Nematology 2004 Vol 6(5) 623-632 Forum article

Revised hypotheses for phylogenetic homology of thestomatostylet in tylenchid nematodes

James G BALDWIN lowast Erik J RAGSDALE and Daniel BUMBARGER

Department of Nematology University of California Riverside CA 92521 USA

Received 26 July 2004 revised 6 September 2004Accepted for publication 6 September 2004

Summary ndash Molecular phylogenetic systematics suggest that Tylenchida share an immediate ancestor with Cephalobina Thisrelationship has implications contradicting classical views for evolution of the stomatostylet and homology with the cephalobid stomaEmerging evidence from comparative TEM and 3D modelling is the basis for hypothesising that the tylenchid stylet specifically thecone and shaft is homologous with cuticle associated with arcade syncytia of the cephalobid gymnostom furthermore the stylet knobsand associated m1 protractors are prostegostom The guiding apparatus through which the stylet moves is lined by epidermal syncytiaand is homologous with the cephalobid cheilostom Junctional complexes associated with the epidermal syncytia of the cheilostom andadjacent gymnostom arcade cells occur in both cephalobids and tylenchids but in the latter the membrane complexes are folded andmodified as the guide ring Testing of the hypothesis requires clearer phylogenetic resolution as well as additional ultrastructural anddevelopmental observations of representatives of tylenchids and outgroups

Keywords ndash Cephalobina cheilostom development evolution fine structure gymnostom stegostom stoma Tylenchida

Tylenchida Thorne 1949 including most species ofplant-parasitic nematodes are characterised by adaptationof the stoma as a protrusible stomatostylet of extracellu-lar collagenous cuticle enclosing a narrow lumen Typi-cally the stylet consists anteriorly of a tapering cone fol-lowed by a cylindrical shaft and one dorsal and two sub-ventral stylet knobs from which protractor muscles extendanteriad The stylet moves through a cuticle-lined chan-nel (guiding apparatus) consisting anteriorly of a vestibule(the hub of a hexaradiate framework) and posteriorly of avestibule extension the stylet is attached to the channelnear the base of the vestibule extension by a junctionalcomplex specialisation of folded cell membranes (guidering) The stylet is structurally and functionally linked tothe pharynx which is a source for digestive enzymes andmuscular pumping during feeding the stylet lumen is theconduit through which enzymes can be injected into thehost and host cytoplasm ingested (Dropkin 1969)

Evolution of the highly specialised tylenchid feedingsystem is pertinent to broader questions of the evolu-tion of plant parasitism (Baldwin et al 2004) The stylethas been previously proposed to have evolved through amorphological transformation series including the openstomas of Cephalobina Rhabditina and Diplogastrina

lowast Corresponding author e-mail jamesbaldwinucredu

(Thorne 1961 Andraacutessy 1962 1976 Goodey 1963 DeGrisse 1972) These classical proposals of morphologicalevolution have included attempts to suggest homologiesof specific rhabdia (thickenings of stoma linings) amongthese stomas to components of the stomatostylet andguiding apparatus Stomatostylet components ascribed torhabdia include the framework vestibule vestibule exten-sion cone shaft and knobs (for terminology see Fig 1 inBaldwin and Hirschmann 1976) Thorne (1961) for ex-ample suggested that among the cephalobid rhabdia thecheilorhabdion is homologous to the tylenchid frameworkwhereas the prorhabdion corresponds to the stylet conethe meso- and metarhabdia to the shaft and the telorhab-dion to the stylet knobs (Fig 1)

A broadly held classical view of phylogenetic rela-tionships consistent with the morphological transforma-tion from the open stoma to the tylenchid stomatostyletwas that of a unique shared ancestry of Diplogastrinaand Tylenchida (Steiner 1933 Thorne 1961 Goodey1963 Andraacutessy 1976 Maggenti 1981) but this viewis inconsistent with SSU rRNA phylogenies which sup-port a Diplogastrina-Rhabditina clade that excludes Ty-lenchida Instead Tylenchida share a unique clade withCephalobina Andraacutessy 1974 (Blaxter et al 1998 Dolin-

copy Koninklijke Brill NV Leiden 2004 623Also available online - wwwbrillnl

JG Baldwin et al

Fig 1 Diagrammatic representation of hypotheses of homology of components of the stoma of Cephalobina to the stomatostylet ofTylenchida A lsquoCephalobus-Typersquo of Andraacutessy (1962) with regions designated identical to those of Thorne (1961) for lsquoEucephalobusrsquoB lsquoTylenchus-Typersquo of Andraacutessy represented by left column labels and lsquoRotylenchusrsquo of Thorne (1961) represented by right columnlabels C Cephalobid with terminology designations of De Ley et al (1995) represented by left column labels and those of Goodey(1963) represented by right column labels D Tylenchid stomatostylet with terminology designations of Goodey (1963) representedby left column Terminology represented by right column labels show homologies proposed herein (Abbreviation dgo = dorsal glandorifice)

ski et al 2001) partly consistent with the earlier hypoth-esis of Siddiqi (1980) Subsequent phylogenies includingmany additional taxa and other genes further support thatthe Tylenchida and Cephalobina are sister taxa (SubbotinThomas and Nadler pers comm) and thus suggest thatthe stomatostylet arose from a narrow open stoma likethat which characterises extant Cephalobina thereby ef-fectively rejecting previous hypotheses of homology

