct with two rows of four cells has also been ob-served The spermatheca and oviduct of an excised go-nad forms the corner of an angle between ovary anduterus The spermatheca of the studied Pratylenchoidesspecies comprises 12 variously shaped cells the sper-matheca cells of P ritteri being more rounded and pro-truding The uterus is constructed from three rows of sixto seven columnar cells

RADOPHOLUS SIMILIS (FIG 2E)

The ripening zone of the ovary has four groups offour cells The oviduct comprises two rows of four attened cells The spermatheca always contains 12variably positionedcells and is followed by a tricolumellacomprising 12 cells

HIRSCHMANNIELLA LOOFI (FIG 2F)

The ripening zone could not be distinguished fromthe remainder of the ovary The oviduct comprises tworows of four cells The spermatheca cells are indistinctlydelimited and their cell borders are twisted The number

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Fig 1 Partial female reproductive systems of Pratylenchus spp A Pratylenchuscrenatus B P coffeae C P thornei D P penetransE P zeae F P scribneri End of ovary (ova) oviduct (ovi) spermatheca (sp) and uterus (ut) (Scale bars D 10 sup1m)

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Fig 2 Partial female reproductive systems of Pratylenchidae A Zygotylenchus guevarai B Pratylenchoides crenicauda C Pmagnicauda D P ritteri E Radopholus similis F Hirschmanniella loo G Nacobbus aberrans spermatheca region H Nacobbusaberrans uterus closer to the vulva End of ovary (ova) oviduct (ovi) spermatheca (sp) uterus (ut) and uterus closer to the vulva(utv) (Scale bars D 10 sup1m)

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Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

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Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

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Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

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Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

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Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

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Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

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Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

protruding and larger spermatheca cells at one side of thespermatheca Four spermatheca cells make the connectionto the uterus (Fig 1F) The spermatheca of P akkensisSeinhorst 1968 and P vulnus Allen amp Jensen 1951 alsoseems to comprise 12 cells but the number of successfuldissections was too low to obtain reliable results

The uterus of all studied Pratylenchus species consistsof three rows of cells (Da tricolumella) each row beingfour cells long The uterine sac follows the columnaruterus

ZYGOTYLENCHUS GUEVARAI

Light microscopy

The ovary connects to the oviduct via a ring of eightcells (Fig 2A) Two rows of four cells comprise theoviduct The spermatheca has 12 cells the two cellsconnecting it to the uterus being clearly larger The spermcells are variously shaped and often swollen They are alsofound at the beginningof the uterus which is formed fromthree rows of four cells

Transmission electron microscopy

The description below is based on the posterior branchof the didelphic reproductive system (Fig 4A) with theexception of one picture of the spermatheca taken fromthe anterior branch (Fig 6)

The ovarian wall cells form a thin layer around the germcells and have extensions between some of the germ cellsThese cytoplasmatic nger-like extensionscan even enterthe oocytes (Fig 4B) In the germinal zone the oocytesarranged in a single le have a clear cell membraneand the large variably shaped nucleus contains a darkspherical nucleolus As the oocytes become older theyincrease in size and contain more and more lipid dropletsand yolk

The oviduct (Fig 5) consists of irregularly shapedcells having highly plicated cell membranes In onelongitudinal section four adjacent oviduct nuclei could beseen two nuclei at the same level being observed close tothe spermatheca No preformed lumen was observed Theoviduct cells contain unknown secretions and nuclei withirregularly shaped nuclear membranes lined with densechromatin

The spherical spermatheca (Fig 6) occupies almost thewhole body cavity as observed in a transverse sectionThe spermatheca wall cells are highly attened theirnuclei being oval with dispersed dense chromatin Thevariably shaped sperm cells have a thick head portioncontaining the nucleus and a thinner more elongated

tail The nucleus lacks a membrane and is seen as aprominent mass of chromatin surrounded by clusters ofmitochondria

The columnar uterus cells (Fig 7A B) have plicatedmembranes The cytoplasm is lled with mitochondrianumerous ribosomes and some inclusionswith both heav-ily and slightly stained zones The nucleus contains aprominent nucleolus and some chromatin adjoins the nu-clear membrane A preformed uterus lumen was not ob-served the columnar cells being pressed against eachother and forming lsquomembrane-junctionsrsquo (Fig 7B) as de-scribed by Endo et al (1997 1999)

The columnar uterus is followed by a uterine sac(Fig 8) lled with a nely granular substance Thecuticula of the vagina is continuouswith the ventral liningof the uterus sac while a attened cellular wall lines therest of the uterine sac

