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2009

Evaluation of Some Vulval Appendages in Nematode Taxonomy

Lynn K. Carta United States Department of Agriculture–Agricultural Research Service, [email protected]

Zafar A. Handoo United States Department of Agriculture–Agricultural Research Service, [email protected]

Eric P. Hoberg United States National Parasite Collection, [email protected]

Eric F. Erbe United States Department of Agriculture, [email protected]

William P. Wergin United States Department of Agriculture, [email protected]

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Carta, Lynn K.; Handoo, Zafar A.; Hoberg, Eric P.; Erbe, Eric F.; and Wergin, William P., "Evaluation of Some Vulval Appendages in Nematode Taxonomy" (2009). Faculty Publications from the Harold W. Manter Laboratory of Parasitology. 639. https://digitalcommons.unl.edu/parasitologyfacpubs/639

This Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Comp. Parasitol. 76(2), 2009, pp. 191–209

Evaluation of Some Vulval Appendages in Nematode Taxonomy

1,5 1 2 3 4 LYNN K. CARTA, ZAFAR A. HANDOO, ERIC P. HOBERG, ERIC F. ERBE, AND WILLIAM P. WERGIN 1 Nematology Laboratory, United States Department of Agriculture–Agricultural Research Service, Beltsville, Maryland 20705, U.S.A. (e-mail: [email protected], [email protected]) and 2 United States National Parasite Collection, and Animal Parasitic Diseases Laboratory, United States Department of Agriculture–Agricultural Research Service, Beltsville, Maryland 20705, U.S.A. (e-mail: [email protected])

ABSTRACT: A survey of the nature and phylogenetic distribution of nematode vulval appendages revealed 3 major classes based on composition, position, and orientation that included membranes, flaps, and epiptygmata. Minor classes included cuticular inflations, protruding vulvar appendages of extruded gonadal tissues, vulval ridges, and peri-vulval pits. Vulval membranes were found in Mermithida, Triplonchida, Chromadorida, Rhabditidae, Panagrolaimidae, Tylenchida, and Trichostrongylidae. Vulval flaps were found in Desmodoroidea, Mermithida, Oxyuroidea, Tylenchida, Rhabditida, and Trichostrongyloidea. Epiptygmata were present within Aphelenchida, Tylenchida, Rhabditida, including the diverged Steinernematidae, and Enoplida. Within the Rhabditida, vulval ridges occurred in Cervidellus, peri-vulval pits in Strongyloides, cuticular inflations in Trichostrongylidae, and vulval cuticular sacs in Myolaimus and Deleyia. Vulval membranes have been confused with persistent copulatory sacs deposited by males, and some putative appendages may be artifactual. Vulval appendages occurred almost exclusively in commensal or parasitic nematode taxa. Appendages were discussed based on their relative taxonomic reliability, ecological associations, and distribution in the context of recent 18S ribosomal DNA molecular phylogenetic trees for the nematodes. Characters were found to be distributed across subsets of terminal and phylogenetically distant taxa, demonstrating considerable homoplasy. Accurate definitions, terminology, and documentation of the taxonomic distribution of vulval appendages are important in evaluations of hypotheses for either parallelism and developmental constraint or convergence and adaptation. KEY WORDS: Araeolaimida, Ascaridida, Capillariidae, character analysis, cuticle, Enoplida, functional morphology, lateral field, Oxyuroidea, phylogeny, Plectidae, Rhigonematida, Trichocephalida, Triplonchida, Spirurida.

Cuticular modifications of the external nematode not used randomly. The categories in this survey have vulva are frequently described in different disciplines the potential to promote the understanding of within the nematology community by using similar functional morphology across taxonomic disciplines. terms for distinctly different morphological struc- These terms can be further qualified when other tures, and very different terms for what may be studies are made to examine homology. homologous structures or analogous features with In nematodes the vulva is a cuticle-covered similar cellular-histological dynamics. Morphological opening in the hypodermis from which eggs are laid analyses may fail to resolve phylogeny through at the ventral exterior of adult females or hermaph- confusion over proper character definition, coding, rodites. The shape of the opening is often a transverse or awareness of interdependence or homoplasy slit and occasionally a longitudinal slit or symmetri- (Giribet, 2003). Character terminology can be critical cal pore. Developmental biologists observed that in for phylogenetic analysis and identification linked to the Caenorhabditis elegans model system, the vulva expert systems (Diederich et al., 2000). The need to is composed of 20 cells, epithelial cells and standardize terminology seems to be a universal associated muscles essential for egg laying (Bird concern among nematologists, as exemplified in and Bird, 1991), that connect the hypodermis with the surveys on the extensive variability in cuticle- uterus (White, 1988). However, the division of layering across diverse taxa (Blaxter and Robertson, exterior vulva and internal vagina is warranted for 1998; Decraemer et al., 2003). In vulval appendages, nematode morphology and taxonomy (Maggenti, terms are often used interchangeably, so confusion 1981). For nematodes such as Dorylaimida, it is occurs when images are not readily available. It is our especially important to more precisely define the contention that distinct structures exist that should be vulva as the outer cuticular region of the female described and generally defined so that adjectives are invagination continuous in width with the body cuticle (De Ley et al., 1993). Here, we describe the variations in terminology 3 Retired (e-mail: [email protected]), 4 Retired (e-mail: [email protected]), and and morphology of vulval appendages with a survey 5 Corresponding author of the literature and specific examples. The terms are

