Trichospirura aethiopica n. sp. (Nematoda: Rhabdochonidae) from Malacomys longipes (Rodentia: Muridae) in Gabon, first record of the genus in the Ethiopian Realm

Trichospirura aethiopica n. sp. is described from unidentified tubular structures (pancreatic ducts?) near the stomach of the murid Malacomys longipes Milne-Edwards, 1877 in Gabon. The extremely long and narrow buccal capsule, posterior position of the vulva, unequal spicules and absence of caudal alae readily identified the specimens as belonging to Trichospirura Smith & Chitwood, 1967, but a combination of several characters distinguished them from the described species in this genus. Males of the new species are characterized by the absence of precloacal papillae, the presence of four pairs of postcloacal papillae and a left spicule length of 165–200 μm. With only five nominal and one unnamed species, the host range of Trichospirura extends into the Neotropical, Indo-Malayan and Ethiopian Realms and comprises three classes of vertebrates, Amphibia, Reptilia and Mammalia, suggesting a larger species diversity than that currently recorded. Detection is difficult as predilection sites are often outside the gut lumen. It was noted that, irrespective of their geographic origin, species from mammals share certain characters (shorter left spicule and absence of precloacal papillae) that oppose them to those from amphibians and reptiles. A hypothesis for the origin of Trichospirura in mammals through a remote host-switching event in tupaiids in southern Asia, likely facilitated by the intermediate hosts, and for their subsequent migration to the Ethiopian and finally Neotropical Realm is proposed. Regarding the two species from anurans and saurians in the Antilles, one or two host-switching events are considered equally possible, based on morphological characters.


Introduction
Representatives of the large family Rhabdochonidae Travassos, Artigas & Pereira, 1928 generally parasitize fishes, except for those of one genus, Trichospirura Smith & Chitwood, 1967 (= Freitasia Baruš & Coy Otero, 1968; [12]), which are found in tetrapod vertebrates. Reports of Trichospirura species are rare. Their infection sites are the small intestine, as in most of the rhabdochonids, or are unusual, such as the pancreatic or salivary ducts, or the abdominal cavity, in which the worms are encapsulated. Only four nominal and one unnamed species are recognized in the genus (Tables 1  and 2). This contrasts with the wide host range and geographic distribution of Trichospirura. Three species occur in South and Central America and are parasites of platyrrhinian monkeys [16], of saurians [2] and of anurans [13]; the others are from Malaysia, where they parasitize tupaiid insectivores and chiropterans [5]. The remarkable host range and geographic distribution of Trichospirura are further extended by the discovery of a new species from an African murid. This poses the question of its origin.

