Two new genera of nematode (Oxyurida, Hystrignathidae) parasites of Passalidae (Coleoptera) from the Democratic Republic of Congo

Abstract Two new genera and species parasitizing passalid beetles from the Democratic Republic of Congo are described. Kongonema meyeri gen. n. sp. n. is characterized by having females with the cervical cuticle unarmed, first cephalic annule cone-like and truncate, sub-cylindrical procorpus and genital tract didelphic-amphidelphic. The males of Kongonema meyeri gen. n. sp. n. have the procorpus sub-cylindrical, the dorsal cuticle of the tail end thickened, a single large, median mammiform pre-cloacal papilla and a pair of small, pre-cloacal, sub-lateral papillae at a short distance before the level of the cloaca. Lubanema decraemerae gen. n. sp. n. is characterized by the body markedly fusiform, cuticle unarmed and strongly annulated, procorpus sub-cylindrical, isthmus as a constriction between procorpus and basal bulb, genital tract monodelphic-prodelphic and the posterior end rounded with a very short tail appendage.


introduction
The family Hystrignathidae comprises 27 nominal genera with more than 100 species of monoxenous nematodes specific of the hind gut of passalid beetles. The family shows a mostly Gondwanian distribution, with taxa from North, Central and South America, West Indies, Africa and Australasia (Adamson and Van Waerebeke 1992). Of these areas, the Americas and West Indies present the highest generic and specific diversity.
In Africa, the group still remains neglected with most of the species restricted to their type localities. Théodoridès (1955) described the first African hystrignathids: Artigasia pauliani Théodoridès, 1955 andA. geopetiti Théodoridès, 1955 from Malagasian passalids. Later, Théodoridès (1958) described A. pauliani var. joliveti from the Democratic Republic of Congo. The status of this variety was analyzed by Adamson and Van Waerebeke (1992) who raised it to the rank of species. Baker (1967) recorded Hystrignathus rigidus Leidy, 1850 and Xyo hystrix Cobb, 1898 parasitizing three species of Pentalobus from Ghana. These two latter species had previously been described from North America and Australia, respectively. The main contribution to the knowledge of the family in the region was made by Van Waerebeke (1973), with the description of 14 species of Artigasia Christie, 1934 one of Hystrignathus Leidy, 1850 and the monotypic genus Passalidophila Van Waerebeke, 1973 all from Madagascar. The author also re-described A. geopetiti and recorded three types of males, unable to be assigned to their correct species. Van Waerebeke and Remillet (1982) described Hystrignathus egalis Van Waerebeke &Remillet, 1982 andH. inegalis Van Waerebeke &Remillet, 1982 from Ivory Coast. This paper retakes the study on African hystrignathids, describing two new genera and species parasitizing passalid beetles from the Democratic Republic of Congo.

Material and methods
Several specimens of passalid beetles from the Democratic Republic of Congo (formerly Zaire) were examined in a parasitological survey during a research visit to the Royal Museum of Central Africa, Tervuren, Belgium. Eight specimens of Didimus sp. and two specimens of Erionomus pilosus Aurivillus, 1896 from Katale, Kivu region were included in this study, all collected during the Belgian expeditions to the Congo in the 1930´s and stored in 70% ethanol.
The hosts were dissected by practicing incisions in both pleural membranes and the intestines were extracted and kept in Petri dishes with 70% ethanol. The guts were excised and the parasites removed.
Nematodes were transferred and cleared in glycerine via slow evaporation method (Seinhorst 1959) and mounted in the same medium. The edges of the coverslips were sealed using nail polish. Measurements were made with a calibrated eyepiece micrometer attached to a compound microscope. De Man's ratios a, b, c and V% were calculated. Each variable is shown as the range followed by the mean plus standard deviation in parentheses; the number of measurements is also given. Micrographs were taken with an AxioCam digital camera attached to a Carl Zeiss AxioScop 2 Plus compound microscope. Line drawings were made with the softwares CorelDRAW X3 and Adobe Photoshop CS2 using the micrographs as templates. Scale bars of all plates are given in millimeters.
Some specimens were prepared for SEM as follows: they were dehydrated in a graded ethanol series, critical point-dried, mounted in aluminum stubs and coated in gold. SEM micrographs were taken at an acceleration voltage of 22-25 kV.
