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Home , Spermatogenesis, Spermatozoon

and structure in some Meloidogyne (Heteroderoidea, Meloidogynidae) and a cornparison with those in some cyst nematodes (Heteroderoidea, 13eteroderidae)

Audrey M. SHEPHERDand Sybil A. CLARK Rothamsted Experimental Station, Harpenden, Herts., England.

Sperm structure and development was examined inthe mitotic (obligatory) parthenogens Meloidogyne incognita incognita, M. incognita wartellei and M. arenaria, in the meiotic (facultative) parthenogens M. hapla (race A) and M. graminicola, in M. oryzae (reproductive status unknown), and in the amphimictic species M. acronea. Sperm- atogenesis in Meloidogyne differs from that in the cyst nematodes, inwhich the maturation divisions occur only in the testis of the pre-adult ; cells in al1 phases of spermatogenesis were present in adult Meloidogyne males. The structure of the in M. hapla is discussed. In Meloidogyne, differences in sperm structure between species concerned mainly : the polarisation of the spermatozoa in some intoa main (( body D and a pseudopodium ; persist- ence or otherwise of the fibrillar bodies ; abundance of filopodia, particularly at earlier stages ; state of the nuclear material and size of mature sperm. Sperm of four Meloidogyne species were, like cyst-nematode testicular sperm, unpolarised. The form of the condensed in the sperm nucleus was similar in most Meloidogyne to that in Heterodera. In Globodera and in M. oryzae the form was different. So-called nucleolar bodies, present in the

I Meloidogyne species,were absentfrom sperm in the testis of cystnematodes. Cortical lining the plasma membrane of the sperm, and the absence of membranous , were constant features throughout the two families.

RÉSUM~ Spermatogénèse et structure des spermatozoïdes chez quelques Meloidogyne (Heteroderoidea, Meloidogynidae) compare‘es avec certaines espCces de ne’matodes à kystes (Heteroderoidea, Heteroderidae) La structure et le développement des spermatozoïdes ont été observés chez les espèces à parthénogénèse mito- tique (obligatoire)Meloidogyne incognita incognita,M. incognita wartellei et 114. arenaria, chez les espèces à parthéno- génèse méiotique (lacultative)M. hapa (race A) et M. graminicola, ainsi que chez I’espbce M. oryzae, dont les moda- lités de sont inconnues, et chez l’espèce amphimictique M. acronea. La spermatogénèse chez Meloido- gyne est différente de celle des nématodes à kystes où les divisions de maturation ont lieu dans le testicule du pré- adulte ; chez les mgles de Meloidogyne, des cellules à toutes les phases de la spermatogénèse étaient présentes. La structure de la spermatidechez M. hapla est discutée. Chez Meloidogyne les différences entre espèces dans la struc- ture des spermatozoïdes concernent principalement : la (( polarisation O entre corps principal du spermatozoïde et pseudopode ; la persistance ou la disparition des corps fibrillaires ; l’abondance des filopodes, particulièrement aux stades les plusjeunes ;l’état du matériel nucléaire et la taille du spermatozoïde Chez mûr. quatre espèces de Meloido- gyne les spermatozoïdes ne sont pas polarisés, demême que les spermatozoïdes testiculaires des nématodesà kystes. Chez de nombreux Meloidogyne l’aspect de la chromatine condensée dans le noyau du spermatozoïde est identique à celui des Heterodera. Chez Globodera et M. oryzae cet aspect est différent. Les corps nucléolaires présents chez Meloidogyne, manquent dans les spermatozoïdes testiculaires des nématodes à kystes. La présence de microtubules corticaux bordant la membrane plasmatique du spermatozoïde, ainsi que l’absence d’organites membraneuses, représentent des caractères constantschez l’une et l’autre familles.

