New records of helminths of Sceloporus pyrocephalus Cope (Squamata, Phrynosomatidae) from Guerrero and Michoacán, Mexico, with the description of a new species of Thubunaea Seurat, 1914 (Nematoda, Physalopteridae)

Abstract A total of 61 specimens of the Red-headed Spiny Lizard Sceloporus pyrocephalus Cope (Phrynosomatidae) collected during the breeding season (June/July 2003, 2004 and 2005) from Western Mexico were examined for helminths. The morphological characterization of the helminths found was made through light microscopy and scanning electron microscopy. Nine taxa of helminths were identified, two cestodes: Mesocestoides sp. and Oochoristica sp., and seven nematodes: Parapharyngodon ayotzinapaensis Garduño-Montes de Oca, Mata-López & León-Règagnon, 2016, Parapharyngodon tikuinii Garduño-Montes de Oca, Mata-López & León-Règagnon, 2016, Parapharyngodon sp., Physalopterinae gen. sp., Skrjabinoptera scelopori Caballero-Rodríguez, 1971, Strongyluris similis Caballero, 1938 and a new species of Thubunaea Seurat, 1914. Larvae of Mesocestoides sp. and Physalopterinae gen. sp. were found in the body cavity and digestive tract, respectively. Excluding the species of Parapharyngodon Chatterji, 1933, S. pyrocephalus is recorded for the first time as a host of the remaining seven taxa of helminths. Additionally, Thubunaea leonregagnonae sp. n. is described and illustrated as a new nematode species, parasite of S. pyrocephalus from Mexico. This new species can be differentiated from the majority of its congeners by the absence of spicules, the particular pattern of caudal papillae in males and the small ratio of oesophagus length:male total body length (0.1–0.16).

leonregagnonae sp. n. is described and illustrated as a new nematode species, parasite of S. pyrocephalus

Introduction
Mexico is ranked as a country with the second highest diversity of reptiles, 864 species of which 493 are endemic to the country (Flores-Villela and García-Vázquez 2014). Lizards are the species-richest reptilian group with 417 species, and among them the Phrynosomatidae are the most diverse family (representing 15.9% of the total lizard diversity from Mexico). Despite this, our knowledge regarding the helminth fauna of this family is limited. Parasite records exist for only 15.3% of the total taxa within the Phrynosomatidae (Paredes-León et al. 2008).
Sceloporus Wiegmann (Phrynosomatidae) is a genus of New World lizards composed of 92 nominal species, of which 59 are endemic to Mexico (Uetz et al. 2016). Particularly, Sceloporus pyrocephalus Cope is an endemic lizard of Western Mexico associated with streams and rivers within tropical deciduous and semi-deciduous forest (Uetz et al. 2016), and distributed along the Pacific coast of the southwestern states of Jalisco, Colima, Michoacán and Guerrero as well as Central Mexico, and Southern Morelos. Despite some efforts to characterize the parasites of this species of lizard (Calisi et al. 2008), current knowledge of its helminth fauna is still far from being complete.
The purpose of the present study is to report on the helminth fauna of S. pyrocephalus, including the description of a new species of Thubunaea Seurat, 1914.

Materials and methods
A total of 61 specimens of S. pyrocephalus were collected during the breeding season from June/July in , 2004 issued to Virginia León-Règagnon by Secretaría del Medio Ambiente y Recursos Naturales, SEMAR-NAT). The specimens were captured by noosing or by hand in 12 localities from Michoacán state, and three localities from Guerrero state, Mexico (Table 1). These localities include tropical-wet and hot-semi-arid climates and elevations ranging from 18-1462 m a.s.l. Lizards were euthanized with an intraperitoneal overdose of pentobarbital sodium. The mouth, peritoneal cavity, and all internal organs were examined for helminths under a stereoscopic microscope. Helminths obtained were counted, fixed, and preserved following the procedure proposed by Lamothe-Ar- gumedo (1997). Nematodes and cestodes were fixed in hot 4% and 10% formaldehyde solution, respectively, and stored in 70% ethanol. For morphological examination, nematodes were cleared in alcohol-glycerol, and mounted on temporary slides. Cestodes were stained with Mayer's paracarmine and mounted on permanent slides using Canada balsam. Preserved specimens were observed under a light microscope. Original drawings were made with an Olympus BX53 microscope equipped with a drawing tube. For scanning electron microscopy (SEM), worms were dehydrated through a graded series of ethanol and then critical point dried with carbon dioxide, coated with a gold/palladium mixture using a Q150R Modular Coating System, and examined in a Hitachi S-2460N microscope and SU1015 SEM (Hitachi). Measurements of the new species are presented in micrometres, unless otherwise indicated; the range is followed by the mean in parentheses. Quantitative descriptors of parasite populations were calculated based on Bush et al. (1997). The helminths were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología (UNAM). Specimens of lizards were deposited in the Colección Herpetológica, Museo de Zoología, Facultad de Ciencias, UNAM (MZFC-HE) and the Collection of the Amphibian and Reptile Diversity Research Center at the University of Texas at Arlington (ARDRC-UTA).

