Redescription of Setaria graberi Shoho in Troncy, Graber & Thal, 1976 (Nematoda: Filarioidea) based on specimens from Redunca arundinum (Bovidae) in South Africa

The filarial onchocercid Setaria graberi Shoho in Troncy, Graber & Thal, 1976 is redescribed from the abdominal cavity of Southern reedbuck, Redunca arundinum (Boddaert), in South Africa, including illustrations and scanning electron micrographs of important morphological features. Morphometric data for this species are provided for the first time. Setaria graberi is characterised by the possession of bifid deirids, and females having a distinctly bulbous tail. The slightly raised peribuccal crown forms a dumbbell-shaped unit with the cephalic elevations in apical view; the dorsal and ventral elevations, spaced 73–115 μm apart in females and 71–93 μm in males, carry two well-separated tips each. In dorsoventral view, the cephalic elevations appear more or less rectangular with a slightly notched apex and are narrow in comparison to the width of the anterior end. They are triangular in lateral view. Four cephalic and four external labial papillae are arranged in a laterally elongated rectangle each. The species is distinguished from other Setaria Viborg, 1795 species that possess bifid deirids or occur in members of the same host genus. The presence of S. graberi in R. arundinum in South Africa constitutes a new host and geographic record.


Introduction
While studying the parasite fauna of artiodactylids in Tanzania between 1964 and 1967, Sachs & Sachs [9] recovered onchocercid specimens belonging to the genus Setaria Viborg, 1795 from the abdominal cavity of a number of hosts, including Common reedbuck, Redunca redunca (Pallas). These specimens were forwarded to Dr. C Shoho, then Animal Health Officer of the FAO in Somalia, for identification. Subsequently, Shoho in 1967 informed his colleagues in a personal communication (quoted by Sachs & Sachs [9]) that the material from R. redunca contained two Setaria species, S. boulengeri Thwaite, 1927 and a yet undescribed species, provisionally named S. graberi. At the time, the description of S. graberi was held back, as Shoho was in the process of preparing a revision of the genus Setaria in African hosts (Shoho, personal communication 2000). Shoho's intention was to publish this manuscript, including the description of S. graberi as a new species, in collaboration with Dr. Sachs at the Tropical Institute in Hamburg. The latter, however, left the institute and the manuscript was never published. A decade later, Shoho made his unpublished manuscript entitled ''Setaria worms from wild animals, mostly ruminants, of Eastern and Southern Africa and some other filarid worms'' available to Troncy et al. [11], when these authors examined material from R. redunca nigeriensis Blaine in the Central African Republic and the Republic of Chad. Troncy et al. [11] accepted S. graberi as described by Shoho as a valid species and assigned their specimens to this taxon, listing Shoho as author. In the absence of a published description, Troncy et al. [11] provided detailed drawings of their specimens of S. graberi and stated that their material corresponded well with the illustrations provided in Shoho's manuscript.
Through the kind facilitation of Prof. S. Uni, Osaka City University, Japan, Dr. Shoho forwarded parts of his unpublished manuscript that dealt with the ''Group of Setaria worms from the Reedbucks, Redunca arundinum Boddaert and Bohor Reedbuck, Redunca redunca bohor Pall.'' to the present authors. According to this part of the manuscript, which does not include any illustrations, the material of S. graberi studied by Shoho comprised one male and one female specimen from Southern reedbuck, R. arundinum (Boddaert), from Pilgrimsrest, Mpumalanga Province (then Transvaal), South Africa, which had been on loan from the National Collection of Animal Helminths (formerly the Onderstepoort Helminthological Collection), and six females and an unspecified number of males from R. redunca from Lake Rukwa, Tanzania; the latter originating from the collection of Sachs & Sachs [9]. Measurements were based on one male and one female from R. redunca, and ''type material (was) preserved at the London School of Hygiene and Tropical Medicine''. Attempts to retrieve these specimens failed In this paper, we describe and illustrate S. graberi from material collected by Boomker et al. [1] from R. arundinum in KwaZulu-Natal, South Africa. Re-examination of said material showed that, in addition to S. bicoronata (Linstow, 1901) Railliet & Henry, 1911 and females of Setaria sp. recorded by Boomker et al. [1], S. graberi was also present. Scanning electron micrographs of important diagnostic characters are provided. We conclude that S. graberi Shoho in Troncy, Graber & Thal, 1976 is a valid species that can morphometrically be distinguished from all other members of the genus. The presence of S. graberi in R. arundinum in South Africa constitutes a new host and geographic record.