191 192 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009 descriptive for categories based on orientation, size, Phylogeny andtissuelocation,andtheyarenotmeanttoimply Three comprehensive alternative trees of nematodes were homology. For example, the prerectum is a repea- recently constructed with 18S small subunit (SSU) rDNA sequences of a large number of similar taxa (Holterman et tably recognizable structure with phylogenetically al., 2006; Smythe et al., 2006; Meldal et al., 2007). Trees of diverse occurrence (Carta and Osbrink, 2005), as are Holterman et al. (2006) and Meldal et al. (2007) were wings in very diverse animals. Therefore, the most chosen as character frameworks for this study based on common and stable vulval appendages defined and taxon coverage, secondary alignment strategies, Bayesian surveyed are mapped onto trees based on current tree-making methods, and better tree resolution. The topology of 1 tree was abstracted from a Bayesian Inference hypotheses of nematode phylogeny. We also have tree created with 339 taxa having 2,471 aligned SSU rDNA refined ecological associations to compare occur- positions from a ClustalW alignment manually edited in rence among species and higher taxa that may accordance with arthropod sequences aligned by secondary support useful biological predictions. Because structure (Holterman et al., 2006). The topology of a second tree was abstracted from a Bayesian Inference tree created orthologous genes can act on homologous or with 212 taxa having 1,167/1,884 aligned SSU rDNA analogous cells (Bolker and Raff, 1996) that characters created from a profile alignment built upon some generate external appendages, insight into their European RNA database sequences aligned according to phylogeny, variability, coordinated occurrence, secondary structure; this alignment was then culled of the and structure are necessary before relevant devel- most ambiguously aligned positions (Meldal et al., 2007). The Holterman tree was supplemented with information on opmental-genetic mechanisms for these natural the placement of Brevibucca Goodey, 1935 and new vulval phenotypes using a candidate gene approach sequence of membrane-bearing Cuticonema Sanwal, 1959 can be considered (Haag and True, 2001). from more ribosomal and nuclear gene characters (Kiontke et al., 2007) that demanded independent placement from the Cephalobidae, and taxonomic categories were based on pre- MATERIALS AND METHODS 2002 taxonomic groupings. The Meldal tree was labelled An extensive literature search was undertaken from with newer taxonomic categories (De Ley and Blaxter, comprehensive reviews (Chitwood and Chitwood, 1950; 2002, 2004; based in part on Blaxter et al., 1998 and Aleshin Maggenti, 1981; Malakhov, 1994) describing Adenophor- et al., 1998) and older, lower level categories as appropriate. ean nematodes (Hope, 1974; Platt and Warwick, 1983, The current trees were made with MacClade version 4.05 1988; Lorenzen, 1994; Warwick et al., 1998), animal- software (Maddison and Maddison, 1992; Sinauer Associ- parasitic nematodes (Skrjabin, 1949, 1951, 1952, 1954), ates, Inc., Sunderland, Massachusetts, U.S.A.) in which plant-parasitic and insect-parasitic nematodes (Hunt, 1993; characters were traced on 2 different tree topologies to Siddiqi, 2000), terrestrial free-living nematodes (Goodey demonstrate the distribution of 3 of the most common vulval and Goodey, 1963; Sudhaus and Fitch, 2001; Andra´ssy, appendages of membranes, flaps, and epiptygmata in higher 1983), as well as numerous individual articles on specific taxa. Life history associations were included in branches on taxa. When a statement is derived from taxa within trees to include 0, free-living; 1, invertebrate commensal (an taxonomic compendia such as those of Siddiqi or Skrjabin, important but overlooked category); 2, invertebrate parasitic; the citation is prefaced with ‘‘in.’’ The online database at 3, animal parasitic; and 4, plant parasitic. www.cabi.org, email: for Nematological and Helmintholo- gical Abstracts from PESTCABWeb, CABI Publishing, RESULTS AND DISCUSSION Wallingford, U.K., also was searched through monthly updates by DialogAlertServices@ Nomenclature anddistribution dialog.com through the National Agricultural Library, Beltsville, Maryalnd, U.S.A., for the following terms through The names ‘‘vulval membrane’’ and ‘‘vulval flap’’ September 2008: lateral vulval membrane, vulval membrane, are based on the precedent set by Siddiqi (2000), vulval fan, vulval flap, vulval dike, vulval alae, and where ‘‘membranes’’ have a primarily lateral orien- epitygma(ta). tation and ‘‘flaps’’ have a primarily parallel orienta- tion relative to the vulval slit. An early general Microscopy approach to terminology for vulval appendages was Some nematodes were processed for differential interfer- ambiguous because lateral membrane was defined as ence light microscopy as either heat-relaxed, live specimens ‘‘a cuticular flap situated on both sides of the vulval (Distolabrellus veechi Anderson, 1983) or 3.2% formalde- slit in some nematodes. See epiptygma, ‘‘vulvar flap’’ hyde-fixed specimens (Aphelenchoides sp. and an unde- scribed species of Mermithidae). Other nematodes (Dis- (Caveness, 1964). Vulval flaps were defined as tolabrellus veechi, C. elegans) were processed for low ‘‘cuticular membranes sited at both ends of the vulva. temperature scanning electron microscopy (LTSEM) in See epiptygma, lateral membrane’’ (Caveness, 1964). distilled water according to standard procedures (Wergin Another general description blurred the distinction et al., 1993; Carta et al., 2003). Photomicrographs were oriented with ventral side facing the top of the plates. Line among what we consider three separate structures of figures from previous publications were traced and modified vulval membranes, vulval flaps, and epiptygmata— or scanned from published drawings. ‘‘Vulval membranes (vulval flaps) occur in some CARTA ET AL.—NEMATODE VULVAL APPENDAGES 193 plant-parasitic Secernentea (Paratylenchus and Ho- (LM) have been described in Cuticonema vivipara plolaimus)’’ (Hirschmann, 1971). A recent compila- Sanwal, 1959 (Sanwal, 1959) (Panagrolaimoidea); tion of designations for nematode morphological Operculorhabditis longespiculosa Khera, 1969 structures for identification by expert systems (Khera, 1969) (Mesorhabditinae) (Fig. 8); and all includes various names for 3 vulval appendages: species of Caenorhabditis. They are quite large in anterior lip of vulva 5 (ventral vulval flap); Caenorhabditis sonorae Kiontke, 1997 and Caenor- epitygmata 5 epiptygmata 5 (lateral membrane); habditis drosophilae Kiontke, 1997 (Kiontke, 1997). lateral vulval flaps 5 vulval membranes 5 vulval Much smaller vulval membranes in C. elegans N2 dikes 5 vulval alae. These authors acknowledge a had visible annulations viewed with LTSEM (Fig. 9). right to be ‘‘wrong’’ about the nature and naming of a Small vulval membranes also were observed with the structure, and the continuing need to resolve SEM in Rhabditis (Oscheius) guentheri Sudhaus and contradictory character concepts for identification Hooper, 1994, described as ‘‘longitudinal cuticular and phylogeny (Diederich et al., 2000). flaps 8–10 mm long … at each end of the vulval slit,’’ Vulval appendages have been studied in some (Sudhaus and Hooper, 1994). An SEM view of a detail among animal-parasitic Strongylida. Sections related free-living rhabditid nematode, Pellioditis through the vulval region of these nematodes reveal 3 pellio (Schneider, 1866) Timm, 1960, showed no distinct types of structures (Hoberg et al., 1993b). vulval appendages (Eveland et al., 1990). Vulval The first includes consistent, symmetrical, lateral membranes also are seen in the relatively primitive cuticular ‘‘fans’’ internally supported by hypertro- Tylenchina such as in Tylodorinae (Siddiqi, 2000), phied struts, and arising from specific regions within e.g., Coslenchus Siddiqi, 1978 (Siddiqi, 1980); the lateral field. They are designated here as vulval Cephalenchus Goodey, 1962 (Golden, 1971) (Sid- membranes. The second comprise inconsistent, diqi, 2000); Pterotylenchus Siddiqi and Lenne´, 1984 irregular, asymmetrical, body wall and cuticular (Siddiqi and Lenne´, 1984); and in one species of ‘‘flaps’’ with a primary anterior to posterior orienta- insect-parasitic Deladenus Thorne, 1941 out of 15 tion parallel to the vulval slit, designated here as species in the genus (Shahina and Maqbool, 1992). vulval flaps. The third includes inconsistent, irregu- Dolichodorus longicaudatus Doucet, 1981 is illus- lar, often lateral asymmetrical cuticular ‘‘inflations’’ trated with a lateral vulval membrane (Doucet, 1981), of amorphous orientation without organized strut and South American species in the related genus support, similar to vulval membranes, and probably Belonolaimus Steiner, 1949 (syn. Ibipora), have unique to the Ostertagiinae, designated as ‘‘cuticular vulval membranes (Doucet and Filisetti, 2000; inflations’’ (Chitwood and Chitwood, 1950). Exam- Siddiqi, 2000). Vulval membranes may be found in ples of these types of appendages in other taxa are the Criconematina, in Paratylenchus Micoletzky, discussed below and illustrated (Figs. 1–19), and 1922 (Raski, 1975a, b; Dolinski et al., 1996), and some are mapped onto phylogenetic trees (Figs. 20, in Hemicriconemoides Chitwood and Birchfield, 21). 1957 as ‘‘vulval sheaths’’ (Dasgupta et al., 1969), where they are prominent in Hemicriconemoides Vulval membranes cocophilus (Loos, 1949) Chitwood and Birchfield, 1957 and Hemicriconemoides wessoni Chitwood and As used here, vulval membranes are semioval, Birchfield, 1957 (Esser and Vovlas, 1990). The approximately symmetrical, cuticular outgrowths vulval membranes of Pratylenchus roseus Zarina lateral and perpendicular to the vulval slit. Annulation and Maqbool, 1998 are unique within this genus or longitudinal indentations are visible on their edges. (Zarina and Maqbool, 1998). In the adenorphorean Vulval membranes are described (Figs. 1, 7–9) with terrestrial mermithid Tunicamermis melelonthae various terms in different taxa in plant parasites as Schuurmans-Stekhoven, Mawson, Couturier, 1963, ‘‘lateral vulval membranes,’’ ‘‘lateral vulval flaps,’’ they are described as wide lips that flank the vulva ‘‘vulval sheaths’’ or ‘‘advulval flaps’’; in bacterial- (Poinar, 1975b). feeding rhabditids as ‘‘opercula,’’ ‘‘ridge-like sculp- tures’’ or ‘‘flaps’’; in insect parasitic mermithids as ‘‘wide lips flanking the vulva’’; in animal-parasitic Vulval flaps Strongylida as ‘‘bilateral vulval fans’’ or ‘‘fin-like The word ‘‘flap’’ in the literature is the most processes’’; and in animal-parasitic Trichocephalida ambiguous term of all, because it has been used to as ‘‘lateral alate membranes.’’ Among the Rhabditida, describe what is designated here as vulval membrane, vulval membranes visible with the light microscope vulval flap, or epiptygma. As used here, vulval flaps 194 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009

