A new species of Torrestrongylus (Trichostrongylidae, Anoplostrongylinae) from Macrotus waterhousii (Chiroptera: Phyllostomidae) in Central Mexico

A new species of nematode, Torrestrongylus tetradorsalis n. sp., is described herein, based on specimens recovered from the small intestine of the leaf-nosed bat, Macrotus waterhousii, from the Biosphere Reserve “Sierra de Huautla” in the state of Morelos, Mexico. The new species is included in Torrestrongylus because it features a bursa of the type 3 – 2, a divided cephalic vesicle with an anterior half in the shape of an umbrella, and a posterior widened half. The new species can be distinguished from the only other congener T. torrei Pérez-Vigueras, 1935 by four key features: first, by the absence of cervical alae in both males and females; second, by the relatively longer second half of the cephalic cap; third, by the configuration of the dorsal ray, that does not have a medial terminal ray, and finally, by the structure of the spicules. This is the second species in the genus, previously known from bats of the families Phyllostomidae and Molossidae in Cuba, and now in Mexico.


Introduction
Species in Anoplostrongylinae Chandler, 1938 (Nematoda: Molineidae) are known to infect xenarthrans and bats [7]. To our knowledge, 28 species distributed in 15 genera are known to occur in microchiropterans. The Phyllostomidae contains 57 genera [23], from which the genus Macrotus includes two species, the Californian leaf-nosed bat, M. californicus Baird, and Waterhouse's leaf-nosed bat, M. waterhousii Gray. The distribution of the latter is disjunct, including a continental and a Caribbean range. The continental range includes areas of western and central Mexico and north-central Guatemala, whereas the Caribbean range includes islands of the Bahamas, Beata, Cayman, Cuba, Hispaniola, and Jamaica [17]. The species has not been found in the Yucatan peninsula or in the coastal plains of the Gulf of Mexico. Waterhouse's leaf-nosed bats are cave dwellers and form groups from a few to as many as 500 individuals; they forage twice during the night, 2 h after sunset, and 2 h before sunrise. As a consequence, some consider them ''gleaning'' insectivores that capture prey such as crickets, grasshoppers, beetles, and sphinx moths from the ground or foliage rather than in flight [1].
During an investigation of helminth parasites of bats in central Mexico, carried out by the research team of the Laboratorio de Parasitología de Animales Silvestres of the Universidad Autónoma del Estado de Morelos (UAEM), several nematodes of the genus Torrestrongylus were recovered from the small intestine of Waterhouse's leaf-nosed bats in the State of Morelos, Mexico. A detailed morphological analysis of this material revealed the presence of a new species, which is described below.

Materials and methods
From August 2012 to July 2013, 36 Waterhouse's leafnosed bats were collected by placing a mist net across the opening of the abandoned ''Mina América'', located in the Biosphere Reserve ''Sierra de Huautla'', municipality of Tlaquiltenango, Morelos, Mexico (18°27 0 47" N, 99°00 0 54" W, 1063 m). Bats were handled and killed following recommendations set forth by the American Society of Mammalogists [16], and collected under Licencia de Colector Científico FAUT -0211 (National Collecting Permit 0211) from the Secretaria del Medio Ambiente y Recursos Naturales (SEMAR-NAT). Parasitological examinations were performed in freshly killed bats, following the recommendations and protocols described in Gardner and Jiménez-Ruiz [8].
A thorough helminthological examination resulted in 219 nematodes of an unidentified species of Torrestrongylus that were recovered from the small intestine of 26 Waterhouse's leaf-nosed bats. The worms collected were washed in saline solution, counted, and fixed in either hot 4% formaldehyde or 70% ethanol. Nematodes were cleared with glycerin and temporarily mounted for morphological examination. Two specimens were progressively dehydrated in a graded ethanol series, dried by critical point drying using CO 2 , affixed to a SEM stub, and sputter-coated with gold palladium. Specimens were imaged on a scanning electron microscope (Hitachi S2460 N, Tokyo, Japan). All measurements provided are in millimeters except where otherwise indicated. Upon identification, all specimens were washed and stored in vials in 70% ethanol and submitted to the Colección Nacional de Helmintos (CNHE) of the Universidad Nacional Autónoma de México (Mexico City, Mexico), the Colección Parasitológica de la Universidad Autónoma del Estado de Morelos (COPAUAEM, Cuernavaca, Mexico), the Harold W. Manter Laboratory of Parasitology (HWML, University of Nebraska, Lincoln, Nebraska), and the United States National Parasite Collection (USNPC, Smithsonian Institution).
Genomic DNA was extracted, isolated, and purified from vouchered nematodes following standard protocols [10,15]. These aliquots were used as a template to amplify the mitochondrial gene cytochrome c oxidase subunit 1 (cox1); the primers and thermal profile used to complete the reactions, as well as the postamplification processing of these fragments, are identical to those described elsewhere [10]. Published sequences of available Anoplostrongylinae and other strongyles were downloaded from GenBank [10], and these include Macuahuitloides inexpectans Jiménez, Peralta-Rodríguez, Caspeta-Mandujano, and Ramírez-Díaz, 2014 Gen-Bank KF425296, and KF425297, HWML68495 and HWML68496, and the species described herein with the accession numbers GenBank KF425294, HWML68493 and GenBank KF425295, HWML68494. The resulting amplicons were aligned with ClustalW (http://www.genome.jp/tools/ clustalw/). The model of evolution HKY + G was selected using the corrected Akaike Information Criterion as implemented in jModelTest [12]. Parsimony and maximum likelihood were used as optimality criteria to reconstruct the phylogeny [18], and a Bayesian inference to estimate the posterior probabilities of the nodes [13]. In PAUP*, the phylogeny was reconstructed by means of a heuristic search with tree bisection reconnection (TBR) branch swapping, 100 random additions of sequences, and 10 trees held at each replicate. One thousand bootstrap replicates were performed using a heuristic search in PAUP*. For the Bayesian inference, five chains were set to run for 10 million generations with resampling every 1000 iterations and a burn-in of 25% of the resulting trees. The remaining trees were used to reconstruct the consensus.
Date of collection: From August 2012 to July 2013. Holotype, allotype, and paratypes were collected in April 2013.
Etymology: The species name, tetradorsalis, refers to the four terminal papillae present in the dorsal ray.

Remarks
The configuration of the cephalic vesicle acts as a reliable character to help discriminate genera in Anoplostrongylinae. Torrestrongylus torrei Pérez-Vigueras, 1935 is characterized by the presence of a cephalic vesicle divided into two parts; the anterior has the shape of an umbrella and the posterior is slightly widened, and it has the appearance of ''handles of a pitcher'' [11]. Based on the structure of the caudal bursa and synlophe, as well as the structure of the cephalic vesicle, the species described herein has been placed in the genus Torrestrongylus. The configuration of the cephalic vesicle is markedly different from the homologous structure in other genera in the subfamily, which in most cases possess ornate and simple cephalic vesicles.
The specimens described herein show remarkable similarities to T. torrei, especially in the configuration of the cephalic vesicle, the presence of a vulvar flap, and the 3 -2 bursal arrangement. In addition, the specimens used in the present description were collected from the same species of bat, Macrotus waterhousii. Torrestrongylus tetradorsalis, however, can be discriminated from T. torrei by the absence of cervical alae in both sexes, and by the absence of a terminal unpaired end in the dorsal ray, as seen in T. torrei (Plate 7 in [11]). Other differences between these two species include: the relatively shorter size of the posterior half of the cephalic cap of T. torrei; the general shape and distal tip of spicules (trifurcated in T. torrei vs. bifurcated in T. tetradorsalis); the larger body size of specimens of T. tetradorsalis; the location of the nerve ring relative to the anterior end, and the larger size of eggs in T. tetradorsalis (Table 1).