Classification at generic level was followed after Adamson and Van Waerebeke (1992). For comparison, one paratype of Passalidophila exceptionalis Van Waerebeke, 1973;deposited  Lateral alae present, from the oesophageal region to a short distance beyond the level of the anus. Posterior ends of the lateral alae rounded, forming lobes. Head bearing eight paired papillae. First cephalic annule cone-like, truncate, barely inflated, about two head-lengths long. Oesophagus consisting of a muscular sub-cylindrical procorpus, its base well set-off from the isthmus. Nerve ring encircling procorpus at its midpoint. Excretory pore post-bulbar. Reproductive system didelphic-amphidelphic. Eggs ovoid, ridged-shelled. Tail filiform and subulate.
Description. Female. Body comparatively robust, widening from the base of the first cephalic annule, maximum body diameter at level of the vulva, then tapering towards anus. Cervical cuticle unarmed, markedly annulated (annuli ca. 5-7 µm). Rest of the body with marked annuli decreasing their width towards the level of the anus. Subcuticular longitudinal striae present. Lateral alae ca. 9 µm wide, from the oesophageal region (ca. 30 µm before the level of the nerve ring) to a very short distance beyond the level of the anus. Posterior ends of the lateral alae rounded, forming short lobes. Head well developed, set-off from body by a single, deep groove and bearing eight rounded, paired papillae. Amphids pore-like, laterally situated. Mouth sub-triangular in shape. First cephalic annule cone-like, truncate, barely inflated, about two head-lengths long. Stoma comparatively long, about three first cephalic annule lengths long, surrounded by an oesophageal collar. Oesophagus consisting of a muscular, sub-cylindrical procorpus, its base slightly wider and well set-off from the short isthmus. Basal bulb subspherical, valve plate well developed. Intestine simple, sub-rectilinear, anterior portion dilated. Rectum short, anus not prominent, as a crescent-like slit. Nerve ring encircling procorpus at about its midpoint. Excretory pore situated at about half of a body width posterior to basal bulb. Genital tract didelphic-amphidelphic, both ovaries reflexed. Anterior ovary reflexed behind the excretory pore, posterior ovary reflexed at about a body width before the anus. Distal flexures of ovaries about one body widthlength long. Oöcytes in single rows. Vulva a median transverse slit slightly displaced to the posterior half of body, lips prominent. Vagina muscular, forwardly directed. Eggs ovoid, bearing eight longitudinal, rough, ridges on the shell. Tail comparatively long, filiform, subulate, ending in a sharp point.
Male. Body shorter than female, comparatively slender, posterior region ventrally curved. Cervical cuticle unarmed. Sub-cuticular longitudinal striae present. Lateral alae from the oesophageal region (about three body-widths posterior to the cephalic end) to the level of the single mammiform papilla (about a body-width before the level of anus). Head not set-off from body. First cephalic annule not developed. Stoma not defined. Oesophagus consisting of a sub-cylindrical procorpus, well set-off from the short isthmus. Basal bulb rounded, valve plate well developed. Intestine simple, anterior portion slightly dilated. Nerve ring encircling procorpus at its posterior half, about 65% of its length. Excretory pore situated at about 1.5 bodywidths posterior to basal bulb. Monorchic. Testis outstretched, arising at a short distance behind the excretory pore. Spicule absent. Dorsal cuticle of the tail region thickened. A single, large, pre-cloacal ventromedian mammiform papilla situated at about a body width before the posterior end. A pair of small, pre-cloacal, sub-lateral papillae situated at a short distance before the level of the cloaca. Tail region becoming sharp visibly from the beginning of the cuticular thickening, until forming a very short tail appendage, its tip rounded. Discussion. There are three genera of hystrignathids the female of which present the cervical cuticle unarmed, procorpus sub-cylindrical and reproductive system digonant: Anomalostoma Cordeira, 1981; Coynema (Coy, García & Alvarez, 1993) Morffe &García, 2011 andVentelia Travassos &Kloss, 1958. The first differs by having the anterior region of the procorpus strongly swollen, surrounding the stoma (Cordeira 1981). The stoma of Kongonema gen. n. is surrounded only by an oesophageal collar, as occur in many hystrignathids. Anomalostoma lacks an evident first cephalic annule vs. conspicuous first cephalic annule of Kongonema gen. n.
Females of Coynema can be segregated by the basal dilatation of its procorpus and the anterior region of the intestine notably inflated, forming a saccular structure (Morffe and García 2011). Both traits are absent in Kongonema gen. n., which procorpus increases its diameter slightly and gradually towards its base and the fore region of the intestine is only moderately inflated, without the saccular structure mentioned above. The oviduct next to the vagina forms a loop in Coynema, instead of the straight oviduct of the present genus.