Materials and methods
Specimens examined during the current study were collected and stored in 70% ethanol. They had been obtained during a previous study by Boomker et al. [1], a survey of the helminth community of R. arundinum in the Eastern Shores Nature Reserve (ESNR), KwaZulu-Natal, South Africa, from 1983 to 1984. The ESNR is situated at the southern end of the Mozambique coastal plain, between 27°51 0 and 28°25 0 S latitude and 32°20 0 and 32°40 0 E longitude [5].
Body length was determined by gently straightening specimens on a ruler and taking the reading using a stereomicroscope. Subsequently, nematodes were cleared in lactophenol and examined using a compound microscope (Olympus BX51) equipped with a drawing tube and digital camera (Olympus DP72). Measurements were taken with the aid of digital imaging software (Olympus cellSens Dimension, version 1.4.1). Drawings were made with the aid of the drawing tube. Microfilariae were dissected from the uterus of one female at the level of the oesophago-intestinal junction and cleared in lactophenol. Specimens for scanning electron microscopy (SEM) were dehydrated through a graded ethanol series and critical point-dried from absolute ethanol. Subsequently, head and tail segments were mounted individually onto conical brass SEM viewing stubs and sputter coated with gold. Samples were viewed and micrographed using a Hitachi S-2500 scanning electron microscope at an accelerating voltage of 8 kV.
Measurements are given as the range, with the mean and the number of measurements taken (n) in parentheses; when available, this number is followed by measurements of a single male and female each from R. r. bohor from Tanzania, described in Shoho's unpublished manuscript (Shoho, personal communication 2000). Measurements are in micrometres, n = 8 for males and n = 9 for females, except when otherwise indicated. Male caudal papillae are numbered following Chabaud & Petter [2].
Collection date: May 1983 to May 1984.

Site of infection: Abdominal cavity.
Material deposited: The specimens have been deposited in the collection of the Muséum National d'Histoire Naturelle, Paris, France, MNHN HEL358 (five males, five females) and the National Collection of Animal Helminths, ARC-Onderstepoort Veterinary Institute, South Africa, NCAH/S/2013/2 (three males, four females).
Prevalence and intensity: In 18 out of 22 (82%) host individuals that were re-examined, with intensity of infection ranging from 1 to 17 and a mean of 4.3 (±4.3).

Description (Figures 1-4)
Long and slender filarial nematodes, off-white in colour. Four cephalic papillae and four external labial papillae arranged in laterally elongated rectangle each ( Figure 4C); amphids approximately on mid-level between cephalic and external labial papillae (Figures 1B and 4C). Oral opening small, oval to round. Peribuccal crown slightly raised, forming dumbbellshaped unit with cephalic elevations in apical view ( Figures 1B and 4C); circular base of dorsal and ventral elevations tapering towards narrow midsection that surrounds oral opening; long axis dorsoventrally orientated ( Figure 1B). Both dorsal and ventral elevations drawn out into two, well-separated, stubby tips in apical view ( Figure 4C); elevations stumplike in median view, with narrow base in relation to width of head, more or less parallel sides and a notched apex ( Figures  1C, 2B and 4B), triangular in lateral view ( Figures 1A, 2A  and 4A). Deirids at level of posterior half of muscular oesophagus, in females usually close to level of vulva ( Figure 1C); bifid, situated on small, rugous, slightly inflated promontories; the two projections elongated, conical, with rounded tips, one slightly shorter, the difference in length more or less pronounced depending on specimen orientation, but most distinct in apical view ( Figure 4D  conical lateral appendages; base of caudal appendages at 24-34 (29) from tail tip and 7-10 (8) long. In a single male of which electron micrographs were taken, the phasmid is distinct at the base of the lateral appendage ( Figure 4C). A single postdeirid situated on left side, at 503-1070 (751) from tail tip ( Figure 3B). Nine precloacal papillae; a single median papilla, slightly anterior to fourth precloacal pair, close to cloaca, and four subventral pairs (pairs 1-4). Six pairs of postcloacal papillae: five pairs anterior to pointed lateral caudal appendages ( Figures 1I and 3B), a small single pair posterior to appendages; pairs 5-8 and pair 10 subventral, pair 9 in laterodorsal position; pair 6 larger than remaining papillae (Figures 1I and 3B).  long, with short vagina vera and well-developed, posteriorly directed vagina uterina that forms a chamber that is joined by ovejector in its posterior third ( Figure 2C). Tail, curved dorsally, 384-545 (483) long, with conical lateral appendages, tip of tail on conspicuous, bulbous swelling ( Figures 1F and 3A, F); base of caudal appendages at 38-47 (43; 47) from tail tip and 9-12 (11) long. Phasmids not seen.

