THE LUNG NEMATODE PARASITES OF THE GENUS RHABDIAS

Bull. Soc. zool. Fr., 2010, 135(1-2) : 109-118. Parasitologie, Biologie, Systématique THE LUNG NEMATODE PARASITES OF THE GENUS RHABDIAS (RHABDIASIDAE): DIVERSITY AND BIOLOGY IN THE CHAMAELEONIDAE (SQUAMATA) AND HYPOTHESES ON THEIR EVOLUTION par N. LHERMITTE-VALLARINO 1, M. BARBUTO 2, K. JUNKER 3, S. WANJI 4, I. INEICH 5, A. SCHMITZ 6 and O. BAIN 7 Presently, fourteen species of Rhabdias are discriminated in the Chamaeleonidae, instead of the two species historically recognized. The mode of reproduction of the parasitic female morphotypes and the free-living phase are elucidated in several species, and compared to those from other host groups. Larval morphological characters suggest that the parasites from Chamaeleonidae form a small group, which might have evolved in these lizards. 1. Nathaly Lhermitte-Vallarino, Muséum national d'histoire naturelle, et CNRS, UMR 7205 OSEB, Département Systématique et Évolution, Parasitologie comparée, 61 rue Buffon, CP52, 75231 Paris Cedex 05. 2. Michela Barbuto, Università degli Studi Milano Bicocca, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. 3. Kerstin Junker, Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110 South Africa. 4. Samuel Wanji, Research Foundation for Tropical Diseases and the Environment, P.O. Box 474, Buea, Cameroon. 5. Ivan Ineich, Muséum national d'histoire naturelle, UMR 7205, Département de Systématique et Évolution, CP 30, 25 rue Cuvier, 75005 Paris Cedex 05. 6. Andreas Schmitz, Department of Herpetology and Ichthyology, Muséum d'histoire naturelle, C.P. 6434, 1211 Geneva 6, Switzerland. 7. Odile Bain (corresponding author), Muséum national d'histoire naturelle et CNRS, UMR 7205 OSEB, Parasitologie comparée, 61 rue Buffon, CP52, 75231 Paris Cedex 05. E-mail : bain@mnhn.fr

110 Bulletin de la Société zoologique de France 135 (1-2) Les nématodes pulmonaires parasites, du genre Rhabdias (Rhabdiasidae) : diversité et biologie chez les Chamaeleonidae (Squamata) et hypothèse sur leur évolution Quatorze espèces de Rhabdias sont actuellement discriminées chez les Chamaeleonidae, au lieu de deux par le passé. Le mode de reproduction des morphotypes femelles parasites et les phases libres sont élucidés chez plusieurs espèces et comparés aux parasites d'autres groupes hôtes. Des caractères morphologiques larvaires suggèrent que les parasites de Chamaeleonidae constituent un petit groupe qui aurait évolué chez ces lézards. Introduction Rhabdias Stiles & Hassal, 1905 is the most diversified genus in the Rhabdiasidae Railliet, 1915, a remarkable family of nematode parasites that have retained primitive morphological and biological characters, such as a simple, straight vagina and a freeliving heterogonic phase (CHABAUD, 1974; ANDERSON, 2000). These nematodes belong to the small clade IVa, which also comprises the better known Strongyloididae Chitwood & McIntosh, 1934, including several species causing disease in humans, parasites of insects and free-living worms (BLAXTER, 2001; DORRIS et al., 2002). Parasitic adults of Rhabdias are lung-dwelling and have a female morphotype. They lay larvated eggs that pass to the faeces via the digestive tract of their host. Infective larvae develop in the free-living female through matricidal endotoky. Species of Rhabdias are restricted to cold-blooded vertebrates, their main hosts being Lissamphibia, especially Anura, and a few groups of Squamata, including the Chamaeleonidae. For decades, Rhabdias from chameleons were assigned to just two species, Rhabdias chamaeleonis (Skrjabin, 1916) in Africa and R. gemellipara Chabaud, Brygoo & Petter, 1961 in Madagascar. This apparent paucity in parasite diversity stood in stark contrast to host diversity, considering that as many as 175 species are currently known in the Chamaeleonidae. Adding the fact that the distribution area of chameleons includes several hot spots of biodiversity, in Madagascar and the equatorial African mountains (MYERS et al., 2000; AMIET, 1987; CHIRIO & LEBRETON, 2007; GOODMAN & BENSTEAD, 2003), such a poor diversity in their lung worms seemed unlikely. Another question was the origin of Rhabdias species in Chamaeleonidae. These lizards are a monophyletic group (TILBURY & TOLLEY, 2009), branching from Iguania in the late Jurassic (HEDGES & VIDAL, 2009). Would their Rhabdias show particular affinities with those parasitic in Afrotropical anurans? Or would they present similarities with the few Rhabdias species that had recently been reported from other iguanian lizards, the Agamidae, a sister group of Chamaeleonidae, or the South American Polychrotidae?