Figures 1–6. Comparative structure of vulval appendages. 1. Vulval membrane of unidentified mermithid (Mermithidae). 2. Protruded vulva with unseparated lateral cuticular seam from unmated female of Distolabrellus veechi (SEM). 3. Persistent copulatory sac of mated female, D. veechi. 4. Persistent copulatory sac of Aphelenchoides sp. 5. Vulval cuticular inflation of Hyostrongylus rubidus (after Hoberg et al., 1993a). 6. Vulval flap, Ostertagia mossi (after Hoberg et al., 1993b). are mild-to-extreme modifications of the anterior and Malek, 1968); and variably sized flaps in vulval lip that show up as overhanging extensions of Bursaphelenchus Fuchs, 1937 (Nickle et al., 1981), cuticle oriented parallel and anterior to the vulval slit where they exist in approximately one third of the (Figs. 6, 10–12). Vulval flaps were described as such known species (in Yin et al., 1998; Braasch, 2001). A among the primarily plant- or insect-parasitic Ty- small vulval flap has sometimes been observed in the lenchida and Aphelenchida, including the prominent tylenchid weevil parasite Elaeolenchus partheno- structures of Rhadinaphelenchus J. B. Goodey, 1960 nema Poinar, Jackson, Bell, and Wahid, 2002 after (Nickle, 1970); a sclerotized, jointed flap in Aphe- it enters the insect but not in the infective female lenchoides vigor Thorne and Malek, 1968 (Thorne (Poinar et al., 2002). Vulval flaps are found in some CARTA ET AL.—NEMATODE VULVAL APPENDAGES 195

Figures 7–19. Comparative structure of vulval appendages. 7. Vulval membrane of Cooperia neitzi (after Hoberg et al., 1993c). 8. Vulval membrane of Operculorhabditis longespiculosa (after Khera, 1969). 9. Vulval membrane of of Caenorhabditis elegans. 10. Vulval flap of Ostertagia occidentalis (after Hoberg et al., 1993b). 11. Vulval flap of Epsilonema espeeli (after Verschelde and Vincx, 1994). 12. Vulval flap of Bursaphelenchus pinasteri (after Baujard, 1990). 13. Epiptygma of Steinernema ritteri (after De Doucet and Doucet, 1990). 14. Epiptygma of Hoplolaimus seinhorsti (after Sher, 1963). 15. Epiptygma of Morulaimus geniculatus (after Sauer, 1965). 16. Epiptygma of Deontostoma californicum (after Hope, 1974). 17. Double epiptygma of Plesiorotylenchus striaticeps (after Elekc¸iog˘lu, 2000). 18. Protruding vulvar appendage of Trichocephalus skrjabini (after Skrjabin, 1954). 19. Vulval ridges of Cervidellus baujardi (after Mounport et al., 2002). 196 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009

