Monanema joopi n. sp. (Nematoda, Onchocercidae) from Acomys (Acomys) spinosissimus Peters, 1852 (Muridae) in South Africa, with comments on the filarial genus

Monanema joopi n. sp. is described from blood drawn from the heart of the murid Acomys (Acomys) spinosissimus in South Africa. It is characterised by a non-bulbous cephalic extremity, shared with only one of its five congeners, and a cylindrical tail with caudal alae and a spicular ratio of 2.7 in the male. As is typical for the genus, microfilariae are skin-dwelling. They are 185 to 215 micrometres long and have no refractory granules beneath their sheath. A key to the species of Monanema is presented and an amended generic description, based on the six currently known species, is proposed. Species of Monanema are primarily lymphatic and the low intensity of infection with M. joopi n. sp. in blood from the heart, might suggest that not all adults settle in the heart cavities. One might also consider that other, more susceptible rodents serve as hosts for this parasite as well. To date, the geographic range of Monanema includes North America, Africa and Australia, each with representatives of a different lineage. Given the present hypotheses on the evolutionary origin and subsequent migrations of rodents, we expect the origin of Monanema to be in the Palearctic-Oriental region.


INTRODUCTION
D uring an on-going ecological study investigating the ectoparasite and helminth assemblages of murids in South Africa, tiny filarial worms were collected from blood taken from the heart of the Spiny mouse Acomys (Acomys) spinosissimus (Peters, 1852). They represented a new species of Monanema Anteson, 1968 (Onchocercidae), a genus parasitic in rodents of which the microfilariae are not in the blood, but in the skin (Ko, 1972;Bianco & Muller, 1977;Bianco et al., 1983;Bain et al., 1985).
In this paper, we give a morphological description of the new species and provide a synthetic analysis of the morphology and biology of all currently known species of Monanema. As a result, the definition of the genus was amended, and a hypothesis on its evolution proposed.

A comys (A.) spinosissimus was recovered from
Goro Game Reserve (22° 58' S, 29° 25' E) in the Limpopo Province in South Africa. Filarial worms were fixed in 70 % ethanol. For morphological studies, worms were cleared in lactophenol and examined under a Wild compound light microscope equipped with a drawing tube. Caudal papillae are tentatively numbered following Chabaud & Petter (1961). Cross-sections were cut with a razor blade in order to study internal structures such as chords, muscles and oesophagus. Measurements were taken Original contribution Parasite, 2012, 19, 331-340 from drawings and are given in micrometres unless otherwise specified. The width of the buccal capsule was taken as its external diameter, measured at the base. Microfilariae were dissected from the uteri close to the vagina and cleared in lactophenol for further study. As earlier studies on Monanema indicated that the ear lobes are a predilection site for its skindwelling microfilariae (Ko, 1972;Muller & Nelson, 1975;El Bihari et al., 1977;Bianco et al., 1983;Bain et al., 1985;Wanji et al., 1990Wanji et al., , 1994, ear snips were taken from the frozen carcasses of all infected animals and teased apart in lactophenol to check for microfilariae. Specimens have been deposited in the collection of the Muséum National d'Histoire Naturelle (MNHN), Paris, France (accession numbers 340 -347 YU). Nomenclature of small mammals follows Wilson & Reeder (2005). Additional organs of some of the hosts that were infected with filariae could be examined at a later stage, using frozen carcasses. This was possible only in cases where these organs had not been dedicated to other studies. The frozen liver, lungs and wall of the caecum-colon of five, two and four animals, respectively, were examined, since adults had been reported from these sites in previous studies (Webster, 1967;Muller & Nelson, 1975;El Bihari et al., 1977;Bain et al., 1986;Wanji et al., 1990).