It is generally accepted that there are three criteria bywhich to propose and evaluate hypotheses of homologyconjunction similarity and congruence (Patterson 1988)Conjunction and similarity are primarily used to proposeinitial hypotheses of homology while congruence withother hypotheses such as through a well-supported phylo-genetic tree is a means by which to test homologies Sim-ilarity as a criterion for proposing hypotheses of homol-ogy can be further categorised as compositional topo-

logical developmental functional or genetic (Panchen1994) Classical views on stoma morphology are basedupon relatively unsophisticated observations of compo-sitional and topological similarity of secreted structures(the rhabdia and stylet components) Previously limitedresolution of light microscopy and lack of developmen-tal information prevented more detailed and meaning-ful interpretations of homology Furthermore the classi-cal hypotheses of stylet homology fail a test of congru-ence with well-supported phylogenetic trees making analternative hypothesis necessary Here we collate and re-evaluate previous TEM together with new informationinto a more viable and testable hypothesis of homology asit pertains to the evolution of the stomatostylet

The proposal to extend interpretations of stoma ho-mologies in Cephalobina to Tylenchida is a crucial step inunderstanding evolution of the stomatostylet Fine struc-

624 Nematology

Homology of the tylenchid stomatostylet

ture and developmental observations are providing newinsight for interpreting cellular organisation of the stomaof Cephalobina in relation to additional Secernentea (DeLey et al 1993 1995 Baldwin amp Eddleman 1995Baldwin et al 1997 Dolinski et al 1998 Dolinski ampBaldwin 2003) These are more recently being extendedthrough 3D modelling of electron micrographs electrontomography 4D developmental observations and confo-cal microscopy (Bumbarger amp Baldwin unpubl Burramp Baldwin unpubl) Key to understanding homologieswith the stomatostylet is that the stoma of Cephalobinaincludes posterior to the cheilostom a cuticle-lined re-gion nearly invisible by light microscopy that is not sur-rounded by pharynx (stegostom) but instead by a thinstack of two arcade syncytia (gymnostom) (Baldwin ampEddleman 1995 De Ley et al 1995) homologues ofthese arcade syncytia and thus the gymnostom are wellsupported in Rhabditina and Diplogastrina (Albertson ampThomson 1975 Wright amp Thomson 1981 Baldwin etal 1997 De Ley amp Blaxter 2002)

Topological and developmental information on styletcomponents (framework vestibule vestibule extensioncone shaft and knobs) that could be informative for es-tablishing homologies has not yet been adequately re-searched There is a body of classical literature on finestructure of several tylenchid species that predates andthus excludes interpretation that considers fine structuralunderstanding of stoma characters and their homolo-gies in other Secernentea (De Grisse 1972 Baldwin ampHirschmann 1976 Shepherd amp Clark 1976 Chen ampWen 1980 Shepherd et al 1980 Endo 1985 Endoamp Nickle 1993 Endo et al 1997) These studies pro-vide a starting point for proposing new hypotheses ofhomologies of the stomatostylet components with partsof the stoma as also understood from fine structure inCephalobina

Comparative observations homologies

We hypothesise that the tylenchid stylet specificallythe cone and shaft is homologous with the cuticlelining of the gymnostom of Cephalobina Unlike extanthypotheses that compare the stylet to the entire stoma

In this paper we use Secernentea for continuity with previousliterature We acknowledge however a strong justification fora clade Chromadorea within which the clade Secernentea (synOrder Rhabditida sensu De Ley amp Blaxter 2002) is embedded(De Ley amp Blaxter 2002)

including all of the stegostom herein only the styletknobs are proposed to be homologous with the liningof the anterior end of the cephalobid stegostom Thisanterior region was designated by De Ley et al (1995)as the prostegostom and equivalent to the prostom ofThorne (1961) and Andraacutessy (1962) and the prostom+ mesostom of Goodey (1963) (Fig 2) The tylenchidvestibule associated cephalic framework and vestibuleextension are surrounded by epidermal syncytia as isthe cheilostom of Cephalobina A guide ring surroundingthe stylet shaft consists of junctional complexes betweensyncytia These complexes are reminiscent of and maybe homologous with the ring of junctional complexesbetween the epidermal syncytium of the cheilostom andthe arcade syncytium of the gymnostom of Cephalobina(Van de Velde et al 1994 Baldwin amp Eddleman 1995De Ley et al 1995)