Revue Nimatol. 6 (1) : 17-32 (1983) 17 A.M. Shepherd CE S.A. Clark

Differences betweenthe structure of sperm of Themales were fixed in 6% glutaraldehydein cystnematodes (Heterodera and Globodera) (Shep- cacodylate buffer at pH 7.2 for 22 h in an ice bath. herd, Clark & Kempton, 1973) in the Tylenchida and The posterior two thirds or so was then excised and of Aphelenchoides (Shepherd & Clark,1976) in the cut into at least two pieces ; each piecewas embedded . hphelenchida (Siddiqi, 1980) are fundamental. How- in 1% agar and returned to the cold fixative for an ever,more examples from each group are needed additional 2 h. They were then thoroughly rinsed in to confirm that thesebasic structural differences buffer,post-fixed in 1% Os04 in veronalacetate arecharacteristic of therespective groups as a bufferfor 3 h, dehydrated in an alcohol series and whole. For comparison with cyst-nematode spermwe infiltratedand embedded in Spurr low visc0sit.y selected Meloidogyne, representing a family (Meloi- resin.Longit>udinal and transverse serial sections dogynidae) close tothe Weteroderidae, andwe (60 mm) were cut and mounted on large-slotgrids examinedsperm structure and development in coated with a necoloidine film. Sections were stained several species to see if theyvaried. This selection on the grids using lead citrate, either alone or after included meiotic (facultative) and mitotic (obligatory) uranylacetate. Some were also pretreatedwith parthenogens, and one species (M. acronea) thought potassium permanganate (Soloff, 1973). to be entirely amphimictic (Page, pers. comm.). Themain object of thisstudy was to compare sperm morphology ; wehave not attempted to Observations interpret genetical differences. The ultrastructure of cytologicaldifferences betweenthe meiotic and mitoticraces of M. hapla duringspermatogenesis MITOTIC ( OBLIGATORY) PARTHENOGENS was described by Goldstein andTriantaphyllou (197Sb), andsperm development in M. hapla by M. incognita incognita. Goldstein andTriantaphyllou (1980). We briefly describedsperm development in M. incognita Al1 stages of sperm development are found in the incognita previously(Shepherd & Clark,1979) ; adult male ganad, the tip of which is usually about here we describe it fully, and comment on the other midway dong thebody. species where differences exist,. The males ofal1 the Spermatogonia. Two or threeintermingled rows speciesexamined are telogonic andusually monor- of spermatogonial cells (sg)(Fig. la) extendfrom chic (althoughsome individuals may have two testes), the testis tip for about 150Fm. The cells are packed andtheir are usually straight but may be closely togetherand wecould notdistinguish a reflexed. definiterachis. The testis Wall [tw) is very thin in this region. Eachspermatogonium has a large, amoeboid, membrane-bound nucleus (nu) occupying Materials and methods much of the ce11 and containinga prominent, electron- dense,spherical nucleolus (nl). The spermatogonia, Adult males of the'various Meloidogyne spp. were whichusually number about 50-70,are about obtained from the sources and populations expressed 6 km in diameter and are rich in ribosomes (ri) and in Table 1, kept in a greenhouse at Rothamsted. mitochondria (m) ; Golgi bodies (gb) arealso plentiful.

Table 1

Species Source Cultured on No. specimens cut

M. acronea Coetzee Ngabu, Malawi sorghum 7 M. arenaria (Neal) origin unknown coleus 7 M. graminicola Golden & Birchfield Bangladesh rice 9 M.hapla (race A) Chitwood Norfolk, England tomato 4 M. incognita incognita (Kofoid & White) origin unknown tomato 11 M. incognita wartellei Golden & Birchfield N.C. State Univ., U.S.A. soybean 7 M. oryzae Maas, Sanders & Dede Surinam rice 2

18 Revue Nimatol. 6 (1) : 17-32 (1983) Fig. 1. M. incogrzita incognita adult male. a : L.S. testis tipshowing spcrmatogonia (sg) with large amoeboid nucleus (nu), spherical nucleolus (nl), nuclear membrane (nm), ribosomes (ri), Golgi bodies (gb), mitochondria(m), smooth endoplasmicrecticulum (ser)and cytoplasmic spherical bodies (csb). x 7 300 ; b : T.S. testis showing a primary (psc) at prophase, with chro- mosomes (chr) condensing andnuclear membrane (nm) almostdispersed. contains fibrillar bodies (fb) and many ribosomes (ri). x 9 200 ; c : L.S. testis showing primary sperma- tocyte(psc) at metaphase with (chr) alignedalong the metaphase plate ; also spindle fibres (sf) and centriolar complex (ce). x 7 300 ; d : T.S. testis showing spermatid (st) with homogeneous nucleus (nu),fibrillar bodies (fb)and mitochondria (m) anda residual body (rb) with many ribosomes (ri), joined by abscission neck (abn) containing contractile ring of fibrils (Cr). x 7 500. Abbréviations : abn : abscission neck ; bmu : body Wall muscle ; ce : ; chn : chromatin ; chr : ; cmt : cortical microtubules ; Cr : contractile ring ; csb : cytoplasmic spherical body ; CU : cuticle ; cyt : cytoplasmic ; esc : early spermatocyte ; fb : fibrillar body ; fp : filopodium ; gb : Golgi body ;int : intestine ;m : mitochondrion ; mt : microtubules ; mtc : compres- sed microtubules ; nl : nucleolus ; nlb : nucleolar body ; nm : nuclear membrane ; nu : nucleus ; osm : outer sperm membrane ; pm : plasma membrane ; ps : pseudopodium ; psc : primary spermatocyte ; rb : residual body ; ri : ribosomes ; SC : spermatocyte ; ser : smooth endoplasmic recticulum ; sf : spindle fibres ; sg : ; sp : ; spb : sperm body ; st : spermatid ; svw : seminal vesicle Wall ; tn : telophase neck ; tv : electron-translucent vesicle ; tw : testis Wall. A.M. Shepherd & S.A. Clark