RESULTS
O ne to two filarial worms were present in blood drawn from the heart of ten of 139 A. (A.) spinosissimus. The liver, lungs and wall of the caecum-colon of five, two and four of these ten hosts, respectively, did not contain any filariae. Microfilariae were found in ear snips of two of the hosts harbouring adult worms.  Tables I-III) Large parts of the specimens' body were covered with patches of red blood cells. Slender worms, offwhite in colour. Body of both sexes tapering at ends but anterior extremity nearly cylindrical. Head not bulbous. Oesophagus not divided into muscular and glandular part, of nearly uniform diameter, posterior extremity slightly flattened at junction with intestine. Mouth opening tiny, round (Fig. 1B). Buccal capsule minute. Cuticle smooth. Female (Fig. 1A-M; Tables I, III) posterior part narrower than anterior part. Cuticle slightly thickened laterally (Fig. 1C, D, E). Lateral chords thick; in lateral view, ventrally to the oesophagus, a peculiar cellular mass is observed (Fig. 1A), its origin is anterior to the nerve ring; in transverse section, this mass appears to be formed by the lateral chords which are directed ventrally (Fig. 1C), the left and right being joined in the median plane (Fig. 1D). One group of four head papillae observed, likely externolateral, arranged in dorsoventrally elongated rectangle; lateral amphids identified (Fig. 1B). Oesophagus with flattened lumen (not y-shaped; Fig. 1C, D), its posterior extremity at posterior level of vagina.

MONANEMA JOOPI
Vulva a longitudinal slit, at level of posterior half of oesophagus (Fig. 1A). Vagina: a short vagina vera, transverse, with flattened lumen; vagina uterina well-developed, directed posteriorly, with a chamber terminating in a sphincter, composed of epithelial cells (Fig. 1G). Ovijector: very thick, with a few loops, joining vagina near its mid-length. Opisthodelphic. Uteri running parallel. Tail: curved ventrally, especially tip; tip rounded and slightly bulbous, without appendages, often with irregular swellings, rarely lobulated (  Table III): uterine microfilariae folded once or twice, in loose-fitting sheath with obtuse ends; first fold usually in posterior half of body. No refractile granules in egg space between sheath and microfilaria. Body slender, posterior region tapering. Left cephalic hook, 2.5-3 long. No cephalic space, nuclei filling head anteriorly; nuclei terminating in single row at short distance from tip of tail. Terminal nuclei at times difficult to distinguish from fine granular material filling tip of tail. In skin, ten of 13 microfilariae were exsheathed. Dermal microfilariae (n = 4) were 190, 220, 225 and 228 long and 5, 5, 6 and 5 wide. The sheath (n = 2) was 132 and 147 long, respectively and 15 wide.  Table II): posterior curled into four tight coils. Tail elongate, slender and cylindrical in ventral view, tip bulbous, without appendages or projections. Narrow caudal alae present, slightly more pronounced on level of cloaca. Caudal papillae: four pairs of pre-and paracloacal papillae (right papillae atrophied in pairs 2 and 4); pair 5 near ventral line; pairs 6-10 asymmetric, roughly evenly spaced on tail (Fig. 2C, D). Area rugosa on coiled part of posterior region, composed of narrowly spaced transverse bands of longitudinal crests (Fig. 2E), terminating approximately on level of cloacal aperture (Fig. 2D), not extending to tail. Spicules unequal and dissimilar. Left spicule with well cuticularized handle, followed by membranous lamina, ending in short filamentous tip. Right spicule short and robust, well cuticularized, with broad pointed tip and recurved hook. Gubernaculum absent.         (Tables  II, III ;Webster, 1967;Muller & Nelson, 1975;El Bihari et al. 1977;Bain et al., 1986;Spratt, 2008). All these are distinct from the current specimens in a number of characters, listed below and in Tables II & III. In addition, the hypertrophy of the lateral chords, joining ventrally in the anterior region, as seen in the current specimens (Fig. 1C, D), has not been described for any of the above species. Monanema marmotae: head bulbous; external labial and cephalic papillae arranged in two squares; females more than twice as long (67- Bain et al. (1985); f observed on live specimens; g a single specimen measured. Host details and geographic distribution listed in Table II. All measurements in micrometres, unless otherwise specified. tely two to three times longer (411-599 vs 190), spicular ratio about twice as high (5.5-6.7 vs 2.7); microfilariae shorter (117-142 vs 185-215) (Webster, 1967). Monanema australe: head bulbous; tip of female tail not bulbous; male tail twice shorter, conical in ventral view, with prominent paracloacal alae ornamented with small rugosities at base and with three pairs of adcloacal, pedunculate, laterally directed papillae; left spicule longer (231-238 vs 190), right spicule shorter (47-52 vs 68), resulting in higher spicular ratio (4.6-4.9 vs 2.7); microfilariae shorter (125) (Spratt, 2008). Monanema globulosa: head bulbous [we noted discrepancies between the scale bar, illustration and text concerning Fig. 1 in Muller & Nelson (1975)]; females shorter (7.6-16 mm vs 24.5-30.8 mm), with two pairs of small appendages on tail; male tail conical in ventral view, without caudal alae; caudal papillae more symmetrically arranged, with three regular pairs of precloacal papillae; left and right spicules shorter (112-144 and 44-50 vs 190 and 68, respectively), right spicule without hook; microfilariae shorter (135-150 vs 185-215), 10-11 refractory granules beneath sheath (Muller & Nelson, 1975). Monanema martini: head bulbous; females shorter (20.5-22.5 mm vs 24.5-30.8 mm), tip of tail with several conical projections; caudal papillae grossly symmetrical; area rugosa extending to mid-tail; both left and right spicule shorter (135 and 130, and 52 and 45 vs 190 and 68, respectively), right spicule with double hook; microfilariae larger (235-263 vs 185-215), 11-26 refractory granules beneath sheath . Monanema nilotica: external labial and cephalic papillae arranged in two squares; female tail longer (208-272 vs 120-180); male tail conical in ventral view, without caudal alae; all caudal papillae paired and symmetrically arranged; two precloacal pairs; spicular ratio higher (3.6 vs 2.7); microfilariae larger (203-235 vs 185-215), 13-34 refractory granules beneath sheath (El Bihari et al., 1977).  Anteson (1968, unpublished thesis;in Chabaud & Bain, 1976) for the species then known as Ackertia marmotae Webster, 1967because, contrary to Ackertia Vaz, 1934, its male has several pairs of postcloacal papillae. The genus Monanema was accepted by Anderson & Bain (1976), Chabaud & Bain (1976 and Bain et al. (1982). In the characters of Monanema listed by these authors, the undivided oesophagus and caudal papillae on the tail were consistent, but other characters (buccal capsule, spicular ratio, head shape) changed, to accommodate the increasing number of Original contribution Parasite, 2012, 19, 331-340 species described. Taking into account the characteristics of the six presently described species, we propose the following amended generic definition for Monanema: buccal capsule small (1.7-6 long, 4.4-10 wide); oesophagus not divided into muscular and glandular part; vulva on level of posterior half of oesophagus; vagina large with chamber and sphincter; absence of caudal appendages on tip of male tail; 7-10 pairs of caudal papillae (Fig. 3), disposed anterior to cloaca and, grossly equidistant, along length of tail; first pair of postcloacal papillae close to midventral line; spicular ratio 2.7-6.7; microfilariae folded in loosefitting sheath with obtuse extremities; microfilariae skin-dwelling.  (Ko, 1972), M. globulosa Muller, 1983) andM. martini (Bain et al. 1985;Petit et al., 1988). It is thus expected that ixodids will also transmit M. joopi n. sp. and the remaining two species. Two ixodid ticks were collected from A. (A.) spinosissimus examined during the current study, Rhipicephalus simus Koch, 1844 and Rhipicephalus follis Dönitz, 1910. While they are likely vectors, their role in the life cycle of M. joopi n. sp. remains to be confirmed. Sites of infection for the species of Monanema are diverse, including the lungs, heart cavities, lymphatics of liver and caecum-colon. For a given species, as for example M. martini, filariae can settle in different places, the lymphatics and, less commonly, in the pulmonary arteries (Wanji et al., 1990;Vuong et al., 1991). In fact, it seems that species of Monanema are primarily lymphatic as suggested by the works of Wanji et al. (1990) who also demonstrated that lymphatic infective larvae and adults could be passively drawn back to cardio-pulmonary sites by the lymph flow when altered (Bain & Babayan, 2003). The low intensity of infection seen in M. joopi n. sp., the high number of aborted eggs in females and the fact that on no occasion males and females were recovered together, suggests that not all adults settle in the heart cavities. However, no adults were recovered from any of the other sites examined (liver, lungs, caecumcolon). This also suggests that other, more permissive murids might contribute to the maintenance of M. joopi n. sp. in nature. Monanema has few representatives but a wide geographic distribution (Tables II, III ;Spratt, 2008). The host range of Monanema is restricted to rodents, with one species in a Nearctic sciurid and five species in murids, of which four in Africa and one in Australia. The trends of morphological evolution in the Dipetalonema line (Chabaud & Bain, 1976), indicate that a bulbous head, head papillae that are arranged in a dorsoventrally elongated rectangle [in M. martini as early as in the infective stage (Bain & Chabaud, 1986)], a high spicular ratio, pairs of caudal papillae that are asymmetrically arranged and a reduced number of caudal papillae are evolved characters. We thus propose the following hypothesis for the relationships among the species of Monanema. The single parasite in a sciurid, M. marmotae, represents a line with a combination of primitive and derived characters (Webster, 1967). Among the species parasitic in murids, the four species in Africa form another line, sharing the primitive character of a small spicular ratio (≤ 3.6), but diversified with respect to other characters: in West and West-Central Africa, M. martini (Bain et al., 1985;Original contribution Parasite, 2012, 19, 331-340 unpublished data); in South Africa, M. joopi n. sp.; in East Africa, M. nilotica (El Bihari et al., 1977); and M. globulosa from mountains in Kenya (1,500 meters of altitude). In Australia, M. australe represents a third evolutionary lineage (Spratt, 2008).

IDENTIFICATION KEY TO THE SPECIES
The Afrotropical region has the highest number of species but is unlikely the place of origin of Monanema. Rodents most likely originated from Eurasia (Wilson & Reeder, 2005;Jansa et al., 2009). During the Miocene/Pliocene, murids dispersed into Africa via the Arabian Peninsula (Winkler, 1994) and into Australia, via South-East Asia and New Guinea (Godthelp, 2001). Marmots, on the other hand, first arose in North America and spread into Eurasia via the Bering land bridge (Steppan et al., 1999). The origin of Monanema is probably the Palaearctic-Oriental region. If a protocol for the detection of dermal microfilariae was included in helminth diversity studies, more species of Monanema would likely be discovered. Particularly, the important finding by Spratt (2008) of a species in an Australian murid, Melomys Thomas, 1922, reveals that representatives of Monanema can be expected in the Indomalayan and Australasian region. To date, only the 12s rDNA sequence of M. martini is known (Ferri et al., 2011). In future, additional molecular studies might help to elucidate the phylogenetic relationships within this genus and to establish its taxonomic position within the Filarioidea.
Filariae with dermal microfilariae are rarely detected. Even in animals as well-studied as dogs and rodents, several new species have been reported in the past ten years, for example Onchocerca lupi (Sréter & Szell, 2008) and Cercopithifilaria spp. (Otranto et al., 2011). This is examplified in Monanema in which only six species have been described in over 40 years, although their current geographical and host distribution suggests a much higher diversity.