Research and Reviews ill Parasitology, 55 (4): 209 215 (1995) 1995 Asociaci6ndeParasit6logos Espaiioles (A.P.E.) PublishedbyA.P.E. Printed in Barcelona. Spain TWO NEW SPECIES OF THE GENUS OSWALDOCRUZIA TRAVASSOS, 1917 (NEMATODA: TRICHOSTRONGYLINA: MOLINEOIDEA) PARASITIZING SPANISH AMPHIBIANS B. BEN SLIMANE I, J. LLUCH 2 & M.C. DURETTE-DESSET' 'Laboratoire de Protozoologie et Parasitologie Comparee E.P.H.E. and Laboratoire de Biologie Parasitaire Protistologie Helminthologie, URA No. 114, Museum National d'histoire Naturelle, 6/ rue Buffon, 75231 Paris Cedex 05, France 2Departament de Biologia Animal, Facultat de Ciencies Biologiques, Universitat de Yalencia, Dr. Moliner 50,46100 Burjassot= Valencia, Spain Received27 June 1995; accepted21 September1995 REFERENCE:BENSLlMANE(B.), LLUCH(J.) & DURETTE-DESSET (M.C.), 1995.- Two new species of the genus Oswaldocrutia Travassos, 1917 (Nematoda: Trichostrongylina: Molineoidea) parasitizing Spanish amphibians. Research and Reviews in Parasitology, SS (4): 209-21 S. AIlSTRACT: In a collection of Oswaldocruzla species parasitizing amphibians from Selva de Oza in Spain, three species were identified: O. guye tanti Ben Slimane, Durette-Desset et Chabaud (1993) and O. hispanica n. sp. coparasites of Rana temporaria, and O. galeanoae n. sp. parasite of Bufo bufo. Up to the present, O. guyetanti was only known in Besancon, France. 0. hispanica n. sp. and O. galeanoae n. sp. are closely related to O. filifonnis (Goeze, 1782). O. hispanica n. sp. is differentiated by cervical alae composed of two latero-ventral ridges but not separated by a small one and by the absence of chitinous reinforcement in all the ridges. O. galeanoae n. sp. is differentiated by the absence of cervical alae, by interrupted ridges all along the body and by the distal processes of the spicular blade. The species of Oswaldocruzia parasitizing amphibians from Western Europe are closely related, but presenting many differentiations concerning the tips of the spicules and mainly the synlophe in the oesophageal region, as well as also the cervical alae. KEYWORDS: ematoda, Molineoidea, Oswaldocrutia hispanica n. sp., Oswaldocruzia galeanoae n. sp., amphibians, Spain. INTRODUCTIO The French fauna of Oswaldocruzia species parasitizing amphibians has been recently found to be more diversified than previously supposed. Three species have been identified (BE SUMANE, DURETTE-DESSET & CHABAUD, 1993): O. filifonnis (Goeze, 1782), O. duboisi Ben Slimane, Durette-Desset et Chabaud, 1993 and O. guyetanti Ben Slimane, Durette-Desset et Chabaud, 1993, Another species, 0, bialata (Molin, 1861) is known farther East, in Bulgaria, Among the genus Oswaldocruzia, the geographic distribution appears to be more determinant than parasite specificity. For these reasons, it seemed worthwhile to look for possible speciations in the Iberian fauna, MATERIAL AND METHODS The material was stored in 70 ethanol, and deposited in the Helminthological Collections of the Museum National d'histoire Naturelle de Paris (M..H.N). The study of the synlophe is based on the method of DURETTE- DESSET(1985), the nomenclature of the synlophe in the oesophageal region follows BE SLlMA E, DURETTE-DESSET& CHABAUD (1993), that of the caudal bursa follows DURETTE-DESSET& CHA- BAUD(1981), and the arrangement of rays 6 and 8, that of Du- RETTE-DESSET,ASHER& BENSUMANE(1992). RESULTS Oswaldocruzia guyetanti Ben Slimane, Durette-Desset et Chabaud, 1993 Material studied: 3 males, 3 females M.N.H.N 728 MDc; coparasite of Oswaldocruzia hispanica n. sp. Host: Rana temporaria Linnaeus, 1758 (Amphibia: Ranidae). Site: small intestine. Locality: Selva de Oza, Spain. Comments The Spanish specimens possess the main characteristics of O. guyetanti, a species known so far only in Rana sp. from France, particularly: A) concerning the synlophe: the shape of the