A new genus and species of pinworm (Nematoda, Oxyuridae) from the gray mouse opossum, Tlacuatzin canescens

Tlacuatzoxyuris simpsoni n. gen. n. sp. is described from the cecum of the gray opossum, Tlacuatzin canescens, a species endemic to the deciduous dry forest of Mexico. The digestive tracts of four specimens were examined for parasites; three of these were archived in the American Museum of Natural History and one was a live capture. Relative to the other four monotypic genera of pinworms known to infect opossums, the new genus is diagnosed on the basis of a round cephalic plate with a semicircular stoma surrounded by a rim. In addition, males feature a prominent cephalic vesicle not fully developed in females, accounting for sexual dimorphism. The new species includes small worms that feature a conspicuous, not reticulated cephalic vesicle and semicircular stoma and lateral alae with two crests. In addition, the postcloacal cuticle of males features a small area with ornamentation between cloaca and submedial papillae. Finally, both spicule and gubernaculum are relatively short. Although the eggs of Tlacuatzoxyuris n. gen. are unknown, the conspicuous differences in traits used in the diagnosis of genera prompted us to propose a new genus for the new species. This is the first species of Oxyuridae reported in mouse opossums outside South America, and the fifth species of the family occurring in didelphimorph marsupials. This is an example of the usefulness of documenting the diversity of parasites associated with this unique clade of mammals through the examination of preserved tissues.


Introduction
Marsupials are a typical component of the Neotropics [28,30,31]. In the Americas, this group of mammals includes three families in three orders of which Didelphidae (Didelphimorphia: Didelphidae) contains roughly 110 species of small to medium-sized marsupials [11,23,27,32]. These diversified into arboreal, scansorial, terrestrial, and semiaquatic habits [4]. The continuous investigation of the basic biology, distribution and diversity of these marsupials has allowed researchers to document the isolation of some populations, to establish the geographic and taxonomic limits for several genera, and to generate evidence to formally describe new didelphimorph taxa [3,14,16,29].
Perhaps because of their ubiquitous nature and high abundance, most helminthological records are centered in four species of medium-sized opossums [1,19,25], yet very few helminth taxa had been described from small sized marsupials of arboreal habitats [2,5,7,9,13,17,18,20,22,24]. Among those, four monotypic genera of nematodes infecting arboreal and terrestrial mouse opossums have been described in South America. These include Neohilgertia venusti Navone [8,12,15,21]. Most of these species are characterized by their relatively short tail and by the presence of numerous mamelons on the ventral side. The criteria to differentiate each of these monotypic genera include the configuration of the stoma or buccal opening, presence of a cephalic vesicle, as well as the ornamentation of the egg shell. The stoma in the four known species is an important element in the diagnosis of these taxa; it can be surrounded by three "lips", which cover 3-4 pharyngeal teeth. The cephalic vesicle is either absent, or thick, with conspicuous internal vesicles in the females of D. thylamisis. Finally, the eggs have been characterized as either operculated or nonoperculated, and the eggs of G. agilisis feature three longitudinal ridges which are used in the diagnosis of the genus.
Herein, we describe a new species of pinworm found in the large intestine of the gray opossum, Tlacuatzin canescens (Allen, 1893) in the dry forest of southern Mexico. Since it shows clear differences in the configuration of the cephalic vesicle and the stoma with the other four monotypic genera, we propose a new genus to include it. This is the fifth oxyurid species identified in didelphid marsupials and the first one occurring north of the basin of the Amazon river. The finding of this nematode in a marsupial north of the Panama isthmus has implications relative to the dispersal of fauna of South American origin into the North American tectonic plate, which is part of the reciprocal biotic exchange among the two landmasses of the New World known as the Great American Biotic Interchange.