STYLET KNOBS STEGOSTOM

In Cephalobina the stegostom is characterised by atriradiate lumen lined by thickened cuticle (Fig 3A) Eachapex of the lumen is associated with a marginal cellelongate in the anterior-posterior axis and between theapices are three sectors occupied by a stack of radialmuscle cells in the dorsal and two subventral positions(Figs 2A 3A) The stack of muscle cells defines fromanterior to posterior the pro- meso- meta- and telo-stegostom (De Ley et al 1995) (Figs 1 2A) In theprostegostom each sector includes a single interradial cellm1 posteriorly in the mesostegostom similar interradialcells are designated m2 In contrast the sectors posteriorto these each include a pair of adradial muscle cells in apattern that apparently extends into the procorpus (De Leyet al 1995 Dolinski et al 1998)

As in Cephalobina the anteriormost cells of the stegos-tom in Tylenchida include three elongate marginal cellsThese are separated by three large interradial cells thestylet protractor muscles (Fig 3B C) The protractormuscles designated m1 (defining the prostegostom) at-tach to the thickened cuticle lining of the stoma vizthe three stylet knobs (Figs 2B 3B C) The protractormuscles of Tylenchida differ from the radial muscles ofCephalobina in that they show a complex pattern of ante-rior branching and attach near the junction of the bodywall and cephalic framework (Baldwin amp Hirschmann1976) (Figs 2B 4D) They also differ by possessing a

We use epidermal as a synonym of hypodermal For a reviewof terminology see Bird and Bird (1991 p 4)

Vol 6(5) 2004 625

JG Baldwin et al

Fig 2 Diagrammatic representation of the components of the cephalobid stoma (A) in relation to the tylenchid stomatostylet (B)Colours corresponding between A and B represent a hypothesis of homology Broken red lines indicate the presence of junctionalcomplexes broken brown lines represent tonofilaments attaching arcade syncytia to stomastomatostylet cuticle lsquoArsquo is adapted fromBaldwin and Eddleman (1995) and lsquoBrsquo is modified and partially redrawn from Baldwin and Hirschmann (1976)

broad noncontractile region that includes the nucleusand which extends posteriorly into the procorpus (Bald-win amp Hirschmann 1976) vs a much narrower poste-rior process as in those of Cephalobina (Dolinski et al1998) Posterior to the tylenchid stylet knobs a second set

of radial muscles m2 (defining the mesostegostom) at-taches to the narrow pharyngeal lumen but extends an-teriad as narrow muscular processes closely associatedwith protractors Curiously m2 comprises a pair of adra-dial cells on the dorsal side whereas on each subventral

Fig 3 Transmission electron micrographs through the anterior stegostom of representatives of Cephalobina and Tylenchida Sectionsare transverse unless otherwise indicated A Zeldia punctata female with the stomatal lumen (l) surrounded by three interradial muscles(m1) separated by three marginal cells (mc) (t = tonofilaments) Arrows denote junctional complexes dorsal is toward the top of thefigure B Meloidogyne incognita male near junction of stylet shaft (ss) with stylet knobs (sk) the former flanked by arcade syncytia(a) The three protractor muscles (m1) are separated by narrow marginal cells (mc) (Abbreviations l = stylet lumen m2 = anteriorcontractile region of m2 Arrows denote junctional complexes dorsal is toward the top of the figure) C Stylet knobs (sk) of Mincognita male (Abbreviations l = lumen m1 = protractor muscles mc = marginal cells t = tonofilaments Dorsal is toward thetop of the figure) D Stylet shaft (ss) of M incognita male surrounded by junctional complexes (arrows) between syncytia includingarcade (a) and epidermis (es) (Abbreviations m1 = protractor muscles) E Longitudinal section through region corresponding to lsquoDrsquoincluding stylet shaft (ss) surrounded by junctional complexes (arrows) associated with adjacent arcade syncytia (a) Figures D and Eare printed from TEM negatives parts of which are used in Baldwin and Hirschmann (1973) and for B C and E parts of which areused in Baldwin and Hirschmann (1976)

626 Nematology

Homology of the tylenchid stomatostylet

Vol 6(5) 2004 627

JG Baldwin et al

628 Nematology

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

Homology of the tylenchid stomatostylet

ture and developmental observations are providing newinsight for interpreting cellular organisation of the stomaof Cephalobina in relation to additional Secernentea (DeLey et al 1993 1995 Baldwin amp Eddleman 1995Baldwin et al 1997 Dolinski et al 1998 Dolinski ampBaldwin 2003) These are more recently being extendedthrough 3D modelling of electron micrographs electrontomography 4D developmental observations and confo-cal microscopy (Bumbarger amp Baldwin unpubl Burramp Baldwin unpubl) Key to understanding homologieswith the stomatostylet is that the stoma of Cephalobinaincludes posterior to the cheilostom a cuticle-lined re-gion nearly invisible by light microscopy that is not sur-rounded by pharynx (stegostom) but instead by a thinstack of two arcade syncytia (gymnostom) (Baldwin ampEddleman 1995 De Ley et al 1995) homologues ofthese arcade syncytia and thus the gymnostom are wellsupported in Rhabditina and Diplogastrina (Albertson ampThomson 1975 Wright amp Thomson 1981 Baldwin etal 1997 De Ley amp Blaxter 2002)

