The vulval pattern of six species of the genus Bursaphelenchus (B. abruptus, B. conicaudatus, B. fraudulentus,
B. luxuriosae, B. mucronatus and B. xylophilus) was studied using scanning electron microscopy. A terminology for the vulval region
structures observed is proposed herein and illustrated by micrographs and line drawings. It was shown that, of the studied species, only
B. mucronatus and B. xylophilus share an identical morphology of the vulval region, all other species differing significantly from each
other and from both B. mucronatus and B. xylophilus. This study indicates the diagnostic potential for variation in vulval morphology
within Bursaphelenchus and it is recommended that such features are recorded in all future descriptions

Mota, Manuel

RYSS, Alexandre

SANTOS, Ana Rita

VIEIRA, Paulo

English

Summary - The vulval pattern of six species of the genus Bursaphelenchus (B. abruptus, B. conicaudatus, B. fraudulentus,  B. luxuriosae, B. mucronatus and B. xylophilus)  was studied using scanning electron microscopy. A terminology for the vulval region structures observed is proposed herein and illustrated by micrographs and line drawings. It was shown that, of the studied species, only B. mucronatus and B. xylophilus share an identical morphology of the vulval region, all other species differing significantly from each other and from both B. mucronatus and B. xylophilus. This study indicates the diagnostic potential for variation in vulval morphology within Bursaphelenchus and it is recommended that such features are recorded in all future descriptions. Keywords -Bursaphelenchus abruptus, Bursaphelenchus conicaudatus, Bursaphelenchus fraudulentus, Bursaphelenchus luxuriosae, Bursaphelenchus mucronatus, Bursaphelenchus xylophilus, SEM. Due to the economic importance of the pine wood nematode (Bursaphelenchus xylophilus (Steiner &amp; Buhrer,  1934) Nickle, 1970, Bursaphelenchus Fuchs, 1937 is one of the most intensively studied genera within the superfamily Aphelenchoidea and currently contains about 100 valid species  Materials and methods In this study, six species were compared  For SEM observations live, mature, inseminated females were collected separately for each species and placed in a few drops of cold tap water in a glass vial. They were fixed in a mixture of 4% gluteraldehyde-2% formaldehyde for 48 h, post-fixed in 2% OsO 4 overnight, dehydrated in an ethanol series and then critical point drying and sputter coated with gold  © Koninklijke Brill NV, Leiden, 200

Paulo Vieira

Alexander Ryss

Ana Rita Santos

Manuel Mota

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On the vulval morphology of some species of Bursaphelenchus (Nematoda: Parasitaphelenchinae)