Results
A total of nine helminth taxa was found parasitizing S. pyrocephalus: two cestodes and seven nematodes (Table 2). Only Mesocestoides sp. and Physalopterinae gen. sp. were found as larval stages in the body cavity and digestive tract, respectively. The remaining taxa were found as adults located in the intestine. In the following section, we list the helminth species from S. pyrocephalus found during the present study, along with previous records of each parasite taxon in different Sceloporus spp. from Mexico, where applicable.  (Goldberg et al. 1996); S. grammicus Wiegmann in Mexico City ; S. torquatus Wiegmann in Querétaro .
Remarks. Specimens recovered during the present study share certain morphological characters with S. panamaensis Bursey, Goldberg & Telford, 2003 and S. similis such as spicule length and number of caudal papillae. Our specimens were identified as S. similis since they possess two pairs of lateral subterminal papillae at the base of the caudal appendage (Caballero 1938), which is a diagnostic feature of this species, contrary to the three pairs of papillae observed at the base of the caudal appendage in S. panamaensis   (Goldberg et al. 1996); in S. acanthinus Bocourt from Motozintla, Chiapas (Caballero 1951); in S. jarrovi (Goldberg et al. 1996) and S. parvus  from Hidalgo; in S. formosus from Oaxaca ; in S. mucronatus from Puebla ; in S. torquatus from Zacatecas . Skrjabinoptera phrynosoma (Ortlepp, 1922) Schulz, 1927 in S. jarrovi from Guanajuato (Goldberg et al. 1996); in S. spinosus Wiegmann from Actopan, Hidalgo (Caballero 1937); in S. jarrovi from Querétaro (Goldberg et al. 1996), and finally, in S. grammicus and S. variabilis from localities not further specified .

Remarks.
Representatives of the subfamily Physalopterinae use ants and beetles as intermediate hosts, which are part of the diet of S. pyrocephalus. By eating that sort of prey, this group of lizards becomes a potential intermediate or paratenic host of these nematodes (Petri 1950, Schell 1952, Lee 1957, Kabilov 1980.
Remarks. Skrjabinoptera is a genus of nematodes poorly represented around the world with only 10 species described as parasites, mainly of lizards, and only one species recorded from a snake (Rudolphi 1819 Etymology. This species is named in honour of Virginia León-Règagnon (Instituto de Biología, UNAM), who was the mentor of the authors of this paper, and for her valuable contribution to our knowledge of helminth parasites in Mexico.
General description. Medium-sized nematodes, filiform body, cuticle with fine transverse striations along entire body. Males smaller than females. Round cephal-ic plate in both sexes (Figs 1B, 2B). Deirids symmetrical, simple with rounded tip ( Fig. 2A, C), located immediately posterior to nerve ring. Mouth with two round and simple lateral lips, each with three small teeth on its internal surface; each lip bears a lateral amphid, and a pair of sub-median papillae (Figs 1B, 2B). Pharynx short, cylindrical, opening into oesophagus. Oesophagus divided into anterior muscular portion and posterior glandular portion. Excretory pore in anterior region of body, posterior to nerve ring and located at level of division of muscular and glandular oesophagus. Posterior end conical and rounded in both sexes (Figs 1F, H, 2D, F).
Remarks. The family Physalopteridae is composed of three subfamilies: Thubunaeinae Sobolev, 1949, Proleptinae Schulz, 1927and Physalopterinae Railliet, 1893(Chabaud 1975. Thubunaeinae comprises two genera Thubunaea and Physalopteroides Wu & Liu, 1940, both of which are parasites of reptiles and are characterized by the absence of a cephalic ring, the presence of numerous caudal papillae, and an ornamented cuticle forming papillary plates distributed on the surface of the cauda in males (Chabaud 1975). These two genera differ from each other mainly by the symmetry of their cephalic structures; in Thubunaea these structures are symmetrical, while they are asymmetrical in Physalopteroides (Chabaud 1975). Some authors, for example Moravec et al. (1997), considered that the morphological features of Thubunaea and Physalopteroides have rarely been analysed using techniques such as SEM, and that these observations could provide detailed information to assess the validity of these two genera. However, to the best of our knowledge, SEM studies are still scarce in both genera, being available for only three species of Thubunaea (Moravec et al. 1997, Pazoki and Rahimian 2014, Ramallo et al. 2016 and two species for Physalopteroides (Elwasila 1990, Goswami et al. 2016). The specimens described in the present study show a symmetrical cephalic structure, as in Thubunaea.