Topological and developmental information on styletcomponents (framework vestibule vestibule extensioncone shaft and knobs) that could be informative for es-tablishing homologies has not yet been adequately re-searched There is a body of classical literature on finestructure of several tylenchid species that predates andthus excludes interpretation that considers fine structuralunderstanding of stoma characters and their homolo-gies in other Secernentea (De Grisse 1972 Baldwin ampHirschmann 1976 Shepherd amp Clark 1976 Chen ampWen 1980 Shepherd et al 1980 Endo 1985 Endoamp Nickle 1993 Endo et al 1997) These studies pro-vide a starting point for proposing new hypotheses ofhomologies of the stomatostylet components with partsof the stoma as also understood from fine structure inCephalobina

Comparative observations homologies

We hypothesise that the tylenchid stylet specificallythe cone and shaft is homologous with the cuticlelining of the gymnostom of Cephalobina Unlike extanthypotheses that compare the stylet to the entire stoma

In this paper we use Secernentea for continuity with previousliterature We acknowledge however a strong justification fora clade Chromadorea within which the clade Secernentea (synOrder Rhabditida sensu De Ley amp Blaxter 2002) is embedded(De Ley amp Blaxter 2002)

including all of the stegostom herein only the styletknobs are proposed to be homologous with the liningof the anterior end of the cephalobid stegostom Thisanterior region was designated by De Ley et al (1995)as the prostegostom and equivalent to the prostom ofThorne (1961) and Andraacutessy (1962) and the prostom+ mesostom of Goodey (1963) (Fig 2) The tylenchidvestibule associated cephalic framework and vestibuleextension are surrounded by epidermal syncytia as isthe cheilostom of Cephalobina A guide ring surroundingthe stylet shaft consists of junctional complexes betweensyncytia These complexes are reminiscent of and maybe homologous with the ring of junctional complexesbetween the epidermal syncytium of the cheilostom andthe arcade syncytium of the gymnostom of Cephalobina(Van de Velde et al 1994 Baldwin amp Eddleman 1995De Ley et al 1995)

STYLET KNOBS STEGOSTOM

In Cephalobina the stegostom is characterised by atriradiate lumen lined by thickened cuticle (Fig 3A) Eachapex of the lumen is associated with a marginal cellelongate in the anterior-posterior axis and between theapices are three sectors occupied by a stack of radialmuscle cells in the dorsal and two subventral positions(Figs 2A 3A) The stack of muscle cells defines fromanterior to posterior the pro- meso- meta- and telo-stegostom (De Ley et al 1995) (Figs 1 2A) In theprostegostom each sector includes a single interradial cellm1 posteriorly in the mesostegostom similar interradialcells are designated m2 In contrast the sectors posteriorto these each include a pair of adradial muscle cells in apattern that apparently extends into the procorpus (De Leyet al 1995 Dolinski et al 1998)

As in Cephalobina the anteriormost cells of the stegos-tom in Tylenchida include three elongate marginal cellsThese are separated by three large interradial cells thestylet protractor muscles (Fig 3B C) The protractormuscles designated m1 (defining the prostegostom) at-tach to the thickened cuticle lining of the stoma vizthe three stylet knobs (Figs 2B 3B C) The protractormuscles of Tylenchida differ from the radial muscles ofCephalobina in that they show a complex pattern of ante-rior branching and attach near the junction of the bodywall and cephalic framework (Baldwin amp Hirschmann1976) (Figs 2B 4D) They also differ by possessing a

We use epidermal as a synonym of hypodermal For a reviewof terminology see Bird and Bird (1991 p 4)

Vol 6(5) 2004 625

JG Baldwin et al

Fig 2 Diagrammatic representation of the components of the cephalobid stoma (A) in relation to the tylenchid stomatostylet (B)Colours corresponding between A and B represent a hypothesis of homology Broken red lines indicate the presence of junctionalcomplexes broken brown lines represent tonofilaments attaching arcade syncytia to stomastomatostylet cuticle lsquoArsquo is adapted fromBaldwin and Eddleman (1995) and lsquoBrsquo is modified and partially redrawn from Baldwin and Hirschmann (1976)

broad noncontractile region that includes the nucleusand which extends posteriorly into the procorpus (Bald-win amp Hirschmann 1976) vs a much narrower poste-rior process as in those of Cephalobina (Dolinski et al1998) Posterior to the tylenchid stylet knobs a second set

of radial muscles m2 (defining the mesostegostom) at-taches to the narrow pharyngeal lumen but extends an-teriad as narrow muscular processes closely associatedwith protractors Curiously m2 comprises a pair of adra-dial cells on the dorsal side whereas on each subventral