Phylogeny
According to sequences of available taxa, the genetic difference between the two specimens of T. tetradorsalis is 0.5%; the posterior credibility of the clade T. tetradorsalis + M. inexpectans is 1. This clade has moderate bootstrap support using maximum likelihood as an optimality criterion, but no support using parsimony (Fig. 27).

Discussion
Other members of Anoplostrongylinae that show similarities to T. tetradorsalis include Anoplostrongylus paradoxus (Travassos, 1918) Boulenger, 1926 and some species of Tricholeiperia Travassos, 1935 [20]. However, T. tetradorsalis cannot be placed in these genera because of the configuration of the cephalic vesicle, caudal bursa, and structure of the synlophe. On the one hand, when compared with A. paradoxus, T. tetradorsalis shows a prominent dorsal ray, more complex spicules, and a relatively equal length of branches of posterior and anterior vestibules. On the other hand, species in Tricholeiperia feature an umbrella-shaped cephalic vesicle, and only Tricholeiperia poeyi Baruš and Valle, 1967 features a divided cephalic vesicle [2]. The cephalic vesicle of this species, however, appears to be formed by an anterior dome-shaped half and a widened posterior half. This configuration is markedly different to the cephalic vesicle present in the other species of the genus [5,9,19,21], which feature a vesicle barely separated from the rest of the body [9,19], or conspicuous expansions divided into two lateral halves [21].
The main difference between species of Tricholeiperia and Torrestrongylus is the configuration of the cephalic cap. In the present study, one of the specimens (GenBank Accession Numbers KF425295, HWML68494) features a poorly developed posterior half of the cephalic vesicle, which suggests it belongs to Tricholeiperia. However, the genetic divergence with T. tetradorsalis (GenBank Accession Numbers KF425294, HWML68493) is 0.5%, which suggests they Figure 27. Relative position of Torrestrongylus tetradorsalis and Macuahuitloides inexpectans as reconstructed in the ML tree used to estimate the posterior probabilities of the branches. The numbers to the right of each node indicate bootstrap support for parsimony and maximum likelihood as optimality criteria and the posterior probability of the branch. Syngamus trachea belongs to Strongyloidea. The tree and data matrix can be retrieved from http://opensiuc.lib.siu.edu/zool_data/4/ belong to the same species. These results show the urgent need for an extensive phylogenetic analysis including other Anoplostrongylinae featuring the umbrella cephalic vesicle, that may allow the evaluation and proposition of reliable characters used in their diagnosis.
Currently, the list of parasitic helminths of M. waterhousii includes five species, two digenetic trematodes including Limatulum aberrans Caballero andBravo, 1950 andL. oklahomense Macy, 1931, and three species of nematodes, Aonchotheca viguerasi (Freitas and Lent, 1937), Litomosoides leonilavazquezae Caballero, 1939 and Torrestrongylus torrei [3,4,11]. The only species of parasite recorded on M. californicus is the nematode Glyptostrongylus collaris [22]; however, this is considered a nomen nudum since no description is associated with the name. Interestingly, the Waterhouse's leafnosed bats have a disjunct distribution, with a continental cluster localized in western Mexico and north-central Guatemala, and a Caribbean cluster that spans several islands in the Caribbean. Torrestrongylus torrei was described from bats in La Habana, Cuba, and T. tetradorsalis was collected in bats from Mexico; therefore, these two localities represent both clusters. Torrestrongylus torrei is known to infect phyllostomid and molossid bats [11,14], which suggests this species may be a generalist. The results of our present investigations suggest that in central Mexico, T. tetradorsalis is restricted to the Waterhouse's leaf-nosed bat. However, the expansion of the survey to other areas in the distribution of this species of chiropteran, as well as the inclusion of additional species from other families, will help in establishing the host spectrum for T. tetradorsalis. Additional specimens from an unidentified species of Torrestrongylus have been reported in two species of phyllostomids in Colombia (Micronycteris schmidtorum and Sturnira bidens) [6]. An examination of those specimens should allow an evaluation of the variation of the characters used in the diagnosis of species in this genus, and in uncovering the diversity of Anoplostrongylinae in Neotropical bats.