Patches of moderatelydense granular material in largeproportion of it,s volume,together with the thecytoplasm (csb) are presumably profiles of mitochondria (which are poorly preserved in Fig. Id). sphericalbodies of somekind, and there is some The ce11 outlinebecomes increasingly complex smoothendoplasmic reticulum (ser) (Fig. la). around this region but not around the residualbody. Throughout this stage thece11 is still bounded only . With the nuclear changes that mark by theplasma membrane but, in very late spermat.ids theinception of thespermatocyte stage, the first the Wall shows signs of thiclrening. In the specimens signs appear of the fibrillar material (fb) characteristic we sectioned there were about a dozen cells at the of most nematode spermat some stage (as illustrated spermatid stage, occupying 35-40ym of the testis. for M. hapla, Fig. 4a). The roughly cuboidal fibrillar Thespermatid, including itsattached residual bodies(Fig. lb,c) grow toabout 0.5-0.6Pm in body,is about 20 pm by 8 ym. ~ diameter. They are not associated withany specialised membranesystem nor are they even contained Spermatozoa. In Our interpretation, the transition within a vesicle but lie free in the cytoplasm from fromspermatid to spermatozoon comes with the their inception. There are usually from 12 to 20 cells sloughing offof the residual body (Shepherd, 1981). invarious phases of thematuration diviasion at. Theearly spermatozoa contain a centralnucleus any one time, occupying 35-40 pm of the length of of homogeneous, very electron-densechromatin the testis, whose Wall (tw) is thicker in this region (nu in Fig. 2b), irregular in shape but with smooth and contains ribosomes and vesicles. Each sperma- outlines and cavities. There are also several spherical t,ocyte is slightly elongated, about 15 ym by 10 pm, satellitebodies, probably nucleolar material (nlb) wit,h many ribosomes (ri) and fibrillar bodies, some (Goldstein & Triantaphyllou,1980), scattered mitochondria(m) and some electron-translucent throughout the cyloplasm. These, too, are homogen- vesicles (tv),whose contents may have been leached eous but less electron-dense and more granular than out during preparation. The outlineof the spermato- the chromatin. The contours of these various bodies cyte becomes complex with thefirst infoldings of the weredetermined byreconstruction from serial boundary membrane that eventually become filopo- sections. The mitochondria(m) andfibrillar bodies(fb) dia (fp) (Fig. lb, c). The ce11 is enclosed only by a areevenly distributed throughout 'the cytoplasm plasmamembrane. The earliest indication of the andthe many filopodia (fp)are free-standing, no start of the maturation division is the condensation longer compacted or possibly even adhering together of thechromatin (chr) within the nuclear mem- as earlier.The outer coat over the whole sperm brane (nm) at early prophase (Fig. lb), followed by surface,including the filopodia,is complex at this dissolution of the nuclear membrane. Fig. lc shows stage (Fig.3f), witrh an inner plasma membrane (pm), metaphase,with fully condensed chromosomes and an outer membrane (osm) that has two dense aligned along the equatorof the spindle. layersseparated by an electron-translucent zone, with an additional dense, fuzzy layer on the outside. . AftertJhe maturation division the -likestructures (cmt) (about 20 nm chromaticmaterial of the nucleusre-organises diam.) in parallel arrays line the inner surface of the into a homogeneous,electron-dense mass (nu) with plasma membrane (Figs 2b ; 3b, f). These look finer no reconstitution of the nuclear membrane (Fig. Id). and less obviously attached to the plasma membrane The ribosomes,Golgi bodies and cytoplasmic spherical thanthose incyst-nematode sperm (Shepherd, bodies, withsome cytoplasm, are segregated from Clark & Kempton,1973), and the fingerprint-like the nuclear region of the spermatid into the residual patterns often noted in tangential sections of Globo- body(rb) (Fig. Id). Acleavage furrow develops dera sperm were not seen in M. incognifa incognita between thetwo portions so thatthey are joined sperm. The only indicationof the parallel arrangement only by an abscission neck (abn) (Fig. Id). This has wasseen insmall areas such as that depictedin similar characteristics to the telophase neck between Fig. 3e. daughter cellsfollowing ce11 division (as seenin Very soon after maturation, al1 mitochondria and M. hapla (tn in Fig. 4d)), with a contractile ring of fibrillarbodies become concentrated in one region fibrils (Cr) seen as a darlrzone justinside the ce11 surrounding the nucleus, and most of the cytoplasm membrane and many microtubules passing through forms a largepseudopodium (ps) (Figs 2a ; 3a). theneck (mt in Fig. 5c, d). Theseoften become The fine structure of thesetwo parts isshown in highlycompressed (mtc) at the cent.re(see Dyson, Figs2b and 3b respectively. Scattered throughout 1978). Theresidual body is eventually discarded the cytoplasm of bothportions are numerous and engulfed by the testisWall as previously described microtubule-likestructures (cyt) similar to those for Globodera (Shepherd, Clark & Kempton, 1973). In in Aphelenchoides sperm(Shepherd & Clark,1976). the spermatid the fibrillar bodies (fb) remain in the In M. incognitaincognita spermthis irregularly- nuclearportion of the ,where theyoccupy a organisedsystem of tubulesappears continuous

20 Reuue Némaiol. 6 (1) : 17-32 (1983) Fig. 2. M. incognita incognita adult male. a : T.S. seminal vesicle showing spermatozoon with nuclear region with dense nucleus (nu), nucleolar bodies (nlb), fibrillar bodies (fb), mitochondria (m) and filopodia (fp), and pseudo- podialregion (ps). x 16 O00 ; b : Nuclearregion of testicularspermatozoon, showing nucleus (nu), nucleolar bodies (nlb),and other organelles, also corticalmicrotubules (cmt) liningperipheral membrane, and tubular elements (cyt) throughout cytoplasm. x 26 000. Abbreviations : see under Fig. 1.