111 Nématode parasite du genre Rhabdias Material and methods Material from chameleons and a few anurans was collected during field trips, obtained from the Muséum national d'histoire naturelle collections, or recovered from hosts seized by customs or purchased from a dealer. Chamaeleo species originated from Senegal and Togo; Trioceros spp. and Rhampholeon spectrum from Cameroon (Volcanic Chain) and Burundi; Rieppeleon brevicaudata from Tanzania; Calumma spp. and Brookesia superciliaris from Madagascar. A single bufonid was from South Africa, two arthroleptids from the Cameroon mountains and a single mantellid from Madagascar. When specimens were recovered alive, part of the material was used to determine their coxi, 12S rdna and 28S rdna gene sequences (FERRI et al., 2009; sequences deposited in the EMBL repository; KUZMIN et al., 2007) and coprocultures were made to obtain the free-living phases. The 16S rdna gene was sequenced for the chameleons from Cameroon for further comparison of host and parasite diversity. Results At present, a total of fourteen Rhabdias species is described from chameleons, nine in Africa and five in Madagascar (LHERMITTE-VALLARINO & BAIN, 2004; LHERMITTE-VALLARINO et al., 2008, 2009a & b, 2010). Molecular analyses performed on four of these confirmed the results of morphological studies. There are numerous discriminative characters, including common ones such as general measurements and ratios, the cuticular body vesicle and oesophagus; characters that received particular attention in our studies were the number and arrangement of head papillae (primitive set of twelve, or reduction to six or four), lips, shape of the mouth, composition of the buccal capsule (one or two segments) (Fig. 1), as well as shape of the intestinal apex and extension of the genital tract. Based on three species, an important feature emerged: buccal capsule size remained constant during adult growth and could thus be used as a reliable diagnostic character. Prevalences, when determinable, varied from 15 to 63% with an intensity of infection from 1 to 25 worms per chameleon. Both sexes were infected. Host size did not seem to influence worm size: in the small Tr. ellioti, R. casiraghii Lhermitte-Vallarino, Barbuto & Bain, 2010 reaches 3 cm, whereas R. kibiraensis Lhermitte-Vallarino, Barbuto & Bain, 2010 does not exceed one centimetre. Coinfection was observed once, with R. casiraghii and R. kibiraensis infecting the same individual of Tr. ellioti. Since body shape at dissection is not a good specific criterion, this concurrent infection was only diagnosed upon completion of the morphological analysis; consequently, the specimen that had been used for molecular analysis could no longer be assigned to either of the two taxa. A given species of Rhabdias generally corresponded to a given chameleon species. Nevertheless, in Cameroon, the same parasite was found in two sympatric Trioceros species from Mont Oku, which perch at slightly different heights in the vegetation. It is, however, noted that parasite-host specificity can be suppressed in experimental infections (CHABAUD et al., 1961).