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Nematology, 2009, Vol. 11(5), 793-798On the vulval morphology of some species of Bursaphelenchus(Nematoda: Parasitaphelenchinae)Paulo VIEIRA 1,∗, Alexander RYSS 2, Ana Rita SANTOS 1 and Manuel MOTA 11 NemaLab-ICAM, Departamento de Biologia, Universidade de Évora, 7002-554 Évora, Portugal2 Zoological Institute RAS, Universitetskaya naberezhnaya 1, St. Petersburg 199034, RussiaReceived: 8 July 2008; revised: 29 January 2009Accepted for publication: 29 January 2009Summary – The vulval pattern of six species of the genus Bursaphelenchus (B. abruptus, B. conicaudatus, B. fraudulentus,B. luxuriosae, B. mucronatus and B. xylophilus) was studied using scanning electron microscopy. A terminology for the vulval regionstructures observed is proposed herein and illustrated by micrographs and line drawings. It was shown that, of the studied species, onlyB. mucronatus and B. xylophilus share an identical morphology of the vulval region, all other species differing significantly from eachother and from both B. mucronatus and B. xylophilus. This study indicates the diagnostic potential for variation in vulval morphologywithin Bursaphelenchus and it is recommended that such features are recorded in all future descriptions.Keywords – Bursaphelenchus abruptus, Bursaphelenchus conicaudatus, Bursaphelenchus fraudulentus, Bursaphelenchus luxuriosae,Bursaphelenchus mucronatus, Bursaphelenchus xylophilus, SEM.Due to the economic importance of the pine wood ne-matode (Bursaphelenchus xylophilus (Steiner & Buhrer,1934) Nickle, 1970), Bursaphelenchus Fuchs, 1937 isone of the most intensively studied genera within the su-perfamily Aphelenchoidea and currently contains about100 valid species (Hunt, 2008). Although molecular tech-niques are now widely used to identify Bursaphelenchusspecies (Hoyer et al., 1998; Ye et al., 2007; Kanzaki et al.,2008), morphology continues to be an important tool innematode systematics (Hunt, 1993, 2008; Braasch, 2001;Ryss et al., 2005; Giblin-Davis et al., 2006). Until now,the vulval pattern variation of Bursaphelenchus spp. hasnot been studied in great detail (e.g., Yik & Birchfield,1981), although for some species the presence of vul-val papillae have been reported and illustrated by SEM(Palmisano & Ambrogioni, 1994; Carletti et al., 2005).It would appear that for species of the xylophilus-group,and also for other species in the genus, this region offerspotentially important morphological features for speciesidentification (e.g., presence of vulval flap, vulval flapshape).∗ Corresponding author, email: pvieira@uevora.ptMaterials and methodsIn this study, six species were compared (Table 1),namely B. abruptus Giblin-Davis, Mundo-Ocampo, Bald-win, Norden & Batra, 1993, B. conicaudatus Kanzaki,Tsuda & Futai, 2000, B. fraudulentus Rühm, 1956, B. lux-uriosae Kanzaki & Futai, 2003, B. mucronatus Mamiya& Enda, 1979 and B. xylophilus. Nematodes of eachspecies were collected from established cultures of Botry-tis cinerea Pars. using the Baermann funnel technique andthen washed several times in distilled water.For SEM observations live, mature, inseminated fe-males were collected separately for each species andplaced in a few drops of cold tap water in a glass vial.They were fixed in a mixture of 4% gluteraldehyde-2%formaldehyde for 48 h, post-fixed in 2% OsO4 overnight,dehydrated in an ethanol series and then critical point dry-ing and sputter coated with gold (Eisenback, 1985) andobserved with a Jeol 35 SEM. For each species, six toten individuals were observed and photographed at differ-ent positions for comparison. Line drawings of the vulvalregion are provided to illustrate each species. The draw-ings were prepared from the SEM images obtained for thespecies used in this study.© Koninklijke Brill NV, Leiden, 2009 DOI:10.1163/156854109X415533Also available online - www.brill.nl/nemy 793P. Vieira et al.Table 1. Bursaphelenchus isolates used in this study.Nematode species n Country Isolate Plant or insect hostB. abruptus 6 USA Ne12/98 Anthophora abrupta Say (Hymenoptera: Anthophoridae)B. conicaudautus 6 Japan BconJ Ficus carica L. (Urticales: Moraceae)B. fraudulentus 8 Germany DE10w Pinus sp. and Picea sp. (Pinales: Pinaceae)B. luxuriosae 6 Japan BluxJ Acalolepta luxuriosae Bates (Coleoptera: Cerambycidae)B. mucronatus 10 Germany DE4w Pinus sylvestris L. (Pinales: Pinaceae)B. xylophilus 10 Portugal Troia Pinus pinaster Aiton (Pinales: Pinaceae)For detailed plant, insect host and species distribution, see Ryss et al. (2005). n = number of mature inseminated females photographedand observed.ResultsTERMINOLOGY OF VULVAL REGION MORPHOLOGYIn order to describe the diversity of the vulval region itis necessary to establish new terms for the observed struc-tures as the vulval morphology has not been studied indetail to date (Fig. 1). The proposed