Fig 3 Transmission electron micrographs through the anterior stegostom of representatives of Cephalobina and Tylenchida Sectionsare transverse unless otherwise indicated A Zeldia punctata female with the stomatal lumen (l) surrounded by three interradial muscles(m1) separated by three marginal cells (mc) (t = tonofilaments) Arrows denote junctional complexes dorsal is toward the top of thefigure B Meloidogyne incognita male near junction of stylet shaft (ss) with stylet knobs (sk) the former flanked by arcade syncytia(a) The three protractor muscles (m1) are separated by narrow marginal cells (mc) (Abbreviations l = stylet lumen m2 = anteriorcontractile region of m2 Arrows denote junctional complexes dorsal is toward the top of the figure) C Stylet knobs (sk) of Mincognita male (Abbreviations l = lumen m1 = protractor muscles mc = marginal cells t = tonofilaments Dorsal is toward thetop of the figure) D Stylet shaft (ss) of M incognita male surrounded by junctional complexes (arrows) between syncytia includingarcade (a) and epidermis (es) (Abbreviations m1 = protractor muscles) E Longitudinal section through region corresponding to lsquoDrsquoincluding stylet shaft (ss) surrounded by junctional complexes (arrows) associated with adjacent arcade syncytia (a) Figures D and Eare printed from TEM negatives parts of which are used in Baldwin and Hirschmann (1973) and for B C and E parts of which areused in Baldwin and Hirschmann (1976)

626 Nematology

Homology of the tylenchid stomatostylet

Vol 6(5) 2004 627

JG Baldwin et al

628 Nematology

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

JG Baldwin et al

Fig 2 Diagrammatic representation of the components of the cephalobid stoma (A) in relation to the tylenchid stomatostylet (B)Colours corresponding between A and B represent a hypothesis of homology Broken red lines indicate the presence of junctionalcomplexes broken brown lines represent tonofilaments attaching arcade syncytia to stomastomatostylet cuticle lsquoArsquo is adapted fromBaldwin and Eddleman (1995) and lsquoBrsquo is modified and partially redrawn from Baldwin and Hirschmann (1976)

broad noncontractile region that includes the nucleusand which extends posteriorly into the procorpus (Bald-win amp Hirschmann 1976) vs a much narrower poste-rior process as in those of Cephalobina (Dolinski et al1998) Posterior to the tylenchid stylet knobs a second set

of radial muscles m2 (defining the mesostegostom) at-taches to the narrow pharyngeal lumen but extends an-teriad as narrow muscular processes closely associatedwith protractors Curiously m2 comprises a pair of adra-dial cells on the dorsal side whereas on each subventral

Fig 3 Transmission electron micrographs through the anterior stegostom of representatives of Cephalobina and Tylenchida Sectionsare transverse unless otherwise indicated A Zeldia punctata female with the stomatal lumen (l) surrounded by three interradial muscles(m1) separated by three marginal cells (mc) (t = tonofilaments) Arrows denote junctional complexes dorsal is toward the top of thefigure B Meloidogyne incognita male near junction of stylet shaft (ss) with stylet knobs (sk) the former flanked by arcade syncytia(a) The three protractor muscles (m1) are separated by narrow marginal cells (mc) (Abbreviations l = stylet lumen m2 = anteriorcontractile region of m2 Arrows denote junctional complexes dorsal is toward the top of the figure) C Stylet knobs (sk) of Mincognita male (Abbreviations l = lumen m1 = protractor muscles mc = marginal cells t = tonofilaments Dorsal is toward thetop of the figure) D Stylet shaft (ss) of M incognita male surrounded by junctional complexes (arrows) between syncytia includingarcade (a) and epidermis (es) (Abbreviations m1 = protractor muscles) E Longitudinal section through region corresponding to lsquoDrsquoincluding stylet shaft (ss) surrounded by junctional complexes (arrows) associated with adjacent arcade syncytia (a) Figures D and Eare printed from TEM negatives parts of which are used in Baldwin and Hirschmann (1973) and for B C and E parts of which areused in Baldwin and Hirschmann (1976)

626 Nematology

Homology of the tylenchid stomatostylet

Vol 6(5) 2004 627

JG Baldwin et al

628 Nematology

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

Homology of the tylenchid stomatostylet

Vol 6(5) 2004 627

JG Baldwin et al

628 Nematology

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

JG Baldwin et al

628 Nematology

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

Homology of the tylenchid stomatostylet

side there is only a single interradial m2 cell (Figs 2B3B) These muscle cells may be discernible with light mi-croscopy (Coomans amp van Bezooijen 1968) thereby al-lowing us to evaluate efficiently these features throughoutTylenchida

Although further investigations of the tylenchid cor-pus may reveal homologies of the metastegostom andtelostegostom we suggest that if present at all these arenot components of the stomatostylet Reviewing TEM ofa moulting tylenchid (Endo 1985) suggests that duringdevelopment the stylet knobs and protractors develop inpharyngeal tissue independent from other more anteriorcomponents of the stylet (our interpretation of publishedmicrographs) Our new interpretation of homology recog-nises the consistent position of the dorsal gland orificeposterior to the stylet knobs and m1 muscles (Tylenchida)and homologue to the stylet knobs (m1 muscle region) inCephalobina (Fig 2) This new hypothesis contrasts withprevious less parsimonious hypotheses where the dorsalgland orifice in Cephalobina occurs well anterior to theputative homologue of the stylet knobs (Thorne 1961Andraacutessy 1962 Goodey 1963) (Fig 1)