Figure 20. Phylogenetic trees for nematodes with vulval appendages (left, vulval membranes; center, vulval flaps; right, epiptygmata; Appendix 1) mapped onto one topology based on 18S SSU-rDNA molecular-based trees (after Holterman et al., 2006). Numbers before taxa signify presence but not necessarily predominance of the following life history characters: 0, free living; 1, invertebrate phoretic or commensal; 2, invertebrate parasitic; 3, vertebrate parasitic; and 4, plant parasitic. aquatic genera of insect parasitic Mermithidae that Travassos, 1914 (e.g., Das and Whitlock, 1960; include Hydromermis Corti, 1902; Lanceimermis Gibbons, 1979; Lichtenfels et al., 1994). Artyukhovsky, 1969 (Nickle, 1972); Limnomermis Daday, 1911; Amphimermis Kaburaki & Imamura, Epiptygmata 1932; and Diximermis Nickle, 1972 (Nickle, 1972). Epiptygmata (singular epiptygma) (Figs. 13–17) Vulval flaps also were noted in a free-living marine are cuticular protrusions on 1 or both vulval lips or nematode, Epsilonema espeeli Verschelde and Vincx, the vaginal wall (Siddiqi, 2000). The smallest 1994 (Desmodoroidea) (Verschelde and Vincx, 1994). epiptygmata seem to be strictly cuticular, but larger In plant parasites (Tylenchoidea), the vulval flap was epiptygmata as currently defined (Baujard et al., described as a ‘‘vulval sleeve’’ in Hemicycliophora 1991a, b) seem to involve cuticle and 1 or more penetrans Brzeski, 1974 (Brzeski, 1974), and ‘‘lip protruding cells. Within plant-parasitic Tylenchida, flap’’ in the Ecphyadophorids (Tylenchoidea) (Siddiqi, epiptygmata are generally small and common as in 2000), such as Ecphyadophora sheri Raski, Koshy and most Hoplolaimoidea and Merliniinae, and some Sosamma, 1982 (Raski et al., 1982). Dolichodoroidea (5 species in Belonolaimus) (Sid- Among animal parasites, vulval flaps are present in diqi, 2000). However, large double epiptygmata are some Trichostrongyloidea, such as Ostertagia oster- described in many populations of Plesiorotylenchus tagi (Stiles, 1892), in which the degree of bovine striaticeps Elekc¸iog˘lu, 2000 (Tylenchina, Tylench- immune response influences the presence, size and ida) (Elekc¸iog˘lu, 2000) and in Scutellonema cave- symmetry of the flap (Michel and Sinclair, 1969; nessi Sher, 1964 (Hoplolaimina, Tylenchina) (Bau- Michel et al., 1972). Vulval flaps also are found jard et al., 1990). These protruding lips were among other species and genera of the Ostertagiinae, described previously with an inner and outer, double including other species of Ostertagia Ransom, 1907 epiptygma composed of exocuticle and mesocuticle, (Fig. 6) and species of Spiculoptertagia (Orloff, but not endocuticle (DeGrisse and Roose, 1975). 1933), Mazamastrongylus Cameron, 1935, and Double epiptygmata also are seen in more distantly Teladorsagia Andreeva and Satubaldin, 1954 (Ho- related Steinernematida within multiple species of berg et al., 1993b). Among Haemonchinae, the Steinernema (in De Doucet and Doucet, 1990; Stock putative sister-group of Ostertagiinae (Hoberg and et al., 2001). Steinernematids were classified under Lichtenfels, 1994; Durette-Desset et al., 1999), a Rhabditida, but more recently they were placed considerable amount of body wall occurs in the flaps within Tylenchina under new infraorder Panagrolai- characteristic of Haemonchus contortus (Rudolphi, momorpha based on 18S rDNA phylogeny (De Ley 1802), H. placei (Place, 1893), and H. similis and Blaxter, 2002, 2004). CARTA ET AL.—NEMATODE VULVAL APPENDAGES 197

Figure 21. Phylogenetic trees for nematodes with vulval appendages (left, vulval membranes; center, vulval flaps; right, epiptygmata; Appendix 1) mapped onto one topology based on 18S SSU-rDNA molecular-based trees (after Meldal et al., 2007). Numbers before taxa signify presence but not necessarily predominance of the following life history characters: 0, free living; 1, invertebrate commensal; 2, invertebrate parasitic; 3, vertebrate parasitic; and 4, plant parasitic.

It is sometimes difficult to determine from population did not possess epiptygmata (Mekete et al., taxonomic descriptions whether hypertrophy of the 2008). Ambiguity also is especially apparent within body wall has occurred within overhanging, asym- structures of Hoplolaimidae and Telotylenchidae. metrical vulval lips, so the distinction between flaps and epiptygmata can be subtle. Within non-Secer- Vulval cuticular inflations nentean nematodes, the exceptional presence of a Irregular ‘‘cuticular inflations’’ (Chitwood and small epiptygma-like structure is found in Deontos- Chitwood, 1950) of variable orientation around the toma californicum Hope, 1974 (Enoplida), which has vulva exist in some individuals within species of a small vulval groove and lip (Hope, 1974). Although Hyostrongylus Hall, 1921 such as Hyostrongylus the structure is asymmetric and seals the vulval rubidus (Hassall and Stiles, 1892) Hall, 1921 (Fig. 5) opening, it would not be large or superficial enough (Hoberg et al., 1993a). They also occur in species of to be considered a flap. Epiptygmata and vulval Longistrongylus Le Roux, 1931, Mazamastrongylus, membranes may be present in the same nematode, as and Camelostrongylus mentulatus (Railliet and in Coslenchus costatus; Antarctenchus hooperi Henry, 1909) (Hoberg et al., 1993c). Within certain Siddiqi, 1980 (Siddiqi, 1980); or Rhabditis species of Spirurida, vulval inflations of the anterior (Oscheius) guentheri (Sudhaus and Hooper, 1994). lip (Digiani, 1999) or of both lips occur, but this However, epiptygmata do not seem to coexist with feature was not included in a species-level parsimony vulval flaps, at least nominally in published descrip- character analysis (Zhang et al., 2003). Cuticular tions. We also are unaware of any studies demon- inflations due to fluid expansion in the medial cuticle strating a transformation between epiptygmata and layer near the bacillary bands in capillariid whip- flaps in individual nematodes. Although small worms (Wright, 1975) are not generally associated cuticular epiptygmata are oriented parallel to the slit, with the vulva, although some of these species have like vulval flaps, their size and presence in a everted vulvae (Lanfredi et al., 1995). nematode population also may be inconsistent within a population as with projecting and nonprojecting epiptygmata in Scutellonema Andra´ssy, 1958 (Mekete Protruding vulvar appendages et al., 2008), but details of this variability were not These are nonartifactual vulval elaborations in given. The vulva, vagina, and uterus seem to be which part of the female reproductive system stretched in an individual in which an epiptygma is not evaginates, with different degrees of exposure of the projecting, so it is unclear whether a proportion of the vulva, vagina, or uterus, depending on species 198 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009

(Lanfredi et al., 1995). They may have variable loides fuelleborni kelleyi von Linstow, 1905 (species shapes, including funnel-shaped, tubular (Lanfredi et in Viney et al., 1991). This species caused potentially al., 1995), or globular (Ching, 1990). These structures fatal human infections in New Guinea and was also are known as ‘‘protruding vulvar folds’’ (Kalia distinguished from the less pathogenic Strongyloides and Gupta, 1984), or even ‘‘noncuticular, transparent fuelleborni fuelleborni von Linstow, 1905 (species in formations’’ (terminology used in Skrjabin, 1954). Viney et al., 1991) in African primates by their These structures that represent partially or fully presence. The paired pits were 90 degrees lateral to everted gonadal tissue are described in adenophorean the vulva, extending the length of 12 distorted capillariid Trichocephalida as large toroidally ‘‘pro- annules (Viney et al., 1991). Small subunit18S rDNA truding membranes’’ as in Capillaria caudinflata sequences demonstrated that the morpho-‘‘subspe- Molin, 1858 (Fig. 18) (Skrjabin, 1954). The vulvar cies’’ from New Guinea actually resided in a separate appendage in Capillaria ohbayashi Justine, 1992 was tree clade, close to Strongyloides papillosus Chang variably present and may not exist in some individuals and Graham, 1957 from various domestic animals within the species (Justine, 1992), probably due to (Dorris et al., 2002). Thus, the pits seemed to be their generation on oviposition (Lanfredi et al., 1995). convergences associated with primate hosts. Host In secernentean pinworms (Heteroxynematidae, Oxy- immunity is associated with reduced body and gonad uroidea) there are ‘‘beak-like’’ structures composed of size in Strongyloides ratti Nigon and Roman, 1952 uterus and surrounding, hypertrophied vulval lips that and damage to Strongyloides stercoralis (Bavay, protrude from the body that are not artifacts of fixation 1876) Stiles and Hassall, 1902 (Wilkes et al., 2004). (Hoberg and Pilitt, unpublished data). Another vulva-associated structure is a cuticular Highly protruding vulval lips were described in saccate protrusion that acts like a spermatophore-like proposed new genera Eminensia Mahajan, 1980 and capsule to catch sperm from males that lack spicules Evaginorhabditis Sultan, Kaul and Chhabra, 1985, (Fu¨rst von Lieven et al., 2005). This was described as a but they were more likely artifacts of fixation in a vulval cuticular sac in related Deleyia Holovachov and relatively weak morphological structure (De Ley, Bostro¨m, 2006 (Holovachov and Bostro¨m, 2006). 1995). Vulval lips also protrude normally even before egg laying in Distolabrellus veechi Anderson, 1983 Copulatory sacs or plugs (Fig. 2), but they often protrude with age in many The copulatory sac (Chitwood, 1929), more rhabditids and some tylenchids, e.g., insect-parasitic recently designated as the copulatory plug (Barker, Deladenus (Chitambar, 1991). The thickness and 1994) is a translucent to opaque protrusion around the protrusion actually seemed to be characteristic of vulva deposited by males during copulation via male species such as Aphelenchus avenae Bastian, 1865 cement glands. This plug secretion is often confused but not of the related Aphelenchus isomerus Ander- with a cuticular vulval membrane (Figs. 3, 4). son and Hooper, 1980 (Anderson and Hooper, 1980). However, upon more extensive survey, multiple sacs Unusual evaginated uteri, in which vulval and body may be found (Chitwood, 1929); the margins are cuticle were left behind, were found in secernentean often irregular (Gerber and Giblin-Davis, 1990); no tylenchid Sphaerularioidea (Siddiqi, 2000) and would cuticular markings are evident; and the sac is sticky to be distinct from all structures mentioned above. bacteria, sperm, or a needle. Spicules and eggs can move through an obscure canal or punctum within the Miscellaneous cuticular vulval appendages sac (Chitwood, 1929). The sacs were observed In females of Cervidellus baujardi Mounport, without bacteria and compared with unmated females Bostrom and Villenave, 2002 (Rhabditida: Cephalo- with many bacteria around the vulva. The caudal alae bidae), protruding tessellate ridges along the body of Pelodera strongyloides (Schneider, 1860) Schnei- had a concave modification perpendicular to each der, 1866 also shaped the final form of the plug side of the vulval slit for the length of 16 annules to (Wagner and Seitz, 1983). The ability of P. form a lateral cuticular ridge. This delimited a strongyloides to dissolve a tunnel within the sac to spindle-shaped window above a sunken vulva region reinseminate the female (Chitwood, 1929) led to (Fig. 19). This structure was unique among species in speculation that the male may have release glands the genus (Mounport et al., 2002) and is here denoted allowing separation (Sarr et al., 1987). Plugs were a ‘‘lateral vulval ridge.’’ variably present in some strains of C. elegans Another peri-vulval cuticle modification of villi- (Maupas, 1900) Dougherty, 1953 (Barker, 1994) filled pits occurred in parasitic females of Strongy- and a few other rhabditids, but they may not be as CARTA ET AL.—NEMATODE VULVAL APPENDAGES 199 obvious at the LM level as in the larger genera. A ette-Desset et al., 1999). Vulval flaps are variable yellow liquid accumulates in the seminal vesicle of among haemonchines and considered unreliable for mating males and is secreted after insemination separating species in Haemonchus Cobb, 1898 and (Barker, 1994). Copulatory sacs may be viewed with Ashworthius Le Roux, 1933 (e.g., Lichtenfels et al., various stains such as those used for plant-parasitic 1994; Dro´z_dz_ et al., 1998), or among the diversity of nematode egg matrices (Southey, 1986). Persistent ostertagiine nematodes in which they are observed. copulatory sacs are prominent in rhabditids Terator- Similarly, vulval membrane-like structures described habditis (Osche, 1952) Dougherty, 1955 (Gerber and as cuticular inflations also are unreliable in the Giblin-Davis, 1990), Cruznema Artigas, 1927 (An- species Hyostrongylus rubidus (Fig. 5) and Hyo- dra´ssy, 1983), Distolabrellus (Anderson, 1983), strongylus kigeziensis Durette-Desset, Ashford, Bu- Pelodera Schneider, 1866, Cephalobus Bastian, tynski, and Reid, 1992 (Hoberg et al., 1993a). Vulval 1865 (Chitwood, 1929), Acrobeloides (Cobb, 1924) flaps and vulval membrane-cuticular inflations are (Jairajpuri and Azmi, 1977), Acrobeles von Linstow, occasionally characteristic of these species, often in 1877 (Steiner, 1929), and in the female generation of different proportions within populations (Gibbons, Heterorhabditis Poinar, 1975 (Poinar, 1975a). Such 1987), but they are not reliable characters. Not only structures, occurring as amorphous, irregular, darkly are the vulval flaps variable in Haemonchus, and the pigmented accumulations capping the vulva also are ostertagiines Hyostrongylus, Ostertagia, Spiculopter- noted among some Strongylida, including species of agia, and Teladorsagia, they are generally variable in Oesophagostomum Molin, 1861. Copulatory sacs occurrence within genera and among conspecifics also have been noted (Sarr et al., 1987) in plant- across the Ostertagiinae. Thus, they are considered parasitic Scutellonema cavenessi Sher, 1964 (De- unreliable even for species-level identification. How- meure et al., 1980), and the marine nematode ever, their variable phenotypes have been used to Desmodora schulzi Vincx, 1983 (Vincx, 1983). characterize ecotype populations that may represent The ambiguity of cuticular and secreted vulval incipient species (Whitlock and Le Jambre, 1981). appendages is partly due to the often-symmetric shape Vulval membranes seem to be consistently present and refractive edge of the copulatory sac. The in a few species within the animal-parasitic Cooper- confusion also may be due to the close phylogenetic iinae (Strongylida), as well as some primitive plant- relationship of nematodes with both structures. One parasitic species within the Tylenchida. Vulval putative vulval membrane of the monotypic genus membranes (described as ‘‘bilateral vulval fans’’) Operculorhabditis Khera, 1969 (Rhabditida) that is are consistent features easily seen with the LM in the briefly described in the literature (Khera, 1969) can be related animal parasites Parostertagia heterospicu- confused even by seasoned taxonomists with the lum Schwartz and Alicata, 1933; Cooperia neitzi copulatory sac of the rhabditids mentioned above Mo¨nnig, 1932 (Fig. 7); Cooperia verrucosa Mo¨nnig, (Fig. 8). Similarly, within the Aphelenchina, in which 1932; and Cooperia okapi Leiper, 1935 (Hoberg et true vulval flaps also occur, an Aphelenchoides Fischer, al., 1993c); and in bacterial-feeding facultative or 1894 species has a copulatory sac similar in appearance phoretic parasites Rhabditis (Choriorhabditis) dubia to a vulval membrane except for the punctum and lack Bovien, 1937 (Osche, 1952) (Sudhaus and Ku¨hne, of annulation (Fig. 4). Copulatory sacs or plugs are 1989) and Rhabditis (Caenorhabditis) sonorae (Sud- common within some females of Rhigonematida in haus and Kiontke, 1996). In Rhabditis (Oscheius) which they may anchor ephemeral spermatophores guentheri (Sudhaus and Hooper, 1994) and Caeno- from the male and exclude other males. Vulval flaps in rhabditis elegans, they are only easily seen with the some species seemed to aid in the protective function SEM. However, in the monotypic genus Operculo- proposed for the plugs (Hunt, 2001). rhabditis Khera, 1969 membranes are described as deciduous, an exceptional condition for cuticular Phylogenetic utility membranes in the literature. These may not be Due to morphological and nomenclatural variabil- cuticular structures, because annular incisures are ity, vulval appendages must be described carefully in not included on the drawing. In addition, variability nematode taxonomy. Among Trichostrongylidae, among the 7 collected females is unknown because occurrence of flaps exhibits homoplasy with some original material was not available, and the species representation among distantly related Cooperiinae has not been found again. In face view, there also and in the putative sister-groups Ostertagiinae + seems to be an exceptional ventral extension Haemonchinae (Hoberg and Lichtenfels, 1994; Dur- connecting the ‘‘membranes’’ (Khera, 1969) that 200 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009 could be persistent copulatory sacs. Sometimes 60 species (Ebsary, 1991); and 1 Cuticonema deciduous copulatory sacs are present in the mor- (Rhabditida) among the entire Panagrolaimoidea phologically similar genus Teratorhabditis. (Sanwal, 1959). The relatively shallow branching Although the presence of an epiptygma-like vuval pattern of vulval appendages also was also seen in flap in Epsilonema espeeli Verschelde and Vincx, 1994 recent species—and generic—level phylogenies. (Chromadorida) is constant, the shape is not Among species of Steinernema Travassos, 1927, (Verschelde and Vincx, 1994). Epiptygmata are fairly epiptygmata arose at least 4 times (Stock et al., common and reliable in some groups of nematodes 2001). Vulval membranes arose in Rhabditidae at with a central vulva and two-armed gonads such as in least 3 times (Sudhaus and Kiontke, 1996). the Dolichodoroidea, Hoplolaimidae, Psilenchidae (Siddiqui, 1986), and Steinernematida (De Doucet Cuticle ridges and lateral field and Doucet, 1990). However, the dolichodoroid Well-developed vulval appendages in some species nematode Melinius adakensis Bernard, 1984 (Teloty- may sometimes be associated with a recognizable lenchidae) has considerable shape variation of epip- pattern of cuticular features such as lateral field tygma either on the dorsal or ventral lip (Bernard, incisures or lateral ridges. Among the 28 species of 1984). In Dolichodoroidea such as Ibipora Monteiro plant-parasitic and insect-phoretic Bursaphelenchus and Lordello, 1977 (Belonolaimidae), epiptygma (Aphelenchida) in European conifers, 4 groups are coexist with vulval membranes (in Siddiqi, 2000). In distinguished in part by 2 to 4, or 6 lateral incisures Rhabditidae (Sudhaus and Hooper, 1994) epiptygma associated with presence and size of vulval flaps. The are less common, but here they also may coexist with largest flaps occur in taxa having 3 lateral lines membranes. However, it was noted that epiptygmata on (Braasch, 2001). The 6 genera within plant-parasitic the posterior lip (exceptionally on the anterior lip) of Merliniinae (Tylenchina) have 6 lateral incisures and Rhabditis (Oscheius) guentheri were not apparent in epiptygmata, but epiptygmata are generally lacking in live, but only fixed material (Sudhaus and Hooper, related genera with fewer incisures (in Siddiqi, 2000). 