112 Bulletin de la Société zoologique de France 135 (1-2) Figure 1 Cephalic characters of Rhabdias spp. from Chamaeleonidae and Anura from Africa and Madagascar. A to E. Heads of five species, in front view. A. R. casiraghii Lhermitte-Vallarino, Barbuto & Bain, 2010 from Trioceros ellioti. B. R. kibiraensis Lhermitte-Vallarino, Barbuto & Bain, 2010 from Tr. ellioti. C. R. brygooi Lhermitte-Vallarino, Barbuto & Bain, 2010 from Brookesia superciliaris. D. R. picardiae Junker, Lhermitte-Vallarino & Bain, 2010 from Amietophrynus gutturalis, Bufonidae. E. R. vencesi Junker, Lhermitte-Vallarino & Bain, 2010 from Boophis madagascariensis, Mantellidae. F & G. Buccal capsule of two species, in lateral view: F. R. kibiraensis. G. R. vencesi. Scale bar: 20 µm. Names of anurans follow Frost (2009). Caractères céphaliques de Rhabdias spp., parasites de Chamaeleonidae et d Anura d Afrique et de Madagascar A to E. Têtes de cinq espèces, en vue apicale. A. R. casiraghii Lhermitte-Vallarino, Barbuto & Bain, 2010 chez Trioceros ellioti. B. R. kibiraensis Lhermitte-Vallarino, Barbuto & Bain, 2010 chez Tr. ellioti. C. R. brygooi Lhermitte-Vallarino, Barbuto & Bain, 2010 chez Brookesia superciliaris. D. R. picardiae Junker, Lhermitte-Vallarino & Bain, 2010 chez Amietophrynus gutturalis, Bufonidae. E. R. vencesi Junker, Lhermitte-Vallarino & Bain, 201 chez Boophis madagascariensis, Mantellidae. F & G. Capsule buccale de deux espèces, vue latérale. F. R. kibiraensis. G. R. vencesi. Barre d échelle : 20 µm (les noms des hôtes sont selon Frost, 2009).

113 Nématode parasite du genre Rhabdias The distribution areas of the various species of Rhabdias were restricted to one of the diverse geographic regions/subregions explored, as were the chameleon hosts. A double case of vicariance was supposed between East and West Africa. Indeed, R. chamaeleonis (Burundi) and R. cristati Lhermitte-Vallarino & Bain, 2008 (Mt Cameroon) have a short, thick oesophagus and a long, thin-walled buccal capsule, whereas R. jarki Lhermitte-Vallarino & Bain, 2004 (Burundi) and R. okuensis Lhermitte-Vallarino & Bain, 2008 (Mt Oku in Cameroon) have a long oesophagus with a thin bulb and a very short, thick-walled buccal capsule. Interestingly, the latter two species perforate the lung wall and their cephalic extremities protrude into the coelomic cavity. No morphological particularities were found to distinguish African from Malagasy Rhabdias. In order to elucidate the mode of reproduction of lung worms in chameleons, ovaries were examined for all fourteen species. In 13 of them, a zone with very small cells among larger ovocytes was identified; its role in generating male gametes was proved when we observed spermatozoa migrating along the ovaries to the oviducts, where they accumulate. Spermatozoa production occurred throughout the whole adult life, but was intermittent and not synchronous between the anterior and posterior ovaries. In one species, R. chamaeleonis, no testis zone was found despite the fact that ovaries were dissected for scrutiny, and no spermatozoa were seen in the oviducts either; instead, in young females, large ovulae with four nuclei were observed. We thus conclude that this species is parthenogenetic (LHERMITTE-VALLARINO & BAIN, 2004). Free-living stages were previously only known from R. gemellipara (cf. CHABAUD et al., 1961); they have now been described from five additional species, all of which undergo heterogonic development. Diagnostic characters were limited in males to the distance between testis bend and anterior extremity; in females, the extension of the genital tract and the number of eggs were characteristic. The majority of eggs aborted and the number of infective larvae produced per female was fixed: a single larva in the three African species parasitic in Trioceros spp., two larvae in R. gemellipara from Calumma brevicornis in Madagascar; it was not fixed, but limited to one or two, in R. mariauxi Lhermitte-Vallarino, Junker & Bain, 2009 from Ri. brevicaudatum. Infective larvae are enclosed in a sheath shown to be a modification of the two larval exuviae of moults 1 and 2. The sheath is thick, especially at the extremities, and has a checkered aspect; its caudal region presents a bilateral notch and bulge. The larva itself possesses longitudinal crests and lateral alae; its caudal extremity is thick, rounded and ornated with a few buds (Fig. 2). Despite their sheath, infective larvae do not resist desiccation. This could explain why no Rhabdias infection has been observed, at least to date, among species of Chamaeleo, which live in less humid a climate than Trioceros, Rhampholeon, Rieppeleon and Calumma. However, survival of infective larvae has been demonstrated in the coelomic cavity of force-fed insects (LHERMITTE-VALLARINO & BAIN, 2004). It is very likely that, in natural transmissions, chameleons mainly become infected when ingesting insect transport hosts.