cervical alae and the absence of ventral ridges at the oesophageal region; B) concerning the caudal bursa: the arrangement of rays 6 and 8 (type IT); C) concerning the spicules: the presence of extra branches on the fork and the shoe (Fig. 1). Therefore some differences can be noted in the number of cuticular ridges in males (65 in France and 46 in Spain), although it appears to be the same number in females (65). Contrary to our knowledge on the majority of the Trichostrongylina, this character can be variable at the intraspecific level in the Oswaldocruzia group (see BEN SUMANE & DURETTE-DESSET, 1993). We consider that the Spanish specimens can be identified as Oswaldocruzia guyetanti. Oswaldocruzia hispanica n. sp. Type-material: holotype male, allotype female M.N.H.N 728 MDa; 2 males, I female M..H.N 728 MDb; eoparasite of O. guyetanti Ben Slimane, Durette-Desset et Chabaud, 1993. Host: Rana temporaria Linnaeus, 1758 (Amphibia: Ranidae).
210 B. BE SUMANE. J. LLUCH & M.C. DURETTE-DESSET d. v.,'"... ~~.<.. ::." ~~.'.- b Fig. 1.- Oswaldocruria guyetanti Ben Slimane, Durette-Desset et Chabaud, 1993: A-F) synlophe in transverse section (A: male, at cervical alae level; B: female, at cervical alae level; C: male, at the oesophago-intestinal junction; D: female, at the oesophago-intestinal junction; E: male, at mid-body; F: female, at mid-body; G-I) male, left dissect spicule (G: extemo-lateral view; H: ventral view; I: dorsal view); J) male, caudal bursa, ventral view. All sections are oriented as in Fig.IB. Scale bars: A-D: 30 urn; E, F: 40 urn; G-I: 50 urn; J: 60 urn. Abbreviations: d.edorsal; v.=ventral; r=right; l=left.
Oswaldocruzia spp. in Spanish amphibians 211 Site: small intestine. Locality: Selva de Oza, Spain. Description Nematodes coiled only in the anterior part (with one spiral); excretory pore situated within the posterior third of the oesophagus; triangular cervical papillae posterior to excretory pore; well developed excretory glands; cervical alae present, lacking chitinous reinforcements (Fig. 2C, D). Cephalic vesicle and dorsal eesophageal tooth present in the head. En face view: buccal aperture triangular with 6 externo-iabial papillae, 4 cephalic papillae and 2 amphids (Fig. 2B). The synlophe has been studied in one male and one female paratypes. In both sexes, the cuticle bears longitudinal ridges uninterrupted along whole length of body. The 78% of ridges in male and 66% in female appear in the oesophageal region. Ridges disappear just anterior to caudal bursa in male and at phasmid level in female. In male, 47 ridges (25 dorsal, 16 ventral, 1 opposite each lateral cord and 2 cervical alae, each composed of 2 latero-ventral ridges more developed than the others) at 250 urn from apex (Fig. 2D). This level corresponds to the widest part of the cervical alae; 51 (25 dorsal, 20 ventral, 1 opposite each lateral cord and 2 cervical alae) at oesophago-intestinal junction (Fig. 2E), 64 (31 dorsal, 31 ventral and 1 opposite each lateral cord) in mid-body (Fig.2F). In female, 37 ridges (20 dorsal, 1I ventral, 1 opposite each lateral cord and 2 cervical alae) at the widest part of cervical alae, 60 (30 dorsal, 28 ventral, 1 opposite each lateral cord) in mid-body (Fig. 2G). Cervical alae appear about 30 urn behind cephalic vesicle. They are 490 urn long and 9 urn wide in male and 360 um long and 11,5 urn wide in female. In transverse section, ridges are orientated perpendicularly to body surface, regularly spaced and same sized, except the more developed cervical alae. Holotype-male: 4800 urn long and 85 urn wide at midbody; cephalic vesicle 70 urn long and 40 urn wide. Nerve ring, excretory pore and cervical papillae 145 urn, 225 f.u11 and 250 urn from apex, respectively. Oesophagus 370 urn long. Caudal bursa with 2-3 pattern which tends towards 2-1-2, i. e. tips of rays 4 being directed