Discussion
Many authors have argued that the inventory of the parasite fauna of host species is of critical importance in biodiversity management and conservation efforts (Brooks and Hoberg 2000, Brooks and McLennan 2002, Funks and Richardson 2002. Parasite studies help to reveal other biological aspects of the hosts such as their natural history and ecology. The close link between parasites and their hosts causes these studies to Table 3. Morphological characters of Thubunaea spp. lacking spicules in males. Measurements in micrometres, unless otherwise indicated. Babero & Matthias, 1967 T. eleodori Ramallo, Goldberg, Bursey, Castillo & Acosta, 2016 T. fitzsimonsi Ortlepp, 1931 T. mobedii
have a cascade effect on our knowledge of the biology of the interaction between host and parasite, as well as with the environment in which this association developed. For this reason, helminthological studies on hosts distributed in areas with high potential for endemism, such as in the present study, are highly relevant for understanding the ecology and evolution of parasite-host interactions. Presently, for S. pyrocephalus, a single study was conducted, evaluating parasite load in conjunction with hormone concentration (Calisi et al. 2008). In this study, only nematodes and cestode larvae were found, without, however, identifying these to species level.
In the present study, nine helminth taxa were found parasitizing S. pyrocephalus: two tapeworms of the order Cyclophyllidea (Mesocestoides sp. and Oochoristica sp.), one nematode of the order Ascaridida (S. similis), three of the order Rhabditida (P. ayotzinapaensis. P. tikuinii, and Parapharyngodon sp.), and three of the order Spirurida (Physalopterinae gen. sp., S. scelopori and T. leonregagnonae sp. n.). Most of these taxa coincide with previous reports on the helminth fauna from lizards in Mexico (Caballero 1938, Telford 1965, Goldberg et al. 1996, Moravec et al. 1997, Paredes-León et al. 2008. Parapharyngodon ayotzinapaensis, P. tikuinii and T. leonregagnonae sp. n. have been recorded as specific nematode species for this lizard. The current results increase from 35 to 40 the number of helminths recorded as parasites of Sceloporus spp. and from 17 to 18 the number of species of this lizard genus for which parasite records are available (Paredes-León et al. 2008).
Oochoristica sp., P. ayotzinapaensis, P. tikuinii, Parapharyngodon sp., S. scelopori, S. similis, and T. leonregagnonae sp. n. use S. pyrocephalus as a definitive host, and Mesocestoides sp. and Physalopterinae gen. sp. were recorded as larval stages. Mesocestoides sp. is a common metacestode found in the body cavity and mesentery of amphibians and reptiles, which serve as paratenic hosts, with carnivorous mammals being the definitive hosts (Santoro et al. 2012). Specimens of Physalopterinae gen. sp. were found in the stomach and large intestine; species belonging to this subfamily are adult parasites of amphibians, reptiles and mammals, so their presence in S. pyrocephalus was probably a result of a recent recruitment.
It has been suggested that reptiles that use a passive feeding strategy (i.e. sitand-wait) have a less diverse and less complex helminth fauna than those with an active searching behaviour (e.g. widely foraging) (Roca 1999), and that these foraging strategies are directly related to the diet of the lizards, with a passive strategy used by predominantly herbivorous lizards and an active search used by carnivorous ones. Nematodes with a direct life cycle, such as pinworms (Oxyurida: Pharyngodonidae), best reveal the differences between herbivore and carnivore hosts. Within the Pharyngodonidae, a family of Oxyuroidea that are characteristic parasites of amphibians and reptiles, are two evolutionary lineages that show a diversification that mirrors that of their hosts' diets. (Roca 1999). Some genera of Pharyngodonidae infect only carnivorous saurian reptiles: Parapharyngodon, Spauligodon Skrjabin, Schikhobalova &Lagodovskaja, 1960, Skrjabinodon, Pharyngodon andParathelandros Baylis, 1930; while the second linage is composed of pharyngodonid parasites of herbivorous iguanids and testudines: Tachygonetria Wedl, 1862, Mehdiella Seurat, 1918, Alaeuris Seurat, 1918, Thaparia Ortlepp, 1933, Ortleppnema Petter, 1966, Ozolaimus Dujardin, 1845, Travassozolaimus Vigueras, 1938and Mamillomacracis Dosse, 1939. Given the particular species of Oxyuroidea found in S. pyrocephalus, along with the presence of S. similis, a typical nematode found in carnivorous reptiles (Núñez 2005), it can be inferred that this lizard is mainly carnivorous. The remaining taxa of helminths found in the present study, including the nematodes T. leonregagnonae sp. n. and S. scelopori, have an indirect life cycle, in which beetles and ants are probably acting as intermediate hosts. Studies in other species of Sceloporus have determined that termites are an important food source for S. gadoviae Boulenger, S. horridus Wiegmann, and S. jalapae Günther (Serrano-Cardozo et al. 2008); hence, termites could likely be the intermediate hosts of the heteroxenous parasites found in S. pyrocephalus.
Since five of the nine parasite taxa recorded for S. pyrocephalus have an indirect life cycle, with arthropods as intermediate hosts, diet might be the predominant factor structuring the helminth fauna in this lizard. This opposes the idea proposed by Aho (1990), who found that most of the parasite species that inhabit the intestine of amphibians and reptiles, are nematodes with a direct life cycle. Studies assessing the helminth fauna associated with Mexican reptiles are of great value to have a better understanding of the factors that influence the ecological dynamics within helminth communities and to establish comparisons between Nearctic and Neotropical populations of hosts.