114 Bulletin de la Société zoologique de France 135 (1-2) Comparison with Rhabdias parasitic in hosts other than Chamaeleonidae Considering the lung worms from Lissamphibia in the Afromalagasy region, only four species had been reported until recently, one from a caecilid (Gymnophiona) in Africa, and the others from Anura (BAYLIS, 1929; BAKER, 1987a; MORAVEC et al., 1987; KUZMIN, 2001). Including the latest species we described from Afrotropical anurans (JUNKER et al., 2010), presently, three species are known from bufonids, two in South Africa, one in Egypt; three species from arthroleptids, one in Tanzania, two in Cameroon; and two species in Madagascar, one from a ptychadenid (introduced from Africa) and one from a mantellid. Specific characters of the parasitic female are the same as for the material from Chamaeleonidae. In particular, it was noted that in both host-groups the head characters displayed were diversified in a similar way: primitive round or derived flattened mouth, either dorso-ventrally or laterally; primitive buccal capsule with two segments, or reduced to one segment (Fig. 1). However, Rhabdias species from Afrotropical anurans were distinct from those from Chamaeleonidae in having a buccal capsule with a smaller diameter (16-25 vs 35) (LHERMITTE-VALLARINO & BAIN, 2004; LHERMITTE- VALLARINO et al., 2008, 2009a,b, 2010); however, exceptions to this pattern are the Figure 2 Characters of the infective larvae of Rhabdias spp. from Chamaeleonidae and Anura from Africa and Madagascar. A. Tail of the larva of R. chamaeleonis from Tr. johnstoni, round extremity with buds (sheath not represented). B. R. vencesi from the mantellid Bo. madagascariensis, conical tail with pointed extremity (inside the sheath). C. Checkered aspect of the sheath, R. brygooi from Br. superciliaris, posterior region. Scale bars: A,B, 15 µm; C, 20 µm. Caractères de la larve infectieuse de Rhabdias spp. provenant de Chamaeleonidae et d Anura (Afrique, Madagascar). A. Queue de la larve de R. chamaeleonis provenant de Tr. johnstoni, extrémité de la queue pourvue de bourgeons (fourreau non représenté). B. R. vencesi provenant de Bo. madagascariensis (mantelle), queue conique à extrémité pointue (dans le fourreau). C. Aspect en mosaïque du fourreau, R. brygooi provenant de Br. superciliaris, région postérieure. Échelles : 15 µm (A,B) ; 20 µm (C).

115 Nématode parasite du genre Rhabdias two small Rhabdias species from Malagasy chameleons, R. gemellipara and Rhabdias sp., with a maximum diameter not exceeding 17 µm (LHERMITTE-VALLARINO et al., 2009b), and R. brygooi, a parasite of the primitive chamaeleonid Brookesia (25-28 µm). When examining the genital tracts of fully gravid females, testis zones were observed in the ovaries of one species and spermatozoa in the oviducts of the others, suggesting that the parasitic stage was not protandric, as reported for some Rhabdias in earlier studies (GOODEY, 1924a,b; BAKER, 1979), but that male gametes were produced during the entire life of the female parasite. The free-living phase was elucidated in one Malagasy species, R. vencesi Junker, Lhermitte-Vallarino & Bain, 2010. As in Rhabdias from Chamaeleonidae and anurans from other geographic regions, this phase is heterogonic. The adults of both sexes are similar to those from chamaeleonids, except for the tail filament, which is longer in R. vencesi. The sheath of the infective larva is checkered. The larva itself has a conical tail with a pointed tip, and no buds (JUNKER et al., 2010; Fig. 2). When comparing species of Rhabdias from Chamaeleonidae with those of other iguanian lizards worldwide, no shared morphological particularities were found. Instead, the three species from Mexican and Central American polychrotids, R. leonae Martínez-Salazar, 2006, R. anolis Bursey, Goldberg & Telford, 2003, and R. nicaraguensis Bursey, Goldberg & Vitt, 2007, had in common with the majority of parasites from anurans the narrow buccal capsule that is longer than wide (BURSEY et al., 2003, 2007; MARTÍNEZ-SALAZAR, 2006). The single species from Agamidae, R. japalurae Kuzmin, 2003 in South-East Asia, had a relatively shallow buccal capsule, similar to that of most species from Chamaeleonidae but narrower (KUZMIN, 2003). No information is available on female parasite reproduction and free-living stages. The ten species of Rhabdias from ophidians are very small, with a relatively large mouth aperture (BAKER, 1978; KUZMIN & TKACH, 2009). Their free-living phase is mainly homogonic, that is eggs laid by the female parasite develop directly into infective larvae, but concurrent heterogonic development was described in some species, e.g. R. agkistrodonis Sharpilo, 1976 and R. elaphe Sharpilo, 1976 (BAKER, 1979; KUZMIN, 1999; KUZMIN & MISKOV, 1999). Morphological analyses of the infective larvae often lack detailed information, but some of the descriptions provide indications that the sheath is checkered and, where available, figures suggest that the tail of the infective larvae is blunt but thinner than in species from Chamaeleonidae (BAKER, 1979; KUZMIN, 1999; KUZMIN & MISKOV, 1999). The approximately 50 species of Rhabdias from the Palaearctic, Nearctic, Neotropical and Oriental regions (BAKER, 1978; BAKER, 1987a; KUZMIN & TKACH, 2009), present morphological features similar to those reported above for the Afromalagasy hosts, meaning a great variability in head morphology, including, in some species, the development of lateral pseudolabia (KUZMIN, 2001; KUZMIN et al., 2003; 2007; MARTÍNEZ-SALAZAR & LEÓN-RÈGAGNON, 2007). The position of the vulva varies from pre-equatorial over equatorial to post-equatorial in the various species (MARTÍNEZ-SALAZAR & LEÓN-RÈGAGNON, 2007). The free-living phase is heterogonic; the number of infective larvae per female varies from one to eight; the

116 Bulletin de la Société zoologique de France 135 (1-2) infective larvae, when described, have a tail that is pointed or round but without buds (BAKER, 1979). Conclusion The fourteen species currently reported from Chamaeleonidae obviously present an incomplete picture of the real diversity of Rhabdias in this host group, the sample size still being very small. In order to answer the question of their monophyly, we have searched for characters that are exclusive to them. Homoplasy of head morphology appears important and the rich diversity of cephalic characters of the female parasites can be used for species identification only. Biological traits such as the mode of reproduction of the female parasites and free-living stages do not distinguish the Rhabdias of Chamaeleonidae from those of Lissamphibia. We highlight here an interesting morphological character of the infective larvae: the tail extremity is invariably thick, round and ornated with buds in species from Chamaeleonidae. This suggests a single acquisition of Rhabdias by ancestral Chamaeleonidae, followed by diversification within this host family. More data are needed to support this hypothesis, but on-going works confirm that infective larvae of species from anurans have a different tail morphology: it is conical and has a pointed extremity (Fig. 2). Life cycles have not been studied in the rare parasites from other iguanians like Polychrotidae and Agamidae, but adult characters suggest a distinct history and, at least for the first representatives, local capture from anuran parasites. The sheath of the infective larva and its checkered aspect appears to be very likely a common trait of the Rhabdiasidae as it was described in two other genera of the family, Pneumonema Johnston, 1916 and Chabirenia Lhermitte-Vallarino & Bain, 2005 (BALLANTYNE, 1991; LHERMITTE-VALLARINO et al., 2005). Acknowledgements This work was supported by the Agence nationale de la Recherche ANR Biodiversity Project Iles Forestières Africaines, IFORA, 2006-2009 and by the Partenariat Hubert Curien, programme Galileo. We thank Ivo Melle Ngwese, our field guide and Renaud Boistel, who provided some material. REFERENCES AMIET, J.L. (1987).- Aires disjointes et taxons vicariants chez les anoures du Cameroun : Implications paléoclimatiques. Alytes, 6, 99-115. ANDERSON, R.C. (2000).- Nematode parasites of vertebrates; their development and transmission. Wallingford, U.K.: CABI Publishing, 2 nd ed., 650 pp. BAKER, M.R. (1978).- Morphology and taxonomy of Rhabdias spp. (Nematoda: Rhabdiasidae) from reptiles and amphibians of southern Ontario. Can. J. Zool., 56, 2127-2141. BAKER, M.R. (1979).- The free-living and parasitic development of Rhabdias spp. (Nematoda: Rhabdiasidae) in amphibians. Can. J. Zool., 57, 161-178.

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