1994). This observation also might apply to some live Among the other genera of Tylenchorhynchinae with parasitic nematodes for which observations and prominent longitudinal lines and ridges, Mulkor- measurements are often not made until material is fixed. hynchus Jairajpuri, 1988 (syn. Dolichorhynchus Mulk and Jairapuri, 1974) has vulval membranes Phylogenetic patterns of individual and and 4 incisures, but there are no membranes in related combinedcharacters Prodolichorhynchus Jairajpuri, 1985 with 2 incisures Associations of vulval appendages of different (in Jairajpuri and Hunt, 1984). nematode taxa can be visualized through the mapping Cuticular ridges are strongly associated with of characters on phylogenetic trees as depicted in appendages in some taxa, in which case appendages Figs. 20 and 21. It was not appropriate to use the may be inversely associated with lateral field ‘‘concentrated changes’’ test for character correlation incisures. Eutylenchus Cobb, 1913 (Tylenchina) has of MacClade because this phylogeny was not fully vulval membranes, with lateral, mid-dorsal, and mid- resolved to reflect the frequent character reversals at ventral ridges and no lateral incisures (Brzeski, the species level. These trees are descriptive; the true 1996), but related Atylenchus Cobb, 1913 lacks incidence of any character correlations may be vulval appendages and ridges but has lateral incisures possible in the future when more detailed phylogenies (Siddiqi, 2000). In South American species of become available among all the relevant taxa. Belonolaimus (syn. Ibipora, Tylenchina), 6 incisures There is a unique acquisition of vulval membranes occur with lateral membranes and small epiptygmata, occurring 5 (Fig. 20) to 7 (Fig. 21) times in the listed whereas in species lacking membranes but having higher level taxonomic categories, of vulval flaps small epiptygmata, only 1 incisure exists (Siddiqi, occurring 5 (Fig. 20) to 6 (21) times, and epiptygmata 2000). Within a recent phylogeny of Caenorhabditis, 5 times (Figs. 20, 21). These structures occur most Caenorhabditis sonorae Kiontke, 1997 had a smooth often in terminal taxa where phylogenetic resolution is lateral field without incisures and moderately devel- generally not available. Examples of exceptional oped vulval membranes (Kiontke, 1997), both of vulval membranes include 1 insect-parasitic tylenchid which were unusual in this genus (Sudhaus and Deladenus species among 15 (Shahina and Maqbool, Kiontke, 1996). In trichostrongylid animal parasites, 1992); 1 plant-parasitic Pratylenchus Filipjev, 1936 there are more midbody synlophe ridges and larger (Zarina and Maqbool, 1998) species among more than vulval membranes in Cooperia nietzi and C. CARTA ET AL.—NEMATODE VULVAL APPENDAGES 201 verrucosa than in C. okapi (Hoberg et al., 1993c). In dent characters is not yet available to assess possible distantly related trichostrongylid stomach worms vulval membrane reversals within this group. (Haemonchinae) possessing large, irregular flaps, For most other taxa, vulval position and appendage Haemonchus similis has 4 more nonlateral ridges at presence is not so closely linked, especially at higher midbody that extend more posteriorly than in related taxonomic levels. Although Cuticonema (Panagrolai- H. placei and H. contortus. These more extensive midae) and many primitive Tylenchina with vulval ridges are associated with a more complex vulval membranes have posterior vulval positions, more structure in which the vulval slit actually resides phylogenetically derived species in Antarctenchus within the flap. In contrast, H. placei and H. (Psilenchidae), Neodolichorhynchus (Mulkorhynchus), contortus have less elaborate flap lobes anterior to and Scutylenchus (Dolichodoridae, Tylenchorhynchi- the vulval slit, and synlophe ridges do not extend dae) have vulval membranes with approximately very far posteriad from the cervical zone (Lichtenfels medial vulvas (in Siddiqi, 2000). Vulval membranes et al., 1994). Variation among haemonchines is in animal-parasitic Trichostrongyloidea are associated apparent, however, because both Haemonchus horaki with posterior vulvas (in Skrjabin, 1952), whereas in Lichtenfels, Gibbons and Boomker, 2001 and related insect-associated Rhabditida, the 4 species with Ashworthius patriciapilittae Hoberg, Abrams, Car- cuticular membranes have medial vulvas. Vulvae are reno and Lichtenfels, 2002 have a synlophe extend- often anteriorly positioned in parasitic Ascarida, ing to near the caudal extremity, and relatively simple Spirurida, and Bunostominae (Strongylida, Ancylos- vulval flaps (Lichtenfels et al., 2001; Hoberg et al., tomoidea) that consistently lack vulval cuticular 2002). The exceptional vulval membranes in Aulo- appendages. In animal-parasitic Oxyuroidea (Oxyur- laimus (Chromadorida) are associated with a fairly ata) in which vulvae may reside in any region of the high number of longitudinal ridges within the genus body, the single instance of a vulval flap was on a (38 within a range of 12–50) (in Shahina et al., 1996). posterior vulva (Skrjabin, 1951). In other oxyurids as These associated ridge, lateral field, and appendage well as capillariids with protruding vulval folds, the features seem to indicate some type of structural vulva is in the anterior part of the body (in Skrjabin, coordination within all these diverse taxa. 1954). Where epitygmata exist in Tylenchida and Besides well-developed cuticular longitudinal ridg- Rhigonematida, the vulva is slightly posterior to es, a tendency toward cuticle loosening is notable midbody. Although animal parasitic Strongyloides especially among plant-parasitic Tylenchidae, Crico- stercoralis (Bavay, 1876) Stiles and Hassall, 1902 nematidae, and Trichodoridae that possess vulval does not possess the primary vulval appendages, the membranes. In Hemicriconemoides, 7/46 species have peri-vulval pits associated with the parasitic female are membranes (designated ‘‘lateral cuticular flaps’’ in associated with a more posterior vulva than the free- Siddiqi, 2000), and a sheath composed of a second living form (Speare, 1989). cuticular layer; both males and females have lateral fields (Esser and Vovlas, 1990). However, there are no Parasitism andecology membranes in related species within the Criconemati- Based on mapping of parasitism onto the small nae and Macroposthoniinae in which females lack subunit (18S) rDNA phylogenetic tree for nematodes, lateral fields and sheaths (Brzeski et al., 2002a, b). associations with plants arose independently at least 3 times, and animal parasitism arose at least 6 times Vulva position (Dorris et al., 1999). When vulval appendages are Within plant-parasitic Paratylenchus Micoletzky, superimposed onto updated trees of related taxa 1922, the position of the vulva is associated with the (Fig. 20, 21 based on Holterman et al., 2006), they degree of development of vulval membranes. A survey seem closely associated with parasitism, phoresis, or (Appendix 2) based on a comprehensive compendium commensalism. In some cases, it has not been clearly of species (Esser, 1992) showed 64/71 Paratylenchus established that vulval appendages are consistent species with vulval membranes, and 23/41 of related features of a species because of limited sampling and Gracilacus Raski, 1962 species with membranes. inability to place specimens in culture. Because some Species that lacked membranes with morphometric vulval appendages represent inconsistent responses to a values for vulval distance from anterior body lengths of host immune challenge, and most are species-level Vulva% less than 80%, and those with ranges more phenomena of parasites, host factors are hypothetical than 80% had well developed appendages. A compre- inducers and selectors of those characters that do hensive phylogeny of these genera based on indepen- become fixed. Environmental estrogens in particular 202 COMPARATIVE PARASITOLOGY, 76(2), JULY 2009 may be involved in fixation of epigenetic characters in (Kolotuev and Podbilwicz, 2004). With this back- vertebrates (Guerrero-Bosagna et al., 2005) and also ground, relevant developmental, behavioral, and may be important in invertebrates. Both Caenorhabdi- ecological factors might be evaluated in taxa with tis and Panagrellus have estrogen receptors (Hood et vulval appendages for testing hypotheses. al., 2000). Although bacterial-feeding Rhabditida are not strict parasites, nearly all have dauer larvae, and like other invertebrate symbionts ‘‘they face the same ACKNOWLEDGMENTS transmission problems and other pressures as those We thank Donna Ellington and Sharon Ochs of the acting on parasites’’ (Poulin, 1998). In Rhabditida, the Nematology Laboratory, and Christopher Pooley, greatest development of vulval membranes is seen in 2 Soybean Genomics and Improvement Lab, USDA– species with a relatively unique preparasitic adaptation: ARS for technical help. Special thanks are extended to Caenorhabditis drosophilae and Choriorhabditis du- Maureen Barr and Paul Sternberg for sharing unpub- bia (Rhabditida, Rhabditoidea) have dauer larvae lished visual information on C. elegans. Mention of requiring fly contact to mature (Kiontke, 1997). These trade names or commercial products in this publication prominent membranes may have evolved in response is solely for the purpose of providing specific to their phoretic associate/facultative host. Host factors information and does not imply recommendation or modulate the degree of flap size in Ostertagia ostertagi endorsement by the U.S. Department of Agriculture. (Rhabditida, Trichostrongylidae) (Michel et al., 1972). The capacity of nematode cuticle to expand between LITERATURE CITED molts (Lee, 2002), in contrast to the rigid cuticle and Aleshin, V. V., O. S. Kedrova, I. A. Milyutina, N. S. saltational growth of insects (Knight et al., 2002) is Vladychenskaya, and N. B. Petrov. 1998. Relation- well suited to localized expansion in an immune ships among nematodes based on the analysis of 18S response. Nematode cuticle also facilitates parasitism rRNA gene sequences: molecular evidence for mono- in its ability to shed and quickly repair the surface coat phyly of chromadorian and secernentean nematodes. Russian Journal of Nematology 6:175–184. after immune assault (Blaxter et al., 1992). Alexopoulos, C. J., C. W. Mims, and M. Blackwell. 1996. Caenorhabditis species with the largest vulval Introductory Mycology, 4th ed. Wiley, New York. membranes have a phoretic association with insects, 868 pp. and those with the smallest are associated with mollusks Anderson, R. V. 1983. 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Copulatory plugs and paternity assurance in the nematode Caenorhabditis elegans. Interference with thigmotropism, or contact-guidance, Animal Behavior 48:147–156. of fungi toward host surface vulnerabilities (Gow, 2004) Baujard, P. 1990. Trois nouvelles espe`ces de Bursaphe- might be one benefit of these appendages toward lenchus (Nematoda: Tylenchida) et remarques sur le competing fungi in the environment. genre (Nemata: Hoplolaimidae) Revue de Ne´matologie 3:167–177. Definitions for specific characters and the taxo- Baujard, P., P. Castillo, M. Doucet, B. Martiny, D. nomic distribution of vulval appendages could be Mounport, and A. Ndiaye. 1991a. Variabilite´ intra- et useful for testing hypotheses of parallelism reflecting interspe´cifique des structures cuticulaires externes dans developmental constraint, or convergence resulting le genre Aorolaimus Sher, 1963 (Nemata: Hoplolaimi- dae). Systematic Parasitology 19:195–213. from adaptation (Yoon and Baum, 2004) such as Baujard, P., D. Mounport, and B. Martiny. 1991b. might occur in nematode–microorganism, host– Study of external cuticular structures in a population parasite, or male mating interactions. Details of the of the nematode Hoplolaimus seinhorsti (Nemata: genetic and histological basis of vulval morphogen- Hoplolaimidae). Afro-Asian Journal of Nematology 1:19–22. esis continue to accumulate in membrane-bearing C. Baujard, P., D. Mounport, B. Martiny, and A. Ndiaye. elegans (Sharma-Kishore et al., 1999; Dalpe´ et al., 1990. Observations en microscopie e´lectronique a` 2005) and appendage-free Pristionchus pacificus balayage sur deux espe`ces du genre Scutellonema CARTA ET AL.—NEMATODE VULVAL APPENDAGES 203