towards anterior of body nearer those of rays 3 than rays 5. Rays 8 arising on root of dorsal ray and overlapped by rays 6 along their median part (type 11).Rays 9 arising on dorsal ray before division of the latter into two branches of which the internal ones are longest (Fig. 2M). Gubernaculum absent, genital cone 20 urn long and 20 urn wide at its proximal part, bearing large zero papilla on anterior lip and two minute papillae 7 on posterior one. Spicules 230 urn long, divided into three main branches: externo-iateral branch or blade, distally divided into 2 processes (Fig. 2K); interno-ventral branch or fork, distally divided at 36% of whole length of spicule (Fig. 21) and interno-dorsal branch or shoe, bearing needle-like process, 56 urn long (Fig. 2L). Allotype-female: 6600 urn long and 130 urn wide at mid-body; cephalic vesicle 60 urn long by 40 urn wide. Nerve ring, excretory pore and cervical papillae 200 urn, 220 urn and 240 urn from apex, respectively. Oesophagus 410 urn long (Fig. 2A). Didelphic. Vulva 2500 urn from caudal extremity. Vagina vera 30 urn dividing vestibule 330 urn long into two equivalent parts. Sphincters both 30 urn long and infundibula both 30 urn long (Fig. 2H). Anterior uterine branch 1200 urn long with 6 eggs; posterior uterine branch 1200 um long with 5 eggs; all eggs at embryonated stage 100 urn long and 50 urn wide. Tail 180 urn long and 50 urn wide at level of anus, with caudal spine 16 urn (Fig. 21). Discussion This species belongs to the Oswaldocruzia species group of the Palaearctic area, which are characterized by the division of the spicular fork above the distal third of the spicule, and by the presence of a ridge opposite each lateral cord. Because of the presence of the cervical alae and the caudal bursa type 11,the Spanish species is close to the following Western European species: O. duboisi Ben Slimane, Durette-Desset et Chabaud,1993, parasite of Rana spp. and Triturus vulgaris; O. guyetanti parasite of Rana spp. and Bufo bufo and O. filiformis (Goeze, 1782) parasite of Bufo bufo and Ranidae. O. filiformis is the most closely related species, with a spicular blade divided into 2-3 processes at its extremity and the cervical alae composed of two latero-ventral ridges separated by a small ridge. This ridge is lacking in the above specimens and there is no chitinous reinforcement of the alae and the ridges. We consider the specimens from Rana temporaria to belong to a new species, Oswaldocruzia hispanica n. sp. Oswaldocruzia galeanoae n. sp. Type-material: holotype male, allotype female M.N.H.N 727 MDa; 2 females M.N.H.N 727 MDb. Host: Bufo bufo (Linnaeus, 1758) (Amphibia: Bufonidae). Site: small intestine. Locality: Selva de Oza, Spain. Description Straight nematodes, excretory pore situated near the end of oesophagus; minute triangular-shaped cervical papillae at same level as excretory pore; well-developed excretory glands; cervical alae absent. Cephalic vesicle and dorsal oesophageal tooth present in the head (Fig. 3B). En face view: buccal aperture
212 B. BE SUMANE, J. LLUCH & M.C. DURETTE-OESSET K Fig. 2.- Oswakiocruzia hispanica n. sp.: A) female, anterior extremity, left lateral view; B) female, head, apical view; C) female, excretory pore and cervical papillae, ventral view; O-G) synlophe in transverse section (0: male, at cervical alae level; E: male, at oesophagointestinal junction level; F: male, at mid-body; G: female, at mid-body); H) female, ovojector, left lateral view;!) female, tail, left lateral view; J-L) male, dissect spicules (1: right one, ventral view; K: right one, externo-lateral view; L: left one, externo-dorsal view); M: male, caudal bursa, ventral view. All sections are oriented as in Fig. 2D. Scale bars: A, H, I, M: SO urn; B: 30 urn; C: 20 urn; D-G, J-L: 40 urn. Abbreviations: d.=dorsal; v =ventral, r.eright, I =Ieft.
Oswaldocruria spp. in Spanish amphibians 213 d. ~ 8 v, Fig. 3.- Oswaldocrutia galeanoae n. sp. A) male, anterior extremity, left lateral view; B) male, head, apical view; C-F) synlope in transverse section (C: male, at the oesophago-intestinal junction level; D: female, at the oesophago-intestinal junction level; E: male, at midbody; F: female, at mid-body); G) female, ovojector, right lateral view; H) female, tail, right lateral view; 1-1) male, dissect spicules (1: left one, ventral view; J: right one, externo-dorsal view); K) male, genital cone, ventral view; L) male, caudal bursa, ventral view; M) female, interrupted cuticular ridges, ventral view; N) male, disappearance of cuticular ridges, right lateral view. All sections are oriented as in Fig. IC. Scale bars: A, F-H, L-N: 60 urn; B, C, I-K: 40 urn; D, E: 50 urn. Abbreviations: d.e dorsal; v.eventral; r.eright; I.=left.
214 triangular with 6 externo-labial papillae, 4 cephalic papillae and 2 amphids. The synlophe has been studied in the holotype male and 1 paratype female. In both sexes, the cuticle bears surface longitudinal ridges irregularly interrupted (see Fig. 3M); 88% of ridges in male and 75% in female appear in the oesophageal region. The ridges disappear just anterior to the caudal bursa in male (Fig. 3N) and at phasmid level in female. There are 71 ridges (male) and 90 (female) at oesophago-intestinal junction (Fig. 3C, D), 80 (male), 119 (female) at mid-body (Fig. 3E, F). Presence of one ridge opposite each lateral cord. Numbers of dorsal and ventral ridges are similar. In transverse section, ridges are orientated perpendicularly to body surface, regularly spaced and of the same height. Holotype-male: 6800 urn long and 160 urn wide at midbody; cephalic vesicle 50 urn long and 40 urn wide. Nerve ring, excretory pore and cervical papillae 160 urn, 350 urn and 370 urn from apex, respectively. Oesophagus 400 urn long (Fig. 3A). Caudal bursa 2-3 pattern which tends towards 2-1-2, i. e. tips of rays 4 being directed towards front of body nearer those of rays 3 and 5. Rays 8 arising on root of dorsal ray and overlapped by rays 6 along their median part (type II). Rays 9 arising on dorsal ray before division of latter into 2 branches of which internal ones are longest (Fig. 3L). Gubernaculum absent, genital cone 20 urn long and 30 urn wide at its proximal part, bearing large zero papilla on its anterior lip and two minute papillae 7 on posterior one (Fig. 3K). Spicule 230 urn long, divided into three main branches: externo-lateral branch or blade, distally divided into 4 processes (Fig. 3J); interno-ventral branch or fork distally divided at 43% of whole length of spicule (Fig. 31) and interno-dorsal branch or shoe bearing needlelike process 90 urn long (Fig. 3J). Allotype-female: 12000 urn long and 200 urn wide at mid-body; cephalic vesicle 70 urn long and 40 urn wide. Nerve ring, excretory pore and cervical papillae 190 urn, 400 urn and 420 urn from apex, respectively. Oesophagus 460 urn long. Didelphic. Vulva 4100 urn from caudal extremity. Vagina vera 50 urn dividing vestibule 420 urn long into two equivalent parts. Sphincters both 50 urn long and infundibula both 20 urn long. Anterior uterine branch 2000 urn long with 9 eggs, posterior uterine branch 2300 urn long with 4 eggs; all eggs at morula stage, 110 urn long and 45 urn wide. Tail 220 urn long and 80 urn wide at level of anus, with caudal spine 15 urn (Fig. 