Revue Nématol. 6 (1) :17-32 (1983) 21 Fig. 3. M. ineognita incognita adult male. a : L.S. showing seminal vesicle filled with mature spermatozoa (sp) with large pseudopodia (ps). x 4 400 ; b : Pseudopodial region (ps) of spermatozoon extending from main sperm body (spb), showing cortical microtubules (cmt) and cytoplasmic tubular elements (cyt). x 30 O00 ; c : Branched filopodium. x 109 O00 ; d : Long portions of filopodia. x 12 400 ; e : Slightly tangential section through sperm coat, showing parallel cortical microtubules. x 76 O00 ; f : Section through periphery of spermatozoon showing plasma membrane (pm), triple-layered outer membrane (osm) with externalfuzzy layer, cortical microtubules(cmt) and cytoplasmic tubular elements (cyt). x 55 000. Abbreviations : see under Fig. 1. Spermatogenesis and sperm structure in Meloidogyne

with the regularly arranged array lining the plasma MEIOTIC (FACULTATIVE) PARTHENOGENS membrane.The two distinct types of (presumed) nuclearmaterial in the spermatozoon are seen in M. hapla (race A). Fig. 2a and b. Most of thevery electron-dense chromatin (nu) is in the centre of the cell. The less As with M. incognitaincoqnita, al1 stages of dense, spherical ‘nucleolar’ bodies (nlb), which look sperm development were present in the adult males granular,are dispersed throughout the cell, often of M. hapla (race A). The spermatogonia (Fig. 4a), aroundthe periphery, and are rarely attached to which at 16 pm x 8-10 pm were larger than those of the chromatin. They look somewhat similar in texture M. incoqnita incoqnita, contained a large, amoeboid, and electron density to the spherical bodies seen in membrane-boundnucleus (nu) ; moderatelydense, thespermatogonial cytoplasm, although perhaps cytoplasmicspherical bodies and smooth ER were slightly more coarse-grained. again present. Some phases of the meiotic sequence The fibrillar bodies. are present in even the most are shown in Fig. 4b-e, progressing to the spernlatid advanced sperm in the seminal vesicle. Their struc- with a highly condensed nucleus lacking an envelope ture issomewhat less compact inspermatozoa (Fig.5a, c). There was no reconstitution of the (Figs. 2b ; 3b) than in the earlier stages and eventu- nuclearmembrane during spermatid development allybecomes rather wispy ‘but they disappear in the adult male specimens we examined. Fibrillar almost completely only in defunct sperm. bodies (fb) were present throughout al1 stages after Thevery numerous filopodia (fpin Fig. 3a) are first appearing in the primary spermatocyte. Fig. 4e mostly confined to the main body of the sperm. The shows late telophase and cytokinesis in the secondary severallayers of theirboundary membrane are spermatocyte.In the telophase neck (tn) there are clearly seen in Fig. 3b and they are often very long many compressedmicrotubules (mtc) of thespindle . (Fig.3d) (portions up to 4 Fm longhave been (sf) within the contractile ring(Cr) cleaving the divid- measured), and sometimes branched (Fig. 3c). ing cell. As mentioned for M. incoqnita irzcogrzita, it The mature sperm are about 12 pm long including appears that acontractile ring also contributesto thepseudopodium, the main body being about the severing of the residual body from the spermatid 6-7 pm in diameter. They are stored, packed quite (Fig. 5c, d). tightlytogether (Fig. 3a), in the seminal vesicle The mature sperm were almost indistinguishable before being passed outat via the glandu- fromthose of M. incogrzita incoqnita, having a larvas deferens, the lumen of whichis normally pseudopodium(ps), although a somewhat smaller occluded. one,distinct from the main body of thesperm (Fig.5e). The overall length of themature sperm M. incoqnita wartellei was about 9 pm, with the main body about 6 pm While spermatogenesis was the sameas in M. inco- indiameter. The only other difference we could gnitaincoqnita, thespermatozoa of M. incognita detectbetween the structure in the two species wartellei (Fig.Sa) were different. With the light was a tendency for M. hapla to develop considerably ,sperm in fixed specimensappear morefilopodia, particularly in the earlierstages, spherical, about 7-8 pm in diameter, andless granular as seen in the spermatocytes in Fig. 4b, compared than“. incognita incoqnifa sperm. In sections they with M. incoqnita irzcognita at the same stage (Fig. lc). appeared slightly elongated, about 8-9 pm x 4-5 Pm. They showedno clear polarisation, the organelles M. graminicola being distributed throughout most of the cell, whose Like M. hapla, M. graminicola has very abundant whole outline was amoeboid. Some fibrillar material filopodia in both early and late stages(fp in Fig. 6a-c). persisted into maturity, although ina fairly dispersed The spermatozoa, which were about 9 Fm X 5 pm, lorm,but it sometimesdisappeared entirely. The showedno clear polarityinto ‘body’ and ‘pseudo- microtubular elements, both cortical and cytoplasmic, podial’ regions. In this they were like M. incoqnita werewell-defined inthis material. The character ruartellei. As with Globodera sperm,they also lost , of the nuclear material was lilre that of M. incoqnita theirfibrillar material immediately on becoming incoqnita. spermatozoa. In this species the chromatin appeared as smaller, sometimes rather diffuse clumps (chn in M. arenaria Fig.6c). The less dense‘nucleolar’ (nlb) material Earlystages in spermatogenesis of M. arenaria wasmostly gathered into a smooth-contoured had fewer incipient filopodia than those of the other central mass, but smaller spherical bodies were also speciesexamined. The spermatozoa (Fig. 7c) were scattered throughout the cytoplasm. The testis was about 10 pm x 8 Fm, and were like those of M. inco- proportionally longer, often occupying three quarters grzita incoqnita. of the body length.