Materials and methods
A single gray mouse opossum was hand trapped in the locality of Cruz Pintada: Morelos: Mexico (18°27 0 58 00 N, 99°2 0 16 00 W, 1016 m) on 10 June 2010. The individual was humanely euthanized and examined for parasites 12 h after trapping, following methods approved by the Institutional Animal Care and Use protocol 09-026 (Assurance Number A-3078-01). Specimens resulting from this examination were fixed in 4% buffered formaldehyde and subsequently transferred to 70% ethanol for storage. In addition, the digestive tracts of three gray opossums and three Mexican mouse opossums (Marmosa mexicana Merriam, 1897) collected in the isthmus of Tehuantepec were requested from the American Museum of Natural History (AMNH) (New York, NY). These tissues were rinsed and cut open, all removed metazoans were washed and saved in 70% ethanol. Collection numbers and locations for these specimens are detailed in Table 1. All 15 pinworms found were cleared in temporary mounts of lactophenol or glycerine. All measurements are given in micrometers and detailed in Table 2; for each character, the range is given first, followed by mean and coefficient of variation [26]. After clearing, two specimens, a male and a female, were washed and then dehydrated progressively in a graded ethanol series, dried to a non-liquid state by critical point drying using CO 2 , attached to an SEM stub, and sputter coated with gold palladium. Specimens imaged with SEM were exposed to a beam of 2-10 kV on a FEI Quanta  Nerve ring  91  106  112  98  100  83  91  85  86  102  87  89  89  73  89  Excretory pore  504  452  480  351  337  322  341  469  418  324  342  286  Vulva  591  538  594  428  425  388  411  505  405  Testis  570  472  474  280  Start area rugosa  718  710  629  569  537  Spicule length  90  88  84  81  88  65  Spicule width  5  6  5  6  5  Gubernaculum  35  50  42  45  35  44  5  4  4  5  4  5  Ovejector  87 86 terminus  166  348  415  363  372  245  271  347  413  52  46  45  42  45  31 fluid preserved guts was deposited in the invertebrate collection of the AMNH.
General: Oxyuridae. Small worms, whitish when alive. Round cephalic plate, semicircular stoma, in terminal depression, surrounded by rim. Lateral alae emerge from cephalic vesicle and runs to tip of tail; alae show two crests.
Male: Prominent, round cephalic vesicle in males. Keel-like area rugosa present, with transverse striations. Four pairs of caudal papillae, two pairs adanal, one pair submedial, posterior to cloaca, one pair terminal. Rough cuticular plate immediately posterior to cloaca. Phasmids oriented dorsally. Spicule and gubernaculum present. Non-salient terminus.
Female: Cephalic cuticle excretory pore and vulva in anterior third of body; thick muscular vagina; didelphic. Site of Infection: Large intestine. Etymology: The species is named after George Gaylord Simpson, American paleontologist who specialized in the study of mammalian evolution. His investigations on the Paleocene fossils from North America, as well as the Neogene fossils of Florida and Patagonia allowed him to formulate the mammalian transitions of the Great American Biotic Interchange.
General: Small worms, whitish when alive; round cephalic vesicle, simple; body width gradually increasing posterior to cephalic vesicle, reaching maximum at midbody, and tapering towards posterior end. Round cephalic plate with four submedial papillae and lateral amphids (Fig. 1). Stoma semicircular, in terminal depression, surrounded by rim (Figs. 1 and 8). Esophagus endowed with three esophageal teeth ( Figs. 1 and  8). Lateral alae emerge from cephalic vesicle and runs to tip of tail; alae taper at level of cloaca and anus in males and females, respectively. Alae show two crests (Fig. 4).

Discussion
The genus Tlacuatzoxyuris can be discriminated from other genera present in the northern Neotropics by the absence of an external terminus in the tail of males and a prominent caudal ala. It can be discriminated from Helminthoxys Freitas, Lent and Almeida, 1937, because Tlacuatzoxyuris feature a cephalic vesicle with no prominent cervical alae; from Heteromoxyuris Quentin, 1973 because males feature 5-7 mamelons as ventral ornamentation, and from Passalurus Dujardin, 1845 in that males are endowed with a spicule. Further, the new genus can be discriminated from members of Trypanoxyuris Vevers, 1923 because species in the latter feature complex labial and buccal structures and no ventral mamelons. Finally, the new genus can be discriminated from the five genera of oxyurids present in Australian marsupials ( Tlacuatzoxyuris simpsoni can be discriminated from all other four species of pinworms infecting mouse and fat-tailed opossums based on the configuration of the stoma and the relatively short spicule and gubernaculum. The stoma of T. simpsoni is separated from the cephalic plate by a terminal rim, it is in a shallow terminal depression and it has the appearance of a very irregular semicircular structure (Figs. 1 and 8).