Materials and methods
In 1996 Dr. G. Dubreuil, Centre International de Recherches Médicales de Franceville, captured rodents in Gabon for virology research. Several animals were fixed in formalin and sent to the Muséum National d'Histoire Naturelle (MNHN), Paris, France, to augment both the collection of mammals and that of zooparasitic nematodes. In a Big-eared swamp rat, Malacomys longipes Milne-Edwards, 1877, rhabdochonid nematodes were recovered during the dissection of tissues near the stomach; several worms were found in the lumen of unidentified tubular structures; these did not seem to be granulomatous reactions of the host, because they possessed a regular wall with an external muscular layer ( Figure 1A); they might have been pancreatic ducts.
All specimens were cleared in lactophenol and examined under a Wild compound light microscope equipped with a drawing tube. Measurements were taken from drawings and are given in micrometres unless otherwise specified. The ovijector length was measured from the vulva to the division of the uteri and includes the vagina, as this structure was not identified in previously described species and there is no clear distinction between vagina and ovijector. In the description, the term buccal capsule, instead of pharynx or vestibule, is used for the tube between the mouth and the oesophagus in order to be consistent with other groups of nematodes.
The nomenclature and classification of small mammals follows Wilson & Reeder [20], that of anurans Frost [8] and that of reptiles Uetz [19]. The classification of biogeographic Realms follows Udvardy [18].  External layer of body cuticle forming a thin sheath with regular transverse salient crests ( Figures 1F, I and 3A). Very thin anterior part, with acute bend at level of distal part of buccal capsule. Body attenuated posteriorly from vulva to tip of tail. Lateral chords narrow; excretory canals not noticed. Excretory pore slightly anterior to proximal end of muscular oesophagus. Nerve ring surrounding buccal capsule in posterior quarter and anterior to excretory pore. Deirids spindle-shaped, generally anterior to nerve ring. Head (Figure 2A, B, E): four groups of latero-median papillae; each group composed of a small but obvious external labial papilla, situated on the internal aspect of the mouth and an external salient cephalic papilla. Depressed amphidial aperture posterior to head papillae ( Figure 2A, B, E). Mouth as wide as apex of body, almost square, with rounded angles ( Figure 2E). Extremely long and thin buccal capsule, well-sclerotized, hardly dilated at anterior end or not at all, depending on orientation (Figure 2A, B); no «muscular ring» (see Moravec & Puylaert [14]) identified in posterior part of buccal capsule.
Female ( Figure 1): didelphic, prodelphic. Vulva preanal and depressed. Ovijector: straight vagina with short vagina vera, followed by a part with thick granulous epithelium and thin external layer of muscles. Two uteri, each beginning with a short narrow part with thick muscular walls, subsequently widening into thin-walled tubes containing embryonated eggs ( Figure 1G, H). Tail long, thin, with conical tip, blunt in ventral view; anus  Moravec & Puylaert (1970) this figure is incorrect and the excretory pore is situated more anteriorly; d sometimes absent; e measured on Figure 2G

Taxonomic discussion
The specimens recovered from M. longipes in Gabon display the typical characters of the rhabdochonid Trichospirura, namely the extremely long and narrow buccal capsule, posterior vulva, unequal spicules and absence of caudal alae [16]. In the two known species examined, three morphological features that went unnoticed in the original descriptions are similar to the material studied herein: the anterior body is often more or less abruptly bent and the vulva is depressed; in addition, a cuticular sheath forming transverse crests was observed in T. leptostoma ( Figure 3J) and T. amphibiophila; it can also be identified on figures of T. willmottae, but was described as ''annelures transversales'' [5]; it might be a shared character, although it has not been reported in T. teixeirai. Despite these similarities, the material described herein can be distinguished from the currently known species of Trichospirura by the following characters (Tables 1 and 2).
Trichospirura amphibiophila: parasitizes Eleutherodactylidae (Anura) in Central America; infection site an abdominal cyst near the liver; excretory pore situated farther anteriorly; buccal capsule longer; two to three times longer left spicule; two pairs of precloacal and five pairs of postcloacal papillae [13].
The following two species from mammals have spicules similar in length and shape to the specimens studied herein and their caudal papillae are all postcloacal as well, but they are distinct in the following characters:  Trichospirura leptostoma: parasitizes Cebidae (Primates) in Brazil; infection site pancreatic ducts; vulva less posterior, ovijector (described herein) three times longer, thin-walled and dilated to form a pouch ( Figure 1E); five or six pairs of postcloacal papillae in the male [16].
Trichospirura willmottae: parasitizes Tupaiidae (Scandentia) in Malaysia; infection site salivary ducts; body length of both sexes twice shorter; males with five pairs of postcloacal papillae, one being near the tail tip. Chabaud & Krishnasamy [5] described large transparent cells in the wall of the distal part of the ovijector.
Trichospirura sp. of Chabaud & Krishnasamy, 1975: parasitizes Vespertilionidae (Chiroptera) in Malaysia; infection site intestine; immature females, which are the only sex known, are much smaller with regard to all measurements presented in Table 2.
The material described from M. longipes represents a new species, for which the name Trichospirura aethiopica is suggested.

Discussion
With only five nominal and one unnamed species, the rhabdochonid genus Trichospirura extends into three Realms, Neotropical, Indo-Malayan and Ethiopian, and its members parasitize three classes of vertebrates, Amphibia, Reptilia and Mammalia. The intestinal T. teixeirai was reported from several lizards, polychrotids, tropidurids and a gekkonid [6,13]. Trichospirura amphibiophila was found in a single species of Eleutherodactylus only, although this eleutherodactylid host genus is highly diversified in Central America and the Antilles [13]; it might be an accidental infection from an unknown host, as the worms, one male and two females, were encapsulated in the abdominal cavity. Trichospirura leptostoma from the pancreatic ducts of the cebid C. (C.) jacchus, was discovered in the Texan laboratory to which these animals had been transported after they had been captured in two widely separated areas in Brazil, the southeastern Tupi Forest area and a vast area north of the Amazon; several animals were examined after approximately one to 16 months in captivity, likely hyperinfected through cockroaches, which were later shown to be the intermediate hosts in animal houses [1,10]; a single specimen was also found in another cebid, Saguinus oedipus (Linnaeus, 1758), in Colombia [16]. The remaining three species were reported only once. Trichospirura willmottae was found in the salivary duct of a single Tupaia glis (Diard, 1820), Trichospirura sp. in the intestine of a single Myotis mystacinus (Kuhl, 1817) and T. aethiopica n. sp. in tubes near the stomach (pancreatic ducts?) of a single M. longipes. The probability of finding these worms is lowered by the fact that their infection sites are often outside the gut lumen.
When Baruš & Coy Otero [2] created Freitasia, they were unaware of the work of Smith & Chitwood [16]. Moravec [12] considered Freitasia a junior synonym of Trichospirura, while at the same time Chabaud [3] differentiated Freitasia from Trichospirura based on the shape of the buccal capsule, the former having a «pharynx dilated anteriorly to form a well-defined buccal capsule», the latter having a «pharynx not or only slightly dilated anteriorly». However, this character does not differ clearly between the two genera [2,16]. It was nevertheless noted during this study that the parasites of mammals can be opposed to the two species from saurians and anurans from Central America (Antilles). Compared to T. teixeirai and T. amphibiophila, the species from mammals have a two to three times shorter left spicule, and precloacal papillae are absent whatever the Realm, Neotropical, Indo-Malayan or Ethiopian, and whatever the host order, Scandentia, Rodentia or Primates (Chiroptera are excluded because the male parasite is not known).
It is generally accepted that the genus Trichospirura was derived through host-switching from Rhabdochona Railliet, 1916, parasites of freshwater fishes [4,5,7,13], and that the necessary adaptations were accompanied by an extraordinary lengthening of the buccal capsule. Excepting Australia, Rhabdochona has a worldwide distribution. Therefore hostswitching might have occurred in each Realm. However, the characters highlighted in this study might suggest a lineage for the parasites of mammals from a remote host-switching event that occurred in Tupaiidae in southern Asia. Murids, which originated in this region, would have been infected before they reached Africa in successive waves during the Miocene, when the two continental masses were joined, approximately 17,21]. Trichospirura leptostoma, with a derived character (ovijector dilated into a chamber), might have originated from the same area, followed by a migration to Africa and finally to the Neotropical region, together with their primate hosts, the platyrrhine monkeys. Indeed, after decades of controversy, it is now well supported that the South American monkeys arrived from Africa by transoceanic migrations in a period (approximately 37-16.8 Mya; [15]) that overlaps with the Miocene era. In the Antilles, the two species from cold blooded vertebrates, a saurian and an anuran host, differ from each other in several important characters: nerve ring at the level of the muscular oesophagus or buccal capsule, excretory pore anterior [14] or posterior to nerve ring, deirids absent or present, gubernaculum present or absent, respectively. A single or two events of host-switching are equally possible.
Host-switching from fishes to other classes of vertebrates was likely facilitated by the intermediate hosts. They are aquatic arthropods, mainly mayflies, for the species of Rhabdochona (first life cycle elucidated by Moravec [11]) and cockroaches for T. leptostoma under experimental conditions [10]. No further life cycles have been studied to date [1]. Hosts that are parasitized by Trichospirura species are insectivorous. This also applies to M. longipes, the host of the new species, which, in addition, lives in an aquatic environment.