Revue Nématol. 6 (1) :17-32 (1983) 23 Fig. 4. M. hapla adult male. a : T.S. testis showing very early primary spermatocyte (esc) with large amoeboid nucIeus (nu), nuclear membrane (nm), spherical nucleoIus (nI), mitochondria (m), incipient fibrillar bodies (fb), Golgi bodies (gb), cytoplasmic sphericalbodies (csb), smoothendoplasmic reticulum (ser) and incipient filopodia (fp). x 14 500 ; b : Testis showing primary spermatocytes (psc) at various phases of . x 4 300 ; c : Early prophase 1 nucleus (nu) of primary spermatocyte, with nuclear membrane (nm) still intact and homologous pairs of chromosomes (chr) condensing around periphery of nucleus. x 13 O00 ; d : Chromosomes (chr) at metaphase 1 in nucleus of primaryspermatoc,yte ; homologous pairslined up dong met,aphaseplate. Showing also centriole (ce)and microtubules of spindle (sf). x 17 600 ; e : The end of telophase II in secondary spermatocyte, with chromatin (chn) recondensing. Telophase neck (tn) contains compressed spindle fibres (sf). x 9 500. Abbreviations : see under Fig. 1.

24 Reuue Ndmatol. 6 (1) : 17-32 (1983) Fig. 5. M. hapla adult male.a : T.S. testis showing spermatids (st) with large residual bodies (rb). Upper spermatid shows abscission neck (abn) with contractile ring of fibrils (Cr). x 9 800 ; b : Enlar- gement of residual body showing smooth ER (ser) and cytoplasmic spherical bodies (csb). x 18 300 ; c : Spermatid (st) showingabscission neck (abn) betweenmain ceIl body andresidual body (rb), similar to telophaseneck in fig. 4d, withcontractile ring (Cr) and compressedmicrotubules (mtc). x 14 800 ; d : Enlargement of abscission neck. x 27 O00 ; e : Spermatozoon, showing the two regions, one containing organelles (spb), the other a pseudopodium (ps) with cortical microtubules (cmt) and cytoplasmic tubular elements (cyt). x 14 000. Abbreviations : see under Fig. 1.

Revue Ndmatol. 6 (1) : 17-32 (1983) 25 Fig. 6. M. graminicola adult male. a : Primary spermatocyte (psc), with nucleus in metaphase 1, also showing many incipient filopodia (fp). x 9 O00 ; b : Spermatid (st) with large residual body (rb) showing abscission neck (abn). Residualbody contains many ribosomes, large Golgi complexes (gb)and electron-translucent vesi- cles (tv). x 8 GO0 ; c : Spermatozoa, one showing largeand small nucleolar bodies (nlb) andratherdiffuse chromatin material (chn). (Slight stain precipitation in nucleolar material.) x 16 100. Abbreviations : see under Fig. 1.