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Helicotylenchus limatus Siddiqi, 1995 (Paraty- 1919) J. B. Goodey, 1960 (Hunt, 1993), Seinura lenchus spp., Hemicriconemoides spp., Pratylenchus (many) (Massey, 1974) roseus Zarina and Maqbool, 1998 Steinernematida,e:Steinernema (some) f: Ecphyadophora espeeli; Elaeolenchus parthe- Rhabditida-Panagrolaimidae,m:Cuticonema nonema Poinar, Jackson, Bell and Wahid, 2002 vivipara Sanwal, 1959 e: small - Histotylenchus histoides Siddiqi, 1971, Rhabditida-Rhabditoidea,m:smallinOscheius Morulaimus arenicolus Sauer, 1966; Neodolicho- guentheri, Caenorhabditis elegans,largerinCae- dorus obtusus (Allen, 1957) Andrassy, 1976; Ty- norhabditis sonore, very large in C. drosophilae lenchorhynchus tuberosus Zarina and Maqbool, and Choriorhabditis dubia Bovien, 1937 and 1994; Merlinius spp., Scutylenchus fici; large - perhaps Operculorhabditis longispiculosa Khera, Hoplolaimus seinhorsti Sher, 1963; some Peltami- 1969 gratus spp. and Scutellonema spp., Plesiorotylenchus Strongylida.f:Ostertagia, Haemonchus, Para- striaticeps Elekc¸iog˘lu, 2000 cooperia spp.; Inflation (irregular): Longiostrongylus, Aphelenchida, Aphelenchoidoidea, f-e: Aphe- Mazamastrongylus, Camelostrongylus m: Cooperia lenchoides vigor Thorne & Malek, 1968, Bursaphe- spp. (3 in Hoberg et al., 1993), Allintoshius nycticeius lenchus (one third of species), Laimaphelenchus Chitwood, 1937 (many), Rhadinaphelenchus cocophilus (Cobb, Paratylenchus CARTA ET AL.—NEMATODE VULVAL APPENDAGES 209