3H). Discussion These specimens belong to the Oswaldocruzia species group of the Palaearctic zone characterized by: A) the division of the spicules into three branches (blade, fork and B. BEN SUMANE, J. LLUCH & M.C. DURETTE-DESSET shoe), with the fork divided above the distal third of the spicules; B) the presence of one ridge in front of each lateral cord; C) the arrangement of rays 6 and 8 of type II. There are two closely related species: O. fulleborni Iwanitsky, 1940, parasite of Bufo viridis in the Ukraine and O. filiformis (Goeze, 1782) parasite of Bufonidae in Western Europe and Hylidae in Switzerland, Corsica and the Canary Islands. Contrary to the opinion of BEN SLlMANE, DURETTE-DESSET& CHABAUD (1993), we suggest that O. fulleborni is a valid species, since it has a synlophe with more numerous ridges than O. filiformis and spicular blade undivided at its tip. The Spanish specimens can be differentiated from O. fullebomi by a spicular blade with undivided tip and fewer cuticular ridges (30-50 versus 80-100). It is distinct from O. filiformis due to the absence of cervical alae and a spicular blade divided into 4 equivalent processes. We consider the specimens from Bufo bufo to belong to a new species, Oswaldocruzia galeanoae n. sp. Dichotomous key to the Palaearctic species of the genus Oswaldocruzia The following dichotomous key can be established for the Palaearctic species of the genus Oswaldocruzia: 1 (12) 2 (5) 3 (4) 4 (3) 5 (2) 6 (7) 7 (6) Arrangement of rays 6, 8 and 9 of type 1. Rays 2 and 3 smaller than rays 5 and 6. Spicule length less than 200 urn, Fork divided within distal third of the spicule. Shoe poorly developed. Caudal bursa heart-shaped. Parasite of Bufo viridis, Ukraine Oswaldocruzia iwanitskyi Sudarikov, 1951 (=0. skrjabini sensu Iwanitsky, 1940 nee Travassos, 1937) Caudal bursa transversely elongated. Parasite of Molge vulgaris, M. palmata, England and Triturus vulgaris, USSR Oswaldocruzia molgeta Lewis, 1928 Rays 2 and 3 longer than rays 5 and 6. Spicule length more than 200 urn. Fork divided above distal third of the spicule. Shoe well developed. Spicular interno-dorsal branch (shoe) longer than two others. Roots of rays 8 far from dorsal ray. Parasite of Rana temporaria, Japan Oswaldocruzia yezoensis Morishita, 1926 Spicular interno-dorsal branch (shoe) as long as two others. Roots of rays 8 arising on the dorsal ray. 8 (9) Cervical alae absent. Parasite of Bufo formosus, Rana rugosa, Japan Oswaldocruzia insulae Morishita, 1926
Oswaldocruzia spp. in Spanish amphibians 9 (8) 10 (11) Cervical alae present. Cervical alae pointed. Ridges present within oesophageal region. Parasite of Rana japonica, R. nigromaculata, Japan Oswaldocruzia socialis Morishita, 1926 11 (10) Cervical alae rounded. Ridges absent within oesophageal region. Parasite of Rana synklepton esculenta, 8ufo sp., Bulgaria Oswaldocrutia bialata (Molin, 1861) 12 (1) 13 (26) Arrangement of rays 6, 8 and 9 of type II or Ill. Arrangement of rays 6, 8 and 9 of type H. 14 (15) Ridges opposite each lateral cord lacking. Heart-shaped caudal bursa. Parasite of Speleomantes genei (Urodel), Sardinia Oswaldocruzia bonai Ben Slimane et Durette-Desset, 1995 15 (14) One ridge opposite each lateral cord present. Caudal bursa transversely elongated. 16 (\ 9) Cervical alae absent. 17 (18) 30-50 uninterruped ridges. Spicular blade with blunt extremity. Parasite of Bufo viridis, Ukraine Oswaldocruzia fulleborni Iwanitsky, 1940 18 (17) 80-100 irregularly interrupted ridges. Spicular blade distally divided into many processes. Parasite of Bufo bufo, Spain Oswaldocruzia galeanoae n.sp. 19 (\ 6) Cervical alae present. 20 (23) Cervical alae poorly-developed, both composed of 2 latero-ventral ridges. 21(22) Cervical alae supported by chitinous reinforcement, both composed of 2 sublateral ridges, separated by very small lateral ridge. Parasite of 8ufo bufo, Rana sp., Hyla sp., Western Europe, Corsica, Canary Islands Oswaldocruzia filiformis (Goeze, 1782) 22(21) Cervical alae without chitinous reinforcement, both composed of 2 sublateral ridges. Parasite of Rana temporaria, Spain Oswaldocruzia hispanica n. sp. 23 (20) Cervical alae well-developed, both mainly composed of 1 hypertrophied latero-ventral ridge. 24 (25) Cervical alae both composed of 1 large triangular ridge supported by strong chitinous spine as 215 wide as ridge itself and surrounded by two small ridges. Presence of ventral ridges within oesophageal region. Parasite of Rana dalmatica, Triturus vulgaris, France. Rana sp., Corfu Oswaldocruzia duboisi Ben Slimane, Durette-Desset et Chabaud, 1993 25 (24) Cervical alae both composed only of 1 large triangular ridge supported by a small chitinous spine located at the apex of the ridge. Absence of ventral ridges within oesophageal region. Parasite of Rana sp., France. Rana temporaria, Spain Oswaldocruzia guyetanti Ben Slimane, Durette-Desset et Chabaud, 1993 26 (13) Arrangement of rays 6, 8 and 9 of type Ill. 27 (28) Presence of extra branches on shoe and fork. Spicular blade distally divided. Parasite of Rana temporaria, Ukraine Oswaldocruzia problematica Iwanitsky, 1940 28 (27) Absence of extra branches on shoe and fork. Spicular blade not distally divided. Parasite of Lacerta vivipara, Anguis, Germany Oswaldocruria skrjabini Travassos, 1937 REFERE CES BEN SUMANE (B.) & DURETIE-DESSET (M.C.), 1993.- Quatre nouvelles especes du genre Oswaldocruzia Travassos, 1917 (Nematoda, Trichostrongyloidea) parasite d' Amphibiens d'equateur. Revue Suisse de Zoologie, 100: 113-136. BE SUMANE (B.) & DURETIE-DESSET (M.C.), 1995.- Oswaldocruzia bonae n. sp. ( ematoda, Trichostrongylina) parasite d'une Salamandre cavernicole de Sardaigne. Parassitologia, 37: 245-248. BEN SUMANE (B.), DURETTE-DESSET (M.C.) & CHABAUD(A.G.), 1993.- Oswaldocruzia (Trichostrongyloidea) parasites d' Amphi biens des Collections du Museum de Paris. Annales de Parasitologie Humaine et Comparee, 68: 88-100. DURETTE-DESSET (M.C.), 1985.- Trichostrongyloid nematodes and their vertebrate hosts: Reconstruction of the phylogeny of a parasitic group. Advances in Parasitology, 24: 239-306. DURETTE-DESSET (M.C.) & CHABAUD (A.G.), 1981.- ouvel essai de classification des Nematodes Trichostrongyloidea. Annales de Parasitologie Humaine et Comparee, 56: 297-312. DURETTE-DESSET (M.C.), NAsHER (A.K.) & BEN SUMANE (B.), 1992.- Oswaldocruria arabica n. sp (Nematoda, Trichostrongyloidea) parasite d'un Bufonidae de la peninsule arabique et remarques sur des especes proches. Bulletin du Museum National d'histoire Naturelle de Paris, 14: 693-703. GOEZE (l.a.e.), 1782.- Versuch einer Geschichte der Eingeweidewiirmer thierischer Korper. Blankenbourg, 11,47. IVANITSKY(S.Y.), 1940.- Materiaux concernant la faune des Helminthes des Vertebres de l'ukraine. Comptes Rendus des Travaux Speciaux de l'lnstitui Veterinaire de Kharkow, 19: 192-155.