PRATYLENCHOIDES WINSLOW 1958 (FIG 2B C D)

The ripening zone of the ovary consists of 16 cells witha ring formed from a variable number of cells connect-ing the ovary to the oviduct The oviduct of P magni-cauda (Fig 2C) and P ritteri Sher 1970 (Fig 2D) com-prises two rows of four cells In P crenicauda Winslow1958 (Fig 2B) only three cells were observed in each rowfor the majority of the observed specimens although anoviduct with two rows of four cells has also been ob-served The spermatheca and oviduct of an excised go-nad forms the corner of an angle between ovary anduterus The spermatheca of the studied Pratylenchoidesspecies comprises 12 variously shaped cells the sper-matheca cells of P ritteri being more rounded and pro-truding The uterus is constructed from three rows of sixto seven columnar cells

RADOPHOLUS SIMILIS (FIG 2E)

The ripening zone of the ovary has four groups offour cells The oviduct comprises two rows of four attened cells The spermatheca always contains 12variably positionedcells and is followed by a tricolumellacomprising 12 cells

HIRSCHMANNIELLA LOOFI (FIG 2F)

The ripening zone could not be distinguished fromthe remainder of the ovary The oviduct comprises tworows of four cells The spermatheca cells are indistinctlydelimited and their cell borders are twisted The number

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Fig 1 Partial female reproductive systems of Pratylenchus spp A Pratylenchuscrenatus B P coffeae C P thornei D P penetransE P zeae F P scribneri End of ovary (ova) oviduct (ovi) spermatheca (sp) and uterus (ut) (Scale bars D 10 sup1m)

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Fig 2 Partial female reproductive systems of Pratylenchidae A Zygotylenchus guevarai B Pratylenchoides crenicauda C Pmagnicauda D P ritteri E Radopholus similis F Hirschmanniella loo G Nacobbus aberrans spermatheca region H Nacobbusaberrans uterus closer to the vulva End of ovary (ova) oviduct (ovi) spermatheca (sp) uterus (ut) and uterus closer to the vulva(utv) (Scale bars D 10 sup1m)

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Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

Vol 5(2) 2003 299

W Bert et al

Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

300 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 1 Partial female reproductive systems of Pratylenchus spp A Pratylenchuscrenatus B P coffeae C P thornei D P penetransE P zeae F P scribneri End of ovary (ova) oviduct (ovi) spermatheca (sp) and uterus (ut) (Scale bars D 10 sup1m)

Vol 5(2) 2003 297

W Bert et al

Fig 2 Partial female reproductive systems of Pratylenchidae A Zygotylenchus guevarai B Pratylenchoides crenicauda C Pmagnicauda D P ritteri E Radopholus similis F Hirschmanniella loo G Nacobbus aberrans spermatheca region H Nacobbusaberrans uterus closer to the vulva End of ovary (ova) oviduct (ovi) spermatheca (sp) uterus (ut) and uterus closer to the vulva(utv) (Scale bars D 10 sup1m)

298 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

Vol 5(2) 2003 299

W Bert et al

Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

300 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

Fig 2 Partial female reproductive systems of Pratylenchidae A Zygotylenchus guevarai B Pratylenchoides crenicauda C Pmagnicauda D P ritteri E Radopholus similis F Hirschmanniella loo G Nacobbus aberrans spermatheca region H Nacobbusaberrans uterus closer to the vulva End of ovary (ova) oviduct (ovi) spermatheca (sp) uterus (ut) and uterus closer to the vulva(utv) (Scale bars D 10 sup1m)

298 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

Vol 5(2) 2003 299

W Bert et al

Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

300 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 3 Schematic presentationof the cellular structure of the female gonoduct within the studied Pratylenchidae

of spermatheca cells varies from ten to 12 The uterus isconstructed from three rows of seven or eight elongatedcells Sperm was not only observed in the spermathecabut also occurred in the uterus up to the level of the secondtriplet of uterus cells

NACOBBUS ABERRANS (FIG 2G H)

The oviduct consists of two rows of four cells whichare partly envelopedby the spermathecaThe spermathecacell boundaries are only partly visible and the spermath-eca region is dif cult to distinguish from the uterus AfterDAPI staining ten to 14 nuclei could be counted in thespermatheca-region although it was impossible to deter-mine the exact number of cells The uterus adjacent to thespermatheca is formed by a long tricolumella with on av-erage 64 cells in each row the cells being larger distallyThe uterus portion closer to the vulva enlarges to form anirregular polycolumella the exact number of cells (morethan 100) being indeterminable

Discussion

Except for N aberrans all species studied show astriking overall similarity in gonoduct construction iean ovary often ending with a ring of cells two rowsof four cells forming the oviduct and a spermathecawith typically 12 cells followed by a tricolumella with16 to 24 cells This overall similarity is an indication

in favour of the coherence of the family Pratylenchidae(with the exceptionof N aberrans) However the cellulargonoduct structure provides no arguments to supportconsideration of this family as a monophyletic group asa similar genital structure was found in some membersof the Belonolaimidaeand Hoplolaimidae(Geraert 1981Chizhov amp Berezina 1988)

OVARY

In the germinal zone of the ovary the wall cells forma thin sheath around the germinal cells with extensionsbetween them Cytoplasmatic contact between germ cellsand epithelial cells appears to be minimal for the Ppenetrans studied herein and for the specimens studiedby Endo et al (1999) In Zygotylenchus on the otherhand an ovarian wall cell extensionwas found penetratingthe germ cell and making cytoplasmatic contact Weassume that these differences between Pratylenchus andZygotylenchus are only a matter of observation Hilgert(1976) attributed a feeding function of the ovarian wallfor the germ cells via cytoplasmatic contact Our resultscorroborate this hypothesis for Zygotylenchus These nger-like wall extensionspresumably have a comparablefunction to the central cytoplasmatic mass extendinginto the ovary of Xiphinema theresiae (Van De Veldeamp Coomans 1988) or the typical rachis-like structurefound for instance in Meloidogyne javanica (Bird ampBird 1991) Transport to the oocytes results in the storageof more and more lipid and yolk and these oocytes

Vol 5(2) 2003 299

W Bert et al

Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

300 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

Fig 4 A Overview TEM study of posterior gonoduct branch of Zygotylenchus guevarai frames approximately locate the TEMphotographs (the spermatheca picture was taken from the anterior branch) B Longitudinal section through ovary of Zygotylenchusguevarai two oocytes surrounded by ovarian wall cells C Detail of ovarian wall cell extension entering an oocyte Abbreviations CuD cuticle ChrWa D chromatin of nucleus ovarian wall cell Cyto D cytoplasmatic ovarian wall extension Int D intestine LD D lipiddroplet NM D nuclear membrane NO D nucleus of oocyte NuO D nucleolus of oocyte NOva D nucleus ovary wall cell O D oocyteOM D oocyte membrane Ova D longitudinal section through ovary OvaC D ovarian wall cell PB D protein body SM D somaticmuscular tissue Sp D spermatheca Ut D uterus Vag D Vagina (Scale bars A D 10 sup1m B D 1 sup1m C D 500 nm)

nally end up in the often empty proximal part of theovary For most of the studied Pratylenchidae this regionwas morphologicallydivergent and comprised 16 slightly

protruding cells most easily observed with LM Endoet al (1999) described the distal part of the ovary asan oviduct with irregularly shaped cells having plicated

300 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 5 A Longitudinal section through oviduct of Zygotylenchus guevarai four adjacent oviduct nuclei are visible two being atthe same level close to the spermatheca B Detail of oviduct a secretion is visible in the two oviduct cells which connect to thespermatheca Abbreviations Cu D cuticle ChrOvi D chromatin of nucleus oviduct cell Int D intestine LD D lipid droplet Novi (12 4) D nuclei of oviduct cells of right cell row Novi (10 30) nuclei of oviduct cells of left cell row NSpr D nucleus sperm cell NSpD nucleus spermatheca cell Ovi D oviduct PCM D plicated cell membrane SecrOvi D secretion within oviduct cell SM D somaticmuscular tissue Sp D spermatheca Spr D sperm cell (Scale bars A D 1 sup1m B D 500 nm)

plasma membranes and muscle laments What they

described as lsquoclosely arranged cellsrsquo (which appear to

function as a valve) the oviduct sensu Endo et al (1999)

must be the ring of eight ovarian wall cells as seen in our

LM studies (Figs 1 2) In their apparent transverse section

(see Fig 12 of Endo et al 1999) a circle of cells is visibleand eight nuclei can be counted

OVIDUCT

The oviduct a relatively short constriction betweenovary and spermatheca and without a visible lumenconsists of two rows of four cells in all the species studied

The oviduct itself is de ned differently in the literature(for an overview see Geraert 1976) We apply the oviductde nition sensu Geraert (1976) ie the few cells that

Vol 5(2) 2003 301

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

Fig 6 A Longitudinal section through spermatheca of Zygotylenchusguevarai B Detail of two adjacent sperm cells AbbreviationsCu D cuticle HSpr D head portion sperm cell LD D Lipid droplet LSp D lumen spermathecaMit D mitochondriaMSpr D membranesperm cell NSp D nucleus spermatheca cell NSpr D nucleus sperm cell PB D protein bodies SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell TSpr D tail portion sperm cell (Scale bars A D 2 sup1m B D 500 nm)

form the constriction between ovary and spermathecaEndo et al (1999) considered the ripening zone ofthe ovary as the oviduct Huettel and Dickson (1981)however illustrated in Radopholus similis an oviductwith approximately 20 cells presumably representing thecombined oviduct and the ring formed by the ovary wallcells Even though these distal ovary cells can form asphincter-like structure this seems to be dependent onnematode maturity being found more often in youngfemales The oviductsensu Geraert (1976) is a remarkablystable structure with a homogeneous morphology in allstudied specimens

SPERMATHECA

The number of spermatheca cells appears to be nearlyconstant within the Pratylenchidae and independent of

whether the species is monosexual (eg P zeae) or bi-sexual (eg P penetrans) Geraert (1973) described forPratylenchus sp a 12-celled spermatheca and we con- rmed this for other Pratylenchus species Chizhov andBerezina (1988) found 12 to 14 cells in the spermathecaof Hirschmanniella sp while we found a variable butslightly lower number Roman and Hirschmann (1969)drew only ten nuclei in the spermatheca of Pratylenchuscoffeae and P scribneri These observations were notmade on dissected gonads but on orcein stained in totomounts Pourjam et al (2000) described from an IranianZygotylenchus population mounted in glycerine a sper-matheca apparently consisting of numerous cells reminis-cent of Meloidogyne and as a consequence a relation-ship with the root-knotnematodeswas suggestedOur LMstudies however showed that the sperm cells often have a

302 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Fig 7 A Longitudinal section through columnar uterus of Zygotylenchus guevarai at spermatheca-uterus transition B Longitudinalsection through columnar uterus of Zygotylenchus guevarai the columnar cells are pressed against each other and form lsquomembrane-junctionsrsquo sensu Endo et al (1997 1999) AbbreviationsCu D cuticle CUt D columnar cells of uterus ChrUt D chromatine in nucleusof uterus cell Int D intestine IUt D inclusion in uterus MJ D membrane junction MUt D membrane uterus cell NUt D nucleus uteruscell SM D somatic muscular tissue Sp Dspermatheca Spr D sperm cell Ut D uterus VE D vesicles with undened content (Scalebars 1 sup1m)

swollen appearance while TEM showed these sperm cellsto be remarkably long and variably shaped As a result it ispossible that Pourjam et al (2000) could have interpretedthe very large sperm cells as spermatheca wall cells

The genital structure of all Pratylenchoides speciesstudied was similar and no differences were found forP magnicauda a species whose systematic positionhas been questioned Amplimerlinius the assumed closerelative of P magnicauda was found to have a slightlydifferent genital structure the axial spermatheca beingbell-shaped with 12 to 14 cells with slightly interlaced cellborders (Bert unpubl) Consequentlyour results supportthe inclusion of P magnicauda in the Pratylenchidae

The shape of the spermatheca has been used in speciesidenti cation within the genus Pratylenchus (see Sein-horst 1968 Loof 1991 Ryss 2002) Our results sug-gest the potential use of spermatheca cell morphology inspecies differentiationalthoughwe only studied the inter-populationvariability of the spermatheca for P penetransFor this species the possession of an asymmetrical sper-matheca with a protruding corner containing small cellson the ovary side was a constant character

UTERUS

The uterus in all species studied consists of three rowsof cells and is hence named a tricolumella This is in con-

Vol 5(2) 2003 303

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

Fig 8 A Longitudinal section through columnar uterus uterine sac and vagina of Zygotylenchus guevarai B Detail of vagina anduterine sac Abbreviations Cu D cuticle Con Va D constrictor vaginae CVS D cellular outline of uterus CuVS D cuticular ventraloutline of uterine sac Dil Va D dilator vaginae Dil Vu D dilator vulvae Int D intestine Nint D nucleus intestinal cell NMD nucleusmuscle cell NUt D nucleus uterine cell SM D somatic muscular tissue US D uterine sac Vag D vagina VE D vesicle with undenedcontent (Scale bars 1 sup1m)

cordance with the majority of the literature describing thecellular arrangement of the uterus within the suborder Ho-plolaimina(sensu Siddiqi 2000) On the other hand Sein-horst (1968) and Endo et al (1999) mention a quadricol-umella in their gonad studies of Pratylenchus This is notbased on their own observationshowever but was respec-tively taken from Wu (1967) and Coomans (1962) Theaccount by Dickerson (1962) of the developmentof a 20-celled quadricolumella in Pratylenchus crenatus is strik-

ing but although his illustration suggests the presence offour cell rows in the uterus only three rows were actuallydrawn

In the studiedPratylenchidaespecies the number of col-umella cells is restricted (max 24 cells) except in Nacob-bus aberrans where the number of uterus cells exceeds300 This could be an adaptation of the mode of repro-duction associated with a sedentary way of life In thisaspect N aberrans is similar to the Heteroderinae and

304 Nematology

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

Comparative morpho-anatomical studies of the female gonoduct within the Pratylenchidae

Meloidogyninae(Bert et al 2002) although the structureof the complete genital system does not match with anyrepresentative of the Heteroderinae or Meloidogyninae Asimilar spermatheca-like structure has not been observedor reported in the literature for any other Nematoda Thediffering gonoduct structure of Nacobbus in contrast tothe overall similarity of the other studied genera is anargument in favour of dividing the Pratylenchidae intoonly two subfamilies (Nacobbinae and Pratylenchinae)as was done by Luc (1987) and not into four subfami-lies (NacobbinaePratylenchinaeHirschmanniellinaeandRadopholinae)as outlined by Siddiqi (2000)

Our TEM observation suggests that the columella cellsmight have a functional role in providing secretions thatcontribute to egg shell formation (Coomans 1962 Birdamp Bird 1991 Endo et al 1999) We observed theproduction of secretions in the uterus and the presenceof secretory granules which appear to merge with thedark-appearing outer layer of the egg shell althoughthe intermediate excretion phase as described by Hilgert(1976) has not been established

LM OF DISSECTED ORGANS VS TEM

De ning and specifying the different gonoduct unitswas based on LM of dissected gonads (Geraert 19761981) This technique is relatively easy and the three di-mensional structure is well preserved However interpre-tation may be speculativeas morphologicaldiscriminationbetween the cells of adjacent gonoduct parts can be dif -cult On a sub-cellular level and with the aid of TEMthese morphological differences are much more evidentand it seems that the interpretation based on LM is justi- ed

Acknowledgements

The authors thank Gerrit Karssen Lieven Waeyen-berge Nancy de Sutter Alex Ryss Alberto TrocolliGamal Elbadri and Ebrahim Pourjam for their generoussupply of specimens We are grateful to Gerrit Karssenfor critically reading this manuscript

References

AL-BANNA L WILLIAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG amp CAP GB (1992) Systematics of Nacobbusthe false root-knot nematode In Gommers FJ amp Maas PW(Eds) Nematology from molecule to ecosystem Proceedingsof the Second International Nematology Congress 11-17August 1990 Veldhoven The Netherlands Invergowrie UKEuropean Society of Nematologists pp 104-112 [Abstr]

BALDWIN JG LUC M amp BELL AH (1983) Contributionto the study of the genus Pratylenchoides Winslow (Nema-toda Tylenchida) Revue de Neacutematologie 6 111-115

BERT W KARSSEN G VAN DRIESSCHE R amp GERAERTE (2002) The cellular structure of the female reproductivesystem within the Heteroderinae and Meloidogyninae (Ne-matoda) Nematology 4 953-964

BIRD AF amp BIRD J (1991) The structure of nematodes 2ndedition New York USA Academic Press 316 pp

CHITWOOD BG amp CHITWOOD MB (1950) An introduc-tion to nematology BaltimoreMD USA Monumental Press213 pp

CHIZHOV VN amp BEREZINA NV (1988) [Structure and evo-lution of the genital system in female nematodes of the or-der Tylenchida 2 Primarily didelphic species] Zoologich-esky Zhurnal 67 485-490

COOMANS A (1962) Morphological observations on Roty-lenchus goodeyi Loof amp Oostenbrink 1958 II Detailed mor-phology Nematologica 7 242-250