terms for the variousvulval structures are listed in Figure 2. The only structurehitherto widely used in diagnostics of Bursaphelenchusspecies is the vulval flap, i.e., a posteriorly directed cuti-cular extension of the anterior vulval lip. The flap shapevaries within different species. The posterior margin ofthe flap is herein referred to as the vulval flap limb. Theposterior vulval lip (= vulval labium) may bear a pair ofvulval papillae. Between the anterior lip (vulval flap) andthe posterior lip (vulval labium) of some species there isa zone which we call the vulval prelabium as it is locatedanterior to the labium yet posterior to the slit which leadsinto the vaginal vestibulum. The latter continues (under-neath the vulval flap) into the vagina, its entrance beingnamed the gonotreme. The vulval area is bordered later-ally by the vulval lateral alae. The vulval lateral alae maybe straight or strongly curved.VULVAL MORPHOLOGY IN THE STUDIED SPECIESThe details of the vulval morphology of all six Bursa-phelenchus species are given below and are illustrated inFigures 1 and 2.i) B. abruptus (Figs 1A; 2A). Vulval papillae absent.Vulval region asymmetrical with vulval flap limb inclinedposteriad from right to left, i.e., entrance directed to theright (Note: in the original description of B. abruptus(Giblin-Davis et al., 1993) the vulval area appears sym-metrical. However, in all six specimens observed in thisstudy, the vulval area displays the same pattern as shownin Figure 1A). Vulval flap massive with somatic annula-tion running across it anteriorly. Vulval lateral alae absent.Prelabium indistinct, not separated from labium.ii) B. luxuriosae (Figs 1B; 2B). Vulval papillae ab-sent. Vulval area symmetrical. Vulval flap moderatelylong, semicircular. Its limb arch-shaped, conically-round-ed. Vulval flap thin with annulation parallel (areolated) tovulval flap limb; annulation not contiguous with somaticannulation. Vulval lateral alae well developed, straight toslightly curved ventrally. Prelabium distinct, not separatedfrom labium by incisure.iii) B. fraudulentus (Figs 1C; 2C). Vulval papillaepresent, prominent. Vulval area symmetrical. Vulval flapmoderately long, limb arch-shaped to rectangular. Vulvalflap thin, annulation parallel to vulval flap limb, notcontiguous with somatic annulation. Vulval lateral alaeindistinct, fused with vulval flap. Prelabium not separatedfrom labium by incisure, prelabium at least twice lengthof labium.iv) B. conicaudatus (Figs 1D; 2D). Vulval papillaepresent, low and flattened. Vulval area symmetrical. Vul-val flap moderately long, semicircular. Its limb arch-shaped, conically rounded. Vulval flap thin, with annu-lation parallel to vulval flap limb, not contiguous withsomatic annulation. Vulval lateral alae well developed,straight to slightly curved ventrally. Prelabium separatedfrom labium by incisure; prelabium at least twice labiumlength.v) B. mucronatus (Figs 1E; 2E) and vi) B. xylophi-lus (Figs 1F; 2F). Vulval papillae present, prominent. Vul-val area symmetrical. Vulval flap extremely long. Its limbarch-shaped to rectangular; lateral edges of flap not reach-ing labium and papillae. Vulval flap thin with annula-tion parallel (areolated) to vulval flap limb, not contigu-ous with somatic annulation. Vulval lateral alae well de-veloped, curved. Labium enclosed by incisure. Prelabium794 NematologyVulval morphology in Bursaphelenchus spp.Fig. 1. Scanning electronic microscope observations of the female vulva region of six different Bursaphelenchus species. A: Bursaphe-lenchus abruptus; B: B. luxuriosae; C: B. fraudulentus; D: B. conicaudatus; E: B. mucronatus; F: B. xylophilus.Vol. 11(5), 2009 795P. Vieira et al.Fig. 2. Female vulval region (ventral view). A: Bursaphelenchus abruptus; B: B. luxuriosae; C: B. fraudulentus; D: B. conicaudatus;E, G: B. mucronatus; F: B. xylophilus; G, H: Female vulva region (ventral and lateral view) showing constituent parts. Abbreviations:ba = body annules; fli = vulval flap limb (labrum limb); gtr = gonotreme; lb = vulval labium (posterior lip); lbi = vulval labiumincisure; lpd = vulval labium posterior depression; pap = vulval papillae; plb = vulval prelabium; val = vulval lateral alae;vf = vulval flap (i.e., vulval labrum or anterior lip of vulva); vfa = vulval flap annuli; vs = vaginal vestibulum. (Scale bars = 5 μm.)796 NematologyVulval morphology in Bursaphelenchus spp.separated from labium by incisure. Prelabium length notgreater than labium length.DiscussionMost of the species studied differ from each otherin vulval morphology. Females of B. abruptus and B.luxuriosae lack vulval papillae on the labium and B.abruptus has no vulval lateral alae, the somatic annulationtherefore continuing across the anterior region of thevulval flap. Conversely, lateral alae are present in B.luxuriosae. Such alae are present in the other studiedspecies