Discussion
The measurements of specimens of S. graberi studied herein correspond closely to those given for ''Setaria graberi n. sp.'' in Shoho's (personal communication, 2000) manuscript. In addition, Shoho refers to the deirids as being a ''bifid spiny formation'', as seen in the present specimens. With reference to cephalic structures, Shoho states that the ''peribuccal ring, surrounding the small round mouth, appears relatively smaller compared with the width of the anterior part of the body'', and describes it as ''appearing as if drawn out dorsally and ventrally''; a description that fits the present specimens well. He also points out that the ''more obtusely ending terminal end of the female, with distinctly rounded knob'' readily distinguishes this species from, amongst others, S. bicoronata. The bulbous tail is a very conspicuous character in the females described herein.
Regarding the male tail, Shoho lists a ''deirid-like formation on the left side at the level of the distal part of the retracted longer spicule'', four pairs of precloacal papillae, plus a median papilla situated close to the anterior border of the cloaca, as well as five pairs of postcloacal papillae, anterior to the ''sharply pointed lateral appendages'', plus two smaller pairs of papillae posterior to the appendages. This corresponds well with the arrangement of the postdeirid and precloacal papillae in the present material, and with the illustration of S. graberi by Troncy et al. [11]. However, as in the latters' illustration, the current specimens possess only six pairs of postcloacal papillae, one of which is situated posterior to the lateral appendages.
The illustrations of Troncy et al. [11] are so detailed and representative of the current material, especially regarding the peribuccal crown and cephalic elevations in lateral, dorsoventral as well as apical view, shape of the female tail and arrangement of caudal papillae in the male, that they are reproduced in this paper. A single exception is the deirids, which are illustrated as having a single tip ( Figure 1D). Their bifid character is difficult to observe in light microscopy specimens, but readily visible using SEM. Neither Shoho nor Troncy et al. [11] comment on the male tail being spirally coiled, but they may have omitted this as a common feature in Setaria males, as well as other filarial worms.
In view of the distinct similarity between the current specimens and those described as S. graberi in Shoho's unpublished manuscript and those illustrated as such by Troncy et al. [11], we conclude that the specimens described herein belong to S. graberi. This is supported by the pronounced differences between the present material and other Setaria species from the same host genus and/or Setaria species possessing bifid deirids (see below). It should also be noted that some of Shoho's material was of the same geographic origin, South Africa, as the specimens studied herein. In the absence of any of the previously described specimens, voucher material for this species has been deposited in two curated collections.
Excepting S. graberi, a further six Setaria species parasitize members of the genus Redunca Smith (see below). Of these, S. hornbyi Boulenger, 1921, S. labiatopapillosa (Alessandrini, 1848) Railliet & Henry, 1911 and S. lamyfortensis Troncy, Graber & Thal, 1968 differ from S. graberi in the possession of deirids with a single tip [4,11]. The configuration of the deirids of S. boulengeri is unknown, and specimens for re-examination could not be obtained. However, Yeh's [14] description of the species differs from that of S. graberi in the distinctly raised peribuccal crown of S. boulengeri and an undivided dorsal as well as ventral elevation.
Similar to S. graberi, the deirids in S. bicoronata and S. pillersi Thwaite, 1927 are bifid [4], but the dorsal and ventral cephalic elevations of S. pillersi are square-shaped, undivided and spaced only 30-40 lm apart, as opposed to the bifid cephalic elevations in S. graberi, that are 71-93 lm apart in males and 73-115 lm in females.
Setaria graberi most closely resembles S. bicoronata in the presence of bifid deirids, but also in the configuration of the cephalic elevations in lateral and dorsoventral view. Examination of the latter structures in apical view, however, reveals distinct differences. While the apical view of the peribuccal crown together with the dorsal and ventral elevations portrays a more or less distinct rectangle in S. bicoronata [11], it is dumbbellshaped in S. graberi. Also, in lateral view, the outer border of the dorsal and ventral elevations extends further posteriad on the head of S. bicoronata than in S. graberi, where the elevations appear perched on the anterior part of the apex ( Figures  1A and 2A). In addition, the conspicuous bulbous swelling of the female tail sets S. graberi apart not only from S. bicoronata, but from all other Setaria species in African artiodactylids.
A further two Setaria species with bifid deirids have been described from hosts other than Redunca, S. saegeri (Le Van Hoa, 1961) from Sylvicapra grimmia Linnaeus in the Congo and S. kabargi Kadenazii, 1948 from Moscus moschiferus Linnaeus in Central Asia [4,13]. However, both species are readily distinguished from S. graberi by the shape of their flat-topped cephalic elevations [4,13].
The following Setaria species have been recorded from hosts of the genus Redunca, including R. arundinum, Mountain reedbuck R. fulvorufula (Afzelius) and R. redunca, in Africa: