New biogeographical and morphological information on Physaloptera ngoci Le-Van-Hoa, 1961 (Nematoda: Physalopteridae) in South-east Asian rodents

During a study of the helminth fauna of 1,643 rodents trapped along the Mekong River (Thailand, Lao People’s Democratic Republic and Cambodia) in 2008–2011, the spirurid nematode Physaloptera ngoci Le-Van-Hoa, 1961 was recovered with an overall prevalence of 2.8%. Based on the original description, it was identified in nine of 23 different Murinae host species and is here reported for the first time from these three countries. A scanning electron microscopy study provides additional morphological data.

Bearing the above-mentioned undetermined species in mind, Physaloptera ngoci is to date the only known species of this genus in Muridae from SEA. Physaloptera ngoci was also described from Vietnam in Rattus norvegicus [5] and subsequently recorded in Mus sp. and Rattus sp. [14].
In the present study, rodents were captured in SEA during 2008-2011 and their helminths, including P. ngoci, recovered.

Materials and methods
Murid hosts were captured at nine different sampling sites along the Mekong River in Thailand, the Lao People's Democratic Republic (PDR) and Cambodia (see Figure 1). The rodents were trapped with baited traps, either locally made or Sherman. At each sampling locality, ten trap lines (composed of ten traps, placed every five metres) were set over a period of 12 nights. Trap lines were moved every four nights. Complementary trapping was carried out in villages and isolated houses, with five traps per house. Rodents were identified by morphology or using species-specific primers and/or barcoding assignments (details available in the Barcoding Tool/ RodentSEA section of the CERoPath project website: www.ceropath.org). Rodents were euthanized and dissected following protocols that maximize animal care, the health and safety of field parasitologists and the generation of quality data.
Viscera were examined under a stereomicroscope and helminth parasites were collected and preserved in 70% ethanol.
Nematodes were cleared in lactophenol and hand-cut sections of the male anterior end were mounted en face for identification. Females were dissected in order to count the number of uteri. Photographs and measurements were taken with a microscope mounted on a Tucsen camera with associated software. Measurements taken from 10 adult individuals of each sex (males from Bandicota berdmorei, Bandicota indica and Bandicota savilei and females from B. berdmorei, B. indica and R. argentiventer) are given in micrometres unless otherwise specified and listed as range and median.
For Scanning Electron Microscopy (SEM) studies, nematodes were fixed in 70% ethanol, critical point-dried, mounted on SEM studs and finally coated with a thin layer of platinum before being examined with a Zeiss DSM 940A Scanning Electron Microscope at an accelerating voltage of 15 kV.

Results and discussion
A total of 1,643 rodents belonging to 23 species were examined and in nine of these species a nematode belonging to the genus Physaloptera was isolated from the stomach and identified as P. ngoci (details of hosts and prevalence are summarized in Table 1). The general prevalence was 2.8%, the highest prevalence being 40% in R. argentiventer (n = 10) from Veal Renh, Cambodia. The observation of structures in the cephalic region, the distribution of the cephalic papillae, the arrangement and number of male caudal papillae, the size of the male spicules, the number of female uteri, the total length and the distance from the anterior end to the vulva all confirm the identification of this helminth as P. ngoci, as per the original description [5]. The cephalic region (Figure 2A-F) bears two semicircular pseudolips, each bearing a pair of papillae, one amphid, a single externolateral tooth and a trident-like internolateral tooth. The arrangement of the 21 caudal papillae in males is as follows: (i) four pairs of pedunculate papillae (one pair precloacal, one adcloacal and two postcloacal) and three single sessile papillae, The morphology of our samples confirms the identification of this nematode as P. ngoci given the similarity of structures and their disposition in the cephalic region, the same number and similar arrangement of male caudal papillae, and the same number of uteri in the females. In addition, our measurements match those provided in the original description: left and right spicule 460 and 300 long, respectively, total length 25 mm in females, distance from the anterior end to the vulva 3.32 mm and egg size 28 · 45 [5].
Physaloptera inermis differs from P. gnoci in the number of male caudal papillae (eight pairs) and longer spicules 2.37 mm [6] (no differences between right and left spicule specified). The total female length is greater 51 mm [6]. The distance from the anterior end to the vulva is not given in the original description. Egg size is similar 26 · 47 [6].
Physaloptera sciuri differs in the number (15) and disposition of male caudal papillae (no sessile precloacal papillae, one unpaired sessile postcloacal papilla and the final pair of postcloacal papillae close to the tail). Spicule lengths are not provided in the original description. The total female length 17-32 mm is similar [13]. The vulva is approximately halfway down the body and we were clearly able to differentiate its position from that in P. gnoci, in which it is more anteriorly. The eggs are smaller 16 · 22 [13].
SEM studies are currently available for four species of Physaloptera: Physaloptera sibirica Petrow & Gorbunow, 1931 in mammals from Spain [9]; Physaloptera bispiculata Vaz & Pereira, 1935 in the South American water rat Nectomys squamipes from Brazil [8]; Physaloptera sp. in an anuran from Argentina [2]; and Physaloptera herthameyerae Lopes et al., 2009 in a marsupial from Brazil [7]. The present study increases the number of SEM studies in Physaloptera to five and is the first such study done on a species from hosts in SEA. This is the first report of P. ngoci from rodents in Cambodia, Lao PDR and Thailand. While its original description was based on specimens from a single host species, the brown rat (R. norvegicus) from Vietnam, several new hosts are listed for P. ngoci in the present study. Its host spectrum now includes several species of the subfamily Murinae; likewise, its known geographic range is expanded to include SEA. Previous reports of Physaloptera spp. in SEA murine rodents can thus probably be assigned to P. ngoci given that our data confirm that this nematode is widespread in SEA.