APPENDIX 2 Membrane size and vulval position after Esser (1992), p. 12

Paratylenchus (Paratylenchus)V%

Anterior, no membrane P.(P). aciculus Brown 1959 68–74 P.(P). acti Eroshenko, 1978 69–73 P.(P). aculentus Brown 1959 71–73 P.(P). costatus (Raski, 1976) Siddiqi, 1986 68–72 P.(P). laocaiensis Nguyen et al., 2004 65–67 P.(P). minisculus Tarjan, 1960 69–81 P.(P). macrodorus Brzeski, 1963 74–81 P.(P). pandatus Raski, 1976 70–78 P.(P). steineri Golden, 1961 75–78 Intermediate, small membranes P.(P). corbetti (Ganguly and Khan, 1990) Brzeski, 1998 75–81 m not prominent P.(P). discocephalus Siddiqi, Khan and Ganguly, 1990 73–80 m indistinct Posterior, large membranes P.(P). arculatus Luc and de Guiran, 1962 78–84 P.(P). dianthus Jenkins and Taylor, 1956 80–88 m large, 4–6 annules P.(P). elachistus Steiner, 1949 82–87 P.(P). flectospiculus Huang and Raski, 1987 81–86 P.(P). holdemani Raski, 1975 84–86 P.(P). microdorus Andra´ssy, 1959 79–86 P.(P). perlatus Raski, 1975 79–86 P.(P.) projectus Jenkins, 1956 83–87 Paratylenchus (Gracilacus) Anterior P.(G). capitatus (Adams and Eichenmuller, 1962) Siddiqi and Goodey, 1964 P.(G). costata Raski, 1986 67–72 P.(G). elegans Raski, 1962 70–74 P.(G). idalima Raski, 1962 71–76 P.(G). latescens Huang and Raski, 1986 68–73 P.(G). marylandica (Jenkins, 1960) Raski, 1962 71–79 P.(G). oostenbrinki Misra and Edwards, 1971 70–73 P.(G). raskii Phukan and Sanwal, 1979 70–74 Posterior P.(G). epacris Raski, 1962 82–87 P.(G). mirus Raski, 1962 80–86 P.(G). robusta (Wu, 1974) Raski, 1976 81–85