26 Reuue Nérnutol. 6 (1) : 17-32 (1983) Spermatogenesis and sperm structure in Meloidogyne

REPRODUCTIVESTATUS UNDETERMINED In Meloidogyne the occupies in general the same proportion of the body length as in the cyst M. oryzae nematodes, averaging 400-500 pm from tip to , M. oryzae is considered by Maas, Sandersand about half to twothirds the nematode’s length. Dede (1978) to be closely related to M. graminicola, Thereare several thousand fully developed sperm butits reproductive status is notyet determined. in the gonoduct of ayoung adult Globodera male. Itssperm (Fig. 7a) somewhat resembled those of Most, of the relatively long acts also as M. graminicola but werelarger (about 15-20 pm a seminalvesicle, housing thefull, complement of x 8-12 Pm). As with M. graminicola, the chromatin spermthat results from most of thegerm cells was less compacted thanin the other species ; in maturing before occurs.Only the posterior M. oryzae it wasmostly dispersed as fine strands c. 50 pm of the duct acts as a valve. In Meloidogyne or granules, or assmall clumps around a central, the whole of the much shorter vas deferens is closed large, smooth-contoured ‘nucleolar’ body (Fig. 7a, b). except to allow the passage of sperm (Shepherd & Smaller ‘nucleolar’ bodies throughout the cyto- & Clark, 1982). The space occupied by the immature plasm were also associated with clumps of chromatin stages leaves room for relatively few fully developed (Fig. 7b). The cytoplasm was frequently vacuolated. sperm (only several hundred) in the gonoduct. Althoughthere was no clear polarity, the nuclear Although a rachis has been clearly demonstrated material and mitochondria were often concentrated in the ovary of Meloidogyne (McClure & Bird, 1976 ; towards the centre, leaving larg:. areas of cytoplasm Goldstein & Triantaphyllou,1978a), and one is free of organelles. seen in the testis of Globodera pre-adult males, we could not recogniseone inthe testis . of the Meloidogyne males. AMPHIMICTICSPECIES (seeIntroduction) The structure of the early stages of the germ cells does not differ greatly between the two families. In M. hcrorzea. most Meloidogyne species the nucleus of the sperma- togonium is very amoeboid inshape (M. acronea Thespermatogonia were smaller (4.5-5.5 pm) in is an exception) whereas in the cyst nematodes it is this species andmore numerous in a cross section oval.Golgi bodies were eitherrare or else poorly of thetestis. Their nuclei were less amoeboid, and preservedin the cyst-nematodematerial but were morelike the ovoidnuclei of thecyst-nematode abundantin the spermatogonia and spermatocytes spermatogonia. of Meloidogyne. In Meloidogyne the incipient fibrillar Latespermatids were sometimes seen joined material appeared first in the primary spermatocytes together (Fig. 8b). Filopodia were abundant in this andwas welldeveloped in al1 subsequentstages species and somefibrillar material was retainedin withthe exception of spermatozoa of three of the the sperrn which, at about 4.5 pm x 3.5 pm, were species. In Globodera it first appeared in the spermatid smallestby far of al1 the species examined.Late and had disappeared in the testicular spermatozoa, sperm (Fig. 8d) tended to be moregenerally amoeboid whereasin Heterodera someremained briefly after ratherthan obviously polarised, although early loss of the residual body. Spermatids were essentially sperm showed some larger pseudopodia (Fig.8c). similar in the three genera. There are greater differences between the sperma- tozoa in the two families, although some similarities Discussion exist.From early maturity the sperm of some Meloidogyne species (M. arenaria, M. hapla and M. A characteristicA of cyst-nematodespermato- incoqnitaincoqnita) aredistinctly polarised into a genesis is the termination ofce11 division at the last nuclearregion and a large pseudopodium ; oLhers moult of the male, so that shortly after the moult (M. graminicola, M. incoqnitauartellei and M. the testis of the adult‘contains only spermatids and oryzae), like the cyst-nematodesperm, are not. spermatozoa.Shepherd, Clark andKempton (1973) M. acrorzea is intermediate, with some indication of suggested thatthis might beassociated with the polarity in early sperm but the mature sperm are apparent inability of the males to feed, a condition moregenerally amoeboid. In unpolarised sperm, in shared,as far as isImown, by Meloidogyne males. both families,fibrillar material either becomes However, al1 stages of spermdevelopment were rather diffuse or dispersesentirely as thesperm found in the testes of adult males of al1 the Meloi- matures. Polarisation occurs only after dogyne species examined, so conservation of energy of the in some nematode species,e.g. Aphelen- resources may not be the major factor we previously choides blastophthorus (Shepherd & Clark, 1976) and thought. Caenorhabditiselegans (Ward & Carrel,1979).

Revue Nimatol. 6 (1) : 17-32 (1983) .27 Fig. 7. a-b M. oryzae adult male. a : L.S. through seminal vesicle showing very large spermatozoa (sp) lacking fibrillar material ; also large spherical nucleolar bodies (nlb) and dispersed chromatin (chn). x 4 400 ; b : Sperma- tozoon, showing large and small nucleolar bodies (nlb), and diffuse clumps and small granules of chromatin ma- terial (chn) ; also remnants of fibrillar material (fb). x 15 400 ; c : M. arenaria adult male. Spermatozoa, showing fibrillar bodies (fb) and pseudopodia (ps). x 8 600. Abbreviations : see under Fig. 1.

28 Reoue Nématol. 6 (1) : 1'7-32 (1983) Fig. 8. a : M. incognitawartellei adultmale. L.S. seminalvesicle withnon-polarised spermatozoa. x 10 400. b-d M. acronea adult male ; b : L.S. testis showing two spermatids (st) with a cornmon residual body (rb). x 8 600 ; c : Early spermatozoa, showing pseudopodia (ps) and some fibrillar material (fb). x 13 O00 ; d : Late spermatozoa near vas deferens. x 9 000. Abbreviations : see under Fig. 1.