DE LEY P amp BLAXTER ML (2002) Systematic position andphylogeny In Lee DL (Ed) The biology of nematodesLondon UK Taylor amp Francis pp 1-30

DICKERSON OJ (1962) Gonad development in Pratylenchuscrenatus Loof and observations on the female genital struc-tures of P penetrans Proceedings of the HelminthologicalSociety of Washington 29 173-176

ENDO BY ZUNKE U amp WERGIN WP (1997) Ultrastruc-ture of the lesion nematodePratylenchuspenetrans (NemataPratylenchidae) Journal of the Helminthological Society ofWashington 64 59-95

ENDO BY ZUNKE U amp WERGIN WP (1999) Ultrastruc-ture of the female reproductivesystem of the lesion nematodePratylenchus penetrans (Nemata Pratylenchidae)Journal ofthe Helminthological Society of Washington 66 155-175

GERAERT E (1973) A comparative study of the structure ofthe female gonads in plant-parasitic Tylenchida (Nematoda)Annales de la Socieacuteteacute Royale Zoologique de Belgique 102171-198

GERAERT E (1976) The female reproductive system in De-ladenus and Hexatylus with a rede nition of the oviduct inthe Tylenchida (Nematoda) Nematologica 22 437-445

GERAERT E (1981) The female reproductive system in nema-tode systematicsAnnales de la Socieacuteteacute Royale Zoologique deBelgique 110 73-86

GERAERT E (1983) The use of the female reproductivesystemin nematode systematics In Stone AR Platt HM ampKhalil LF (Eds) Concepts in nematode systematicsLondonamp New York Academic Press pp 73-84

Vol 5(2) 2003 305

W Bert et al

GERAERT E (1997) Comparison of the head patterns in theTylenchoidea (Nematoda) Nematologica 43 283-294

HILGERT A (1976) [Electron microscopic observations onthe female gonad of Pratylenchus penetrans] MSc ThesisGhent University Belgium 56 pp

HIRSCHMANN H amp TRIANTAPHYLLOU AC (1968) Modeof reproduction and development of the reproductive systemof Helicotylenchus dihystera Nematologica 13 558-574

HUETTEL RN amp DICKSON DW (1981) Karyology andoogenesis of Radopholus similis (Cobb) Thorne Journal ofNematology 13 16-20

KARNOVSKY MJ (1965) A formaldehyde-glutaraldehyde xative of high osmolarity for use in electron microscopyJournal of Cell Biology 27 137A

LOOF PAA (1991) The family PratylenchidaeThorne 1949In Nickle WR (Ed) Manual of agricultural nematologyNew York USA Marcel Dekker pp 363-421

LUC M (1987) A reappraisal of Tylenchina (Nemata) 7 Thefamily Pratylenchidae Thorne 1949 Revue de Neacutematologie10 203-218

MOODY EH LOWNSBERY BF amp AHMED JM (1973)Culture of the root-lesion nematode Pratylenchus vulnus oncarrot disks Journal of Nematology 5 225-226

POURJAM E GERAERT E amp ALIZADEH A (2000) Somepratylenchidsfrom Iran (Nematoda Tylenchina)Nematology2 855-869

ROMAN J amp HIRSCHMANN H (1969) Morphology andmorphometrics of six species of Pratylenchus Journal ofNematology 1 363-383

RYSS AY (2002) Genus Pratylenchus Filipjev (NematodaTylenchida Pratylenchidae) multientry and monoentry keysand diagnostic relationships Zoosystematica Rossica 10 11-25

RYSS A amp STURHAN D (1994) Studies on Pratylenchoidesivanovae Ryss 1980 and P magnicauda (Thorne 1935)Russian Journal of Nematology 2 121-128

SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-510

SIDDIQI MR (2000) Tylenchida parasites of plants andinsects 2nd Edition WallingfordUK CABI Publishing 864pp

SPURR AR (1969) A low viscosity epoxy resin-embeddingmedium for electron microscopy Journal of UltrastructuralResearch 26 31-43

VAN DE VELDE MC amp COOMANS A (1988) Electron mi-croscopy of the germ cells and the ovarian wall in Xiphinema(Nematoda) Tissue amp Cell 20 881-890

WU LY (1967) Differences of spermatheca cells in thegenera Ditylenchus Filipjev 1936 and Tylenchus Bastian1865 (TylenchidaeNematoda) Canadian Journal of Zoology45 27-30

306 Nematology