STYLET CONE AND SHAFT GYMNOSTOM

In Cephalobina the gymnostom is enclosed by thick-ened cuticle surrounded by a stack of two arcade syncytialrings that occur anterior to the stegostom and the pharynx(Fig 2A) (De Ley et al 1995) As in Rhabditina the ar-cade cytoplasm is characterised (between moults) by mul-tiple small vesicles and the near absence of mitochondriaendoplasmic reticulum and other organelles (Wright ampThomson 1981) Junctional complexes occur between thetwo arcade syncytia Posteriorly at the point of attachmentand junctional complex between the second arcade syn-

cytium and the stegostom there is a distinctive electrondense band of tonofilaments (Fig 2A) Delimiting the an-terior end of the gymnostom a junctional complex occursbetween the anterior arcade syncytium and adjacent epi-dermal syncytium of the cheilostom The transition is fur-ther distinguished by an abrupt discontinuity and changein associated cuticle lining of the stoma the cuticle an-terior to this boundary being a labial invagination con-tinuous with the body wall and separate from the cuticlelining the gymnostom stegostom and pharynx (De Leyet al 1995) The cuticle lining associated with the gym-nostom protrudes anteriorly to the gymnostom and intothe cheilostom lumen (Van de Velde et al 1994 Baldwinamp Eddleman 1995 De Ley et al 1995) (Figs 2A 4A)This extension of the cuticle associated with the gymnos-tom protruding into the cheilostom lumen occurs in otherSecernentea and is particularly striking in Diplogastrina(Baldwin et al 1997)

Herein we hypothesise that the cone and shaft ofthe tylenchid stylet together represent the homologueof the cuticle lininglumen of the gymnostom (Fig 1)Although the stylet may extend far anteriad (whereit moves freely through the guiding apparatus) it issignificant that its anterior point of attachment is witharcade syncytial tissue that surrounds the shaft (Figs2B 3D E) As in Cephalobina this arcade syncytialtissue includes vesicles but between moults has very fewmitochondria endoplasmic reticula and other organellesPosteriorly this arcade tissue forms in conjunction withabundant tonofilaments a junctional complex with thestegostom (consisting of protractor muscles and marginalcells) analogous to that observed between the gymnostomand stegostom in Cephalobina (Figs 2 3A-C) Whatis visible with light microscopy as the guide ring that

Fig 4 Transverse transmission electron micrographs through the cheilostom and gymnostom of representatives of Cephalobina andTylenchida A Zeldia punctata during fourth moult showing a portion of the cheilostom (c) and surrounding epidermal syncytium (es)Within the stoma is a portion of the adult and moulting cuticle of the fourth-stage protruding from the more posterior gymnostom (g)Arrow indicates stoma space between the cheilostom and gymnostom cuticle (l = lumen of the gymnostom) B Acrobeles complexusfemale showing vestibule of the cheilostom (c) and its relationship to the hexaradiate framework (f) that is surrounded by epidermalsyncytium (es) Cheilostom lumen (l) encloses an anterior protrusion of the more posterior gymnostom (g) C Anterior terminus ofZeldia punctata female including a portion of the cheilostom (c) surrounding the stoma lumen (l) The very shallow cephalic framework(f) is surrounded by the epidermal syncytium (es) D Anterior portion of vestibule extension (ve) in Meloidogyne incognita maleVestibule extension is enclosed by epidermal syncytium that includes mitochondria (mt) and extends between branches of the protractorcells (m1) that extend anteriorly from the stegostom Arrow indicates stoma space between the cheilostom and stylet cone (sc) cuticle(Abbreviation l = lumen of the stylet cone) E Cephalic framework (f) of M incognita male Vestibule (v) encloses stylet cone (sc)(Abbreviation es = epidermal syncytium) F A portion of the cephalic framework (f) slightly anterior to that shown in D Epidermalsyncytium (hs) includes large mitochondria (mt) Arrow indicates stoma space between the vestibule (v) and stylet cone (sc) Figures Dand E printed from TEM negatives parts of which are also used in Baldwin and Hirschmann (1976)

Vol 6(5) 2004 629

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

JG Baldwin et al

surrounds the stylet shaft is known from TEM to be aring of folded junctional complexes between syncytiaThese are considered analogous to junctional complexescharacterising the gymnostom and form between theepidermal and anterior arcade syncytia as well as betweenthe two arcade syncytia in Cephalobina (Figs 2 3D E)

The stylet cone and shaft originating within the gym-nostom and extending anteriad into the cheilostom as thestylet (gymnostom cuticle) elongates is reminiscent of asimilar morphology in Cephalobina and other Secernen-tea (see above) This interpretation appears to be con-sistent with light microscope observations of stylet for-mation during moulting (Roman amp Hirschmann 1969)and with TEM observations of cone and shaft formationwithin two arcade syncytia These two syncytia lie pos-terior to the developing guiding apparatus (cheilostom)through which the stylet extends but to which it remainsunattached (Endo 1985)

VESTIBULE VESTIBULE EXTENSION CHEILOSTOM

The cheilostom of Cephalobina occurs anterior to thegymnostom and may also enclose the anterior extensionof the gymnostom (Figs 2A 4A) Since the cuticle liningof the cheilostom is continuous with that of the body wallthe underlying tissue is epidermis as is also the case forthe body wall Reconstruction through 3D modelling ofcells and syncytia of the anterior end of Cephalobina sug-gest that the epidermis of the head region primarily con-sists of two highly convoluted and interdigitating syn-cyctial rings (Bumbarger amp Baldwin unpubl) Anteriorlythe cheilostom region of Cephalobina is surrounded by ahexaradiate cuticular framework although in some taxathis framework may be very shallow and may follow thedome shape of the anterior end of the head (Fig 4B C)

In Tylenchida we hypothesise that the cheilostom isrepresented by the guiding apparatus and associated struc-tures including the vestibule framework and vestibuleextension all of which are continuous with the bodywall cuticle The vestibule and framework are similarin form to that of Cephalobina although in Tylenchidathey are typically much more elongate along the anterior-posterior axis (Figs 2 4B-E) The vestibule extension iscontinuous with and extends posteriad to the vestibule(Figs 2B 4D) The syncytium enclosing the guiding ap-paratus includes some large mitochondria and is similarin this respect to the labial epidermis of other Secernen-tea (Wright amp Thomson 1981) (Fig 4D F) It pervades

Equivalent to stomatal wall in Endo (1985)

the region above the framework forming a sheath aroundthe vestibule extension that extends between the anteriorbranches of the stylet protractors (Figs 2B 4D-F)

Conclusions

Relative to the simple stoma of most Cephalobinathe stomatostylet of Tylenchida is extremely complexWhereas homologies are difficult to establish based onlight microscopy alone a re-evaluation of TEM of Ty-lenchida in conjunction with a newer understanding ofthe stoma structure in Cephalobina has provided a ba-sis for plausible hypotheses of homology of cheilostomgymnostom and prostegostom between the two groupsFurther testing of the hypotheses requires clearer phylo-genetic resolution This resolution provides the basis forinterpreting representatives used as morphological modelsto address questions such as that of the independent evo-lution of the stylet in Tylenchida and some Aphelenchida(Baldwin et al 2004) Due to the highly derived mor-phology of the tylenchid stylet (and the possibility that ithas further diverged within the tylenchids) it is proposedthat a choice of taxa basal to the tylenchid clade such asAphelenchus (see Blaxter et al 1998 and Baldwin et al2004 for phylogenies including this genus) a choice thatwould be made in the light of more clearly resolved phy-logenies might be most informative for comparison withCephalobina

Other approaches for testing the hypotheses include3D ultrastructural and developmental reconstruction ofrepresentatives of Tylenchida and outgroups Particu-larly complex tissues include the epidermal and arcadesyncytia their junctional complexes and tonofilament-rich connections with cuticle and the relationship ofall these structures during stomastomatostylet develop-ment It is not known if some cells that participate duringstomastomatostylet formation undergo transformation ordeath in the adult perhaps obscuring their relationship tothe completed structures as seen between moults

Acknowledgements

The authors acknowledge electron micrographs ar-chived and used for comparative reconstruction in theBaldwin laboratory including those from previous workby Drs Dolinski Zhang and Eddleman

630 Nematology

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

Homology of the tylenchid stomatostylet

This work was supported by the US National Sci-ence Foundation grants DEB-0228692 DEB-9712355and DEB-0206569

References

ALBERTSON DG amp THOMSON JN (1975) The pharynxof Caenorhabditis elegans Philosophical Transactions of theRoyal Society of London Series B 275 99-325

ANDRAacuteSSY I (1962) Uumlber den Mundstachel der Tylenchiden(Nematologische Notizen 9) Acta Zoologica Hungarica 8167-315

ANDRAacuteSSY I (1976) Evolution as a basis for the systematiza-tion of nematodes London UK Pitman Publishing 288 pp

BALDWIN JG amp EDDLEMAN CD (1995) Buccal capsuleof Zeldia punctata (Nemata Cephalobidae) an ultrastructuralstudy Canadian Journal of Zoology 73 648-656

BALDWIN JG amp HIRSCHMANN H (1973) Fine structureof cephalic sense organs in Meloidogyne incognita malesJournal of Nematology 5 285-302

BALDWIN JG amp HIRSCHMANN H (1976) Comparative finestructure of the stomatal region of males of Meloidogyneincognita and Heterodera glycines Journal of Nematology 81-17

BALDWIN JG GIBLIN-DAVIS RM EDDLEMAN CDWILLIAMS DS VIDA JT amp THOMAS WK (1997)The buccal capsule of Aduncospiculum halicti (NemataDiplogasterina) an ultrastructural and molecular phyloge-netic study Canadian Journal of Zoology 75 407-423

BALDWIN JG NADLER SA amp ADAMS BJ (2004)Evolution of parasitism among nematodes Annual Review ofPhytopathology 42 83-105

BIRD AF amp BIRD J (1991) The structure of nematodesNew York USA Academic Press Inc 316 pp

BLAXTER M DE LEY P GAREY JR LIU LXSCHELDEMAN P VIERSTRAETE A VANFLETERENJR MACKEY LY DORRIS M FRISSE LM VIDAJT amp THOMAS WK (1998) A molecular evolutionaryframework for the phylum Nematoda Nature 392 71-75

CHEN TA amp WEN GY (1980) Electron microscopy ofthe stomatostylet and esophagus of Criconemoides curvatumJournal of Nematology 12 72-83

COOMANS A amp VAN BEZOOIJEN J (1968) Observations onthe anterior body region of Helicotylenchus pseudorobustusNematologica 14 146-148

DE GRISSE AT (1972) Elektromikroskopische waarnemin-gen aangaande de struktuur van de stekelschede in Tylenchi-den Mededelingen Fakulteit Landbouwwetenschappen Rijk-suniversiteit Gent 34 1109-1115

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

DE LEY P DE GRISSE A VAN DE VELDE MC ampCOOMANS A (1993) Ultrastructure of the stoma in Ty-lopharynx Mededelingen Faculteit LandbouwwetenschappenUniversiteit Gent 58 763-778

DE LEY P VAN DE VELDE MC MOUNPORT D BAU-JARD P amp COOMANS A (1995) Ultrastructure of thestoma in Cephalobidae Panagrolaimidae and Rhabditidaewith a proposal for a revised stoma terminology in Rhabdi-tida (Nematoda) Nematologica 41 153-182

DOLINSKI CM amp BALDWIN JG (2003) Fine structure ofthe stoma of Bunonema sp and Teratorhabditis palmarum(Nematoda) and its phylogenetic significance Journal ofNematology 35 244-251

DOLINSKI C BORGONIE G SCHNABEL R amp BALDWINJG (1998) Buccal capsule development as a considerationfor phylogenetic analysis of Rhabditida (Nemata) Develop-mental Genes and Evolution 208 495-503

DOLINSKI C BALDWIN JG amp THOMAS WK (2001)Comparative survey of early embryogenesis of Secernentea(Nematoda) with phylogenetic implications Canadian Jour-nal of Zoology 79 82-94

DROPKIN VH (1969) Cellular responses of plants to nema-tode infections Annual Review of Phytopathology 7 101-122

ENDO BY (1985) Ultrastructure of the head region of moltingsecond-stage juveniles of Heterodera glycines with emphasison stylet formation Journal of Nematology 17 112-123

ENDO BY amp NICKLE WR (1993) Ultrastructure of thebuccal cavity region and oesophagus of the insect parasiticnematode Heterorhabditis bacteriophora Nematologica 40278-298

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

GOODEY JB (1963) Speculations on the identity of the partsof the tylenchid spear Nematologica 9 468-470

MAGGENTI A (1981) General nematology New York USASpringer-Verlag 372 pp

PANCHEN AL (1994) Richard Owen and the concept ofhomology In Hall BK (Ed) Homology the hierarchicalbasis of comparative biology London UK Academic Presspp 21-62

PATTERSON C (1988) Homology in classical and molecularbiology Molecular Biology and Evolution 5 603-625

ROMAN J amp HIRSCHMANN H (1969) Embryogenesis andpostembryogenesis in species of Pratylenchus (NematodaTylenchidae) Proceedings of the Helminthological Society ofWashington 36 164-174

SHEPHERD AM amp CLARK SA (1976) Structure of theanterior alimentary tract of the passively feeding nematodeHexatylus viviparus (Neotylenchidae Tylenchida) Nemato-logica 22 332-342

SHEPHERD AM CLARK SA amp HOOPER DJ (1980)Structure of the anterior alimentary tract of Aphelenchoides

Vol 6(5) 2004 631

JG Baldwin et al

blastophthorus (Nematoda Tylenchida Aphelenchina) Ne-matologica 26 313-357

SIDDIQI MR (1980) The origin and phylogeny of the nema-tode order Tylenchida and Aphelenchida n ord Helmintho-logical Abstracts Series B 49 143-170

STEINER G (1933) The nematode Cylindrogaster longistoma(Stefanski) Goodey and its relationship Journal of Parasito-logy 20 66-69

THORNE G (1961) Principles of nematology New YorkUSA McGraw-Hill Book Co Inc 552 pp

VAN DE VELDE MC DE LEY P MOUNPORT D BAU-JARD P amp COOMANS A (1994) Ultrastructure of the buc-cal cavity and the cuticle of three Cephalobidae (NematodaRhabditida) Nematologica 40 541-563

WRIGHT KA amp THOMSON JN (1981) The buccal capsuleof Caenorhabditis elegans (Nematoda Rhabditoidea) anultrastructural study Canadian Journal of Zoology 59 1952-1961

632 Nematology