Revue Nimafol. 6 (2) :27-32 (1983) 29 A.M. Shepkerd & S.A. Clark

Nelson and Ward (1980) were able to induce polar- state of the nuclear material ; the size of the mature isation,in C. elegans testicularsperm using the sperm. The smallest sperm (M. acronea) wereonly monensin. about one twentieth the volume of the largest (M. The state of condensation of the chromatin in the oryzae). Some of these characteristics are summarised testicular sperm is similar in n/Jeloidogyne and Hefe- inTable 2. The differencebetween thesperm in rodera, where, in mosLof the species we examined, the two subspeciesof M. incognita is noteworthy. hlso it is a solid, homagaeous rnass ; in M. oryzae, there was a clear distinction between the condition however, it is partially dispersed in granular form. of thenuclear material in M. graminicola and M. In Globodera it passes through a distinctive, beaded- oryzae, where the less electron-dense‘nucleolar’ filamentous phase before consolidating after passage materialwas compacted into the larger mass, of the sperm through the vas deferens. compared with the other specieswhere the denser The spherical bodies, considered by Goldstein and chromatin predominated. Triantaphyllou(1980) to benucleolar bodies, are With Our material of M. hapla, we foundno foundin Meloidogyne species but not in testicular evidence in the adult male testis of a brief reconsti- sperm of thecyst nematodes. In Globodera they tution of the nuclear membrane in the early sper- appearfirst in sperm in the spermatheca of the matid,which Goldstein and Triantaphyllou (1980) female ; in that instance they are attached to the described from the pre-adnlt testis, nor of the han- coarse threads of chromatin. Goldstein and Trianta- sitory nature of the fibrillar material during sperma- phyllou considered them to be RNA on the grounds teleosis. Development proceededas in other nematode of the response of the material to Bernhard’s (1969) spermand culminated in polarised spermatozoa, procedure.This seenls plausible a assumption and not in the unpolarised sperm reported by these althoughthe method used does not differentiate authors. unequivocallybetween ribonucleoprotein and pure Although most of the Meloidogyne species studied (Franke & Falk, 1970). When the material wereparthenogenetic, we foundno morphological beingcharacterised is dispersedin thecytoplasm, abnormalitiesin any of thestages during sperm asin these nematode sperm, rather than within a development, nor any strictly morphological differ- nuclear membrane, there is less certainty of its being ences distinguishingmitotic from meiotictypes. RNA when shown not to be DNA. Our designation This is in agreement with t.he findings of Goldstein of structures as cchromatin’ or ‘nucleolar material’ andTriantaphyllou (1980) for thetwo races of is based only on electron-density following staining M. hapla. Eventhough, unlike in cyst-nematode with leadcitrate and uranyl acetate, unconfirmed males, the facility for continued sperm development by cytochemical tests. existsin Meloidogyne males,individuals in which The ‘cytoplasmic spherical bodies’ seen in sperma- thespermatozoa were degenerating in the seminal tocytesand spermatids of &Ieloidogyne species vesicle were often encountered, and so evidently no (cg. DI. hapla, csb in Figs 4a ; 5a, b) resemble those greatadvantage accrues interms of theeventual described by Kessel(1981) in sperma- number of available sperm. In fact, it seems possible tocytes, where they were associated with smoothER thatthe system evolvedin thecyst nematodes is and annulate lamellae, and were thought to contain the moreefficient, since the malesare at least bothRNA and protein. Kesselsuggested that, guaranteed a relatively large numberof fully develop- Iike nuclear membrane, the system might assemble edsperm, whereas in Meloidogyne males sperm polyribosomes.Annulate lamellae, resembling production mightbecome impaired if energy resources portions of nuclearmembrane, were present in G. were depleted by activity. rostochiensis spermatids (Fig. 2c of Shepherd, Clark It is clear that although there are some differences and Kempton (1973) but not named) but were not in sperm structure and development between these seen in Meloidogyne. two families inthe Heteroderoidea, their basic Sperm of al1 three genera had cortical microtubules, spermstructure is fundamentally similar. Subject although they seemed to differ slightly in the two to confirmation, it seems the Aphelenchida may be families. Intracytoplasmic tubules were not seen in the only group among the plant-parasitic nematodes thecyst-nematode sperm, apart from the more in which the sperm areof the type with membranous typicalmicrotubules closely associatedwith the organelles, as inmost -parasitic orders and nuclear material. inthe rhabditids and enoplids (see Bird,1971 ; Within the genusMeloidogyne, then, thedifferences Shepherd Clark & Kempton,1973 ; Shepherd & between species concerned mainly : the polarisation Clark,1976 ; Shepherd,1981). These ,organelles do of the spermatozoain some ; the persistence or notoccur in any of theTylenchida sperm so far otherwise of the fibrillar material ; the abundance of studied,including Ditylenchusdipsaci and Praty- filopodia,particularly atthe earlierstages ; the lenchuspenetrans, which were cursorilyexamined

30 Revue Nérnalol. 6 (1) : 17-32 (1983) Spermatogenesis and sperm structure in Meloidogyne

Table2 Some characteristics of testicular spermatozoa in the genera Globodera, Heterodera and Meloidogyne

Species and sperm Pbrillarsperm and Species corticalno.nucleolar filopodia cytoplasmic polarised material bodies microtubules microtubulesreproductioemicrotubules statusbodies material polarised

Globodera spp. (am) - - ** - + - Heterodera spp. (am) - ** - + - M acronea (am) ++- *** + + + M. arenaria (mi) + ** - + + M. graminicola (me) - - +** -F + + 11.1. hapla [me) + ** t** L + + incognita incognitaM. incognita (mi) + ** t* + + + incognita amtelleiM. incognita (mi) - ** + + + M. oryzae (?) - - *** + + +

- absent ; + present ; 5, 55, 555 few-many ; am, amphimixis ; mi, mitotic (obligatory) ; me: meiotic (facultative) parthenogenesis

by Shepherd and Clark(1975), nor in the spermof the electronmicroscopy of serialsections. Chrorno- dorylaims Longidorusmacrosolna and Xiplzinema soma, 70 : 131-139. index (Shepherd & Clark, 1980). GOLDSTEIN,P. 62 TRIANTAPHYLLOU,A.C. (1980). We thank Dr. S.B. Jepson for helpfuldiscussion Theultrastructure of spermdevelopment in the and for, with Miss A. Hoole, supplying the Meloido- plant-parasiticnematode fileloidogyne hapla. J. gytze males. We are also grateful Lo Dr. A.G. Trianta- Ultrastruct. Res., 71 : 143-153. phyllou for his comments. KESSEL,R.G. (1981). Origin, distribution and possible functional role of annulate lamellae during sperma- togenesis in . J. Ullrastruct. Res., 75 : 72-96. REFERENCES MAAS, P.W.T.,SANDERS, H. & DEDE, J. (1978). Meloidogyneoryzae n. sp. (Nematoda, Meloidogy- BERNHARD, W. (1969). Anew staining procedure for nidae)infesting irrigated rice in Surinam (South electron microscopical cytology. J. Ultrastruct. Res., America). Nematologica, 24 : 305-311. 27 : 250-265. MCCLURE, M.A. & BIRD,A.F. (1976). The tylenchid BIRD,A.F. (1971). Thestructure of nematodes. New (Nematoda) egg shell : formation of the egg shell York, Academic Press, 318 p. in Meloidogynejavanica. Parasitology, 72 : 29-39. DYSON,R.D. (1978). Ce11 : A molecular NELSON,G.A. & WARD, S. (1980). Vesicle fusion, approaclz. Boston, Allyn & Bacon, 616 p. pseudopodextension and amoeboid motilityare FRANKE,W.W. & FALK, H. (1970).Appearance of induced in nematode spermatids by the ionophore nuclearpore complexes after Bernhard’sstaining monensin. Cell, 19 : 457-464. procedure. Histochemie, 24 : 266-278. SI-IEPHERD,A.M. (1981).Interpretation of sperm GOLDSTEIN,P. & TRIANTAPHYLLOU,A.C. (1978~). developmentinnematodes. Nemalologica, 27 : Occurrence of synaptonemal complexes and recom- 122-125. bination nodules in a meiotic race of lieloidogyne SHEPI-IERD,A.M. & CLARK, S.A. (1975). In : Jones hapla and their absence in a mitotic race. Chromo- F.G.W., Nematology Department. Rep. Rothamsted soma, 68 : 91-100. eep. Stn for 1974 : 173-190. GOLDSTEIN,P. & TRIANTAPHYLLOU,A.C. (1978b). SHEPIIERD,A.M. & CLARK, S.A. (1976).Spermato- Karyotypeanalysis of Meloidogyne hapla by 3-D genesis and the ultrastructure of sperm and of the reconstruction of synaptonemal complexesfrom male reproductivetract of Aphelenchoides blas-

Revue Nématol. 6 (1) : 27-32 (1983) 31 A.M. Shepherd & S.A. Clark

tophthorus (Nematoda : Tylenchida, Aphelenchina). cyst nematodes, Heterodera spp. Nemaiologica, Nematologica, 22 : 1-9. 19 (1973) : 551-560. SIIEPIIERD,A.M. & CLARK, S.A. (1979). In : Jones, SIDDIQI,M.R. (1980). Theorigin and phylogeny of F.G.W., Nematology Department. Rep. Rothamsted thenematode orders Tylenchida Thorne, 1049 exp. Stn for 1978 : 167-189. and Aphelenchida n.ord. Helminth.Abstr. Ser. B. SI-IEPImRn,A.M. & CLARK, S.A. (1980). In : St.one, 49 : 143-170. A.R., NematoIogy Department. Rep.Rothamsted SOLOFP, B.L. (1973).Buffered potassium perman- ezp. Stn for 1979 : 139-151. ganate - uranylacetate - leadcitrate staining SHEPIIERD,A.M. & CLARK, S.A. (1982). In : Stone, sequence for ultrathinsections. StainTechnol., A.R., NematologyDepartment. Rep.Rothamsted 48 : 150-165. exp. Sfnfor 1981, in press. WARD, S. & CARREL, J.S. (1979).Fertilization and SHEPI-IERD,A.M., CLARK,S.A. & KEMPTON,A. (1974). in the nematodc Caenorhabditis Spermatogenesis and sperm ultrastructure in some elegans. Deu. Biol., 73 : 304-321.

AcceptS pour publication le 18 mars 1982.

32 Revue Nèmatol. 6 (1) : 17-32 (1983)