The postcloacal cuticle of males shows ornamentation immediately posterior to the cloaca, just anterior to the pair of submedial papillae (Fig. 2). Finally, both spicule and gubernaculum are relatively short. The eggs of Tlacuatzoxyuris remain unknown.
Tlacuatzoxyuris simpsoni can be differentiated from D. thylamisis based on four main characteristics. First, the relative and absolute size of the spicules and gubernaculum are larger in D. thylamisis than in the homologous structures in T. simpsoni. Second, the cuticle immediately posterior to the cloaca of T. simpsoni has ornamentation limited to a small patch between the cloaca and the submedial papillae (Fig. 2), whereas tubercle-like bumps are present on the surface of the cuticle posterior to the submedial papillae of D. thylamisis (Fig. 13). Third, the cephalic vesicle of females of T. simpsoni is not very well developed and the cuticle is simple, lacking the characteristic complex reticulated network of vesicles seen in the type specimens of D. thylamisis. Finally, the stoma of T. simpsoni is subterminal and surrounded by an irregular cuticle (Figs. 1 and 8), whereas the homologous structure appears to be depressed, covered by cuticular flaps and endowed with esophageal teeth in D. thylamisis (see Figs. 1B, C, and 4A, C in Gardner and Hugot [12].
Tlacuatzoxyuris simpsoni can be readily distinguished from M. dollmeiri in that the latter bears a prominent caudal terminus that emerges from the cuticle, and because the cuticle surrounding the stoma features three lips. The terminus in T. simpsoni is non-salient, a trait shared with the other three monotypic genera. The species described herein features three esophageal teeth, an arrangement that contrasts with the four teeth described in N. venusti. In addition, males of the latter species feature up to six pairs of small precloacal papillae. Finally, T. simpsoni can be discriminated from G. agilisis in that the stoma of the latter presents three or more divisions described as lips, as well as a weakly developed cephalic vesicle. The divisions around the stoma appear to be cuticular flaps that cover the stoma. These are divided in some of the specimens observed with the aid of SEM [8,24].
For didelphimorph-dwelling pinworms, the ornamentation of the eggs has been used as an important characteristic to justify the placement of species in monotypic genera. In particular, eggs in Didelphoxyuris are described as non-operculated, whereas the eggs of Gracilioxyuris show three longitudinal ridges with an anterior operculum. This character is of paramount importance in the definition of Gracilioxyuris [8,24], since both Gracilioxyuris and Didelphoxyuris share similarities on the structure of the cephalic and caudal arrangement of papillae.
No gravid female was collected as a result of our efforts, which yielded a single male from the three screened gray opossums from Tehuantepec and only mature females from the type locality. We predict that the eggs for this species will be operculated as is the case in eggs of M. dollmeiri, N. venusti and G. agilisis. A taxonomic revision based on a reconstruction of the relationships among the opossum-dwelling pinworms is necessary to clarify homologous characters defining the monotypic genera.
The single specimen collected from Oaxaca likely belongs to a different species, based on the characteristics of the spicules, distance of the area rugosa to anterior end and the shape of the cephalic vesicle. However, the characterization of the species requires a description of character variability of the cephalic plate and vesicle, the arrangement of the caudal papillae and configuration of the structures present in females. Thus, we are unable to present a description at this time. Interestingly, the presence of this second pinworm species mirrors the splitting of the marsupial host lineage [3]. The pinworms herein documented infected individuals collected from two different clades that represent the geographic clusters of the Balsas basin and the Tehuantepec Isthmus.
Because there is a bias towards the study of common species, we urge colleagues to document the diversity of metazoan parasites associated with all didelphimorph marsupials. The greatest species diversity of didelphimorph marsupials includes small sized opossums, most of which are arboreal or are restricted to small areas in the tropical forests [4,6]. We urge the examination of preserved mammalian guts available in scientific collections to continue reconstructing the biological inventory of their symbionts, which includes parasites. In addition, we recommend that scientists working in the field should preserve the parasites or the tissues of freshly caught mammals in the field and link both parasite and host through a relational database [10]. The completion of this parasite inventory will give us an opportunity to better understand the expansion of the marsupials and their pathogens as they were both involved in the Great American Biotic Interchange and may be involved in subsequent events of dispersion or translocation.