of the xylophilus-group (although indistinct in B.fraudulentus) and separate the somatic annulation fromthe vulval area. In B. fraudulentus and B. conicaudatus theprelabium is very large, its length being at least twice thatof the labium, whereas in B. xylophilus and B. mucronatusthe prelabium is less well developed and is shorter thanthe labium length. The vulval area of B. fraudulentus isvery small, the lateral alae being fused with the vulvalflap. Conversely, in B. conicaudatus, the vulval regionis distinctly larger with the lateral alae separated fromthe vulval flap. The prelabium in B. fraudulentus is notseparated from the labium by an incisure, whereas such anincisure is present between the labium and prelabium in B.conicaudatus. Distinct differences in vulval morphologybetween B. xylophilus and B. mucronatus females werenot found.This study, although confined to only six species,indicates the diagnostic potential for variation in vulvalarea morphology and it is therefore recommended thatfuture descriptions illustrate this area using the SEM.AcknowledgementsThis research was partly supported by the GRICESproject (Portugal-Russia Cultural Agreement Programme)together with the scientific exchange programme of theRussian Academy of Sciences. The second author waspartially supported by the ‘Unique Fund Collectionsof the Zoological Institute RAS’ (Rosnauka – FederalScience and Innovations Agency 02.518.11.7086-UFKZIN 2-2.20) programme. The authors kindly thank Prof.Kazuyoshi Futai (Kyoto University, Japan) and DrThomas Schröder (Federal Biological Research Centrefor Agriculture and Forestry, Germany) for providing ne-matode isolates and cultures, Prof. Esperança Pina (Fac-uldade de Ciências Biomédica, Universidade Nova deLisboa, Portugal) for the use of the SEM, Mr OctávioChaveiro (Estação Agronómica Nacional, Portugal) fortechnical assistance and Eng. Francisca Figo (Universi-dade de Évora, Portugal) for laboratory assistance.ReferencesBRAASCH, H. (2001). Bursaphelenchus species in conifers inEurope: distribution and morphological relationships. EPPOBulletin 31, 127-142.CARLETTI, B., IRDANI, T., COSI, E., BRANDSTETTER, M.,PENNACCHIO, F., ROVERSI, P.F. & AMBROGIONI, L.(2005). First record of Bursaphelenchus fraudulentus Rühm(Goodey) (Nematoda Aphelenchoididae) in Italy. Redia 88,27-35.EISENBACK, J. (1985). Techniques for preparing nematodes forscanning electron microscopy. In: Barker, K.R., Carter, C.C.& Sasser, N.J. (Eds). An advanced treatise on Meloidogyne,Vol. II. Raleigh, NC, USA, North Carolina State UniversityGraphics, pp. 79-105.GIBLIN-DAVIS, R.M., MUNDO OCAMPO, M., BALDWIN,J.G., NORDEN, B.B. & BATRA, S.W.T. (1993). Descriptionof Bursaphelenchus abruptus n. sp. (Nemata: Aphelenchoi-didae), an associate of a digger bee. Journal of Nematology25, 161-172.GIBLIN-DAVIS, R., KANZAKI, N., YE, W., CENTER, B.J. &THOMAS, W.K. (2006). Morphology and systematics of Bur-saphelenchus gerberae n. sp. (Nematoda: Parasitaphelenchi-dae), a rare associate of the palm weevil, Rhynchophorus pal-marum in Trinidad. Zootaxa 1189, 39-53.HOYER, U., BURGERMEISTER, W. & BRAASCH, H. (1998).Identification of Bursaphelenchus species (Nematoda: Aphe-lenchoididae) on the basis of amplified ribosomal DNA(ITS-RFLP). Nachrichtenblatt des Deutschen Pflanzen-schutzdienstes 50, 273-277.HUNT, D.J. (1993). Aphelenchida, Longidoridae and Tricho-doridae: Their systematics and bionomics. Wallingford, UK,CAB International, 352 pp.HUNT, D.J. (2008). A checklist of the Aphelenchoidea (Nema-toda: Tylenchina). Journal of Nematode Morphology and Sys-tematics 10 (2007), 99-135.KANZAKI, N., AIKAWA, T., MAEHARA, N. & MATSUMOTO,K. (2008). Bursaphelenchus doui Braasch, Gu, Burgermeister& Zhang, 2005 (Aphelenchida: Parasitaphelenchidae), an as-sociate of Monochamus subfasciatus Bates (Coleoptera: Ce-rambycidae) and Pinus densiflora Sieb. & Zucc. Nematology10, 69-78.PALMISANO, A.M. & AMBROGIONI, L. (1994). Aphelen-choidoidea nematodes associated with Pinus spp. Redia 77,225-240.RYSS, A., VIEIRA, P., MOTA, M. & KULINICH, O. (2005).A synopsis of the genus Bursaphelenchus Fuchs, 1937Vol. 11(5), 2009 797P. Vieira et al.(Aphelenchida: Parasitaphelenchidae) with keys to species.Nematology 7, 393-458.YE, W., GIBLIN-DAVIS, R.M., BRAASCH, H., MORRIS, K.& THOMAS, W.K. (2007). Phylogenetic relationships amongBursaphelenchus species (Nematoda: Parasitaphelenchidae)inferred from nuclear ribosomal and mitochondrial DNAsequence data. Molecular Phylogenetics and Evolution 43,1185-1197.YIK, C.P. & BIRCHFIELD, W. (1981). Observations on themorphology of the pine wood nematode, Bursaphelenchusxylophilus. Journal of Nematology 13, 376-384.798 Nematology

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On the vulval morphology of some species of Bursaphelenchus (Nematoda: Parasitaphelenchinae)

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