The males of Kongonema gen. n. resemble their counterparts of Coynema (only close genus where the male is known) by lacking of spicule and by having a similar arrangement of the copulatory papillae: the ventromedian pre-cloacal papilla (typical of Hystrignathidae) and another pair of small sub-lateral pre-cloacal papillae. Kongonema gen. n. differs by having a sub-cylindrical procorpus, without the basal dilation and by lacking the saccular region of the intestine characteristic of Coynema. The posterior end of Kongonema gen. n. forms a short, rounded tail appendage vs. the sharp tail of Coynema.
Description. Female body large, notably robust and fusiform, widening gradually from the base of the first cephalic annule, reaching maximum width near mid-body, then tapering softly towards the posterior end that rounds off abruptly. A comparatively very short, conical tail appendage with its tip rounded arises terminally from the posterior end. Cervical cuticle unarmed, with marked annule (ca. 13 µm wide), extending to the rest of body, until level of the anus. Lateral alae thick, ca. 55 µm wide, extending from the hind third of the procorpus to the level of the anus. Posterior ends of lateral alae almost forming a straight angle with the body axis, slightly convex, their external margins forming a very short lobe. Head bearing eight rounded, paired papil- lae, set-off from body by a single, deep groove. Amphids pore-like, laterally situated. Mouth trirradiate. First cephalic annule cone-like, slightly inflated, its margins convex, about two head-lengths long. Stoma long, about 1.5 first cephalic annule lengths long, surrounded by an oesophageal collar. Oesophagus consisting of a muscular, subcylindrical procorpus, its diameter little increased at its base. Isthmus as a constriction between the procorpus and the large, rounded, basal bulb. Valve plate well developed. Intestine simple, sub-rectilinear, its fore region very inflated. Rectum short. Anus subterminal. Nerve ring encircling procorpus at its posterior half (ca. 60% of its length). Excretory pore located at about the half of a body width behind the basal bulb. Vulva a median trasverse slit, displaced to the posterior half of body, lips less prominent. Vagina muscular, forwardly directed. Genital tract monodelphic-prodelphic. Ovary stout, reflexed at about one third of the body width behind the basal bulb. Oöcytes in a single row, about four times wider than long (ca. 8×2 µm). Eggs large, markedly ovoid, smooth-shelled. Male unknown.
Discussion. The Malagasian genus Passalidophila resembles Lubanema gen. n. by having both the body robust and fusiform, cervical cuticle unarmed and markedly annulated, a similar form of the cephalic end, the lateral alae extending from the level of the procorpus to the anus and the tail short (Van Waerebeke 1973). Differs by having a procorpus which diameter increases towards its joint with the isthmus. Lubanema gen. n. have a more cylindrical procorpus and the isthmus is absent. The tail of Passalidophila is subulate, instead of the current new genus, which presents a very short tail appendage arising from the rounded posterior end. In addition, the ovary of Passalidophila is slender vs. the robust ovary of Lubanema gen. n.
Coronocephalus bears prominent, digitiform oral papillae, instead of the shorter, less developed papillae of Lubanema gen. n. In the latter genus the procorpus meets directly the basal bulb, while Coronocephalus present an isthmus. Glaber differs from Lubanema gen. n. by having the base of the procorpus clavate, instead of the subcylindrical procorpus present in the new genus.
Vulcanonema presents the cephalic end consisting of a narrow cephalic annule separated of the head by a conical region. In opposition, Lubanema gen. n. have the first cephalic annule just after the head. Also, the procorpus of Vulcanonema is subcylindrical, with a basal dilation, absent in the present new genus.
Lubanema gen. n. shows morphological affinities with the Australian genera Anuronema Clark, 1978 andSprentia Clark, 1978 by having the cuticle unarmed and strongly annulated, reduction of the isthmus and the tail. Moreover, the lateral alae of Anuronema extends from the oesophageal region to almost the level of the anus, similar to Lubanema gen. n. The new genus differs from both by its genital tract monodelphic-prodelphic vs. didelphic-amphidelphic, procorpus sub-cylindrical vs. claviform and development of the lateral alae, which in Lubanema gen. n. are very wide and with lobes in the margins at their terminal ends. The procorpus is widely amalgamated with the basal bulb in Anuronema and Sprentia, whereas Lubanema gen. n. presents a well defined constriction separating both structures. Anuronema has a total reduction of the tail appendage (Clark 1978) not observed in Lubanema with a short, conical tail.
Carlosia Travassos & Kloss, 1957 also presents a reduction of the isthmus, a large, slightly inflated first cephalic annule and marked annule in the cervical region (Hunt 1982). It can be easily segregated from Lubanema gen. n. by having a didelphic-amphidelphic genital tract and the annule of the cervical cuticle retrorse, with posterior prolongations forming a double row of spines laterally situated.