Helminths of three species of opossums (Mammalia, Didelphidae) from Mexico

Abstract From August 2011 to November 2013, 68 opossums (8 Didelphis sp., 40 Didelphis virginiana, 15 Didelphis marsupialis, and 5 Philander opossum) were collected in 18 localities from 12 Mexican states. A total of 12,188 helminths representing 21 taxa were identified (6 trematodes, 2 cestodes, 3 acanthocephalans and 10 nematodes). Sixty-six new locality records, 9 new host records, and one species, the trematode Brachylaima didelphus, is added to the composition of the helminth fauna of the opossums in Mexico. These data, in conjunction with previous records, bring the number of taxa parasitizing the Mexican terrestrial marsupials to 41. Among these species, we recognized a group of helminths typical of didelphids in other parts of the Americas. This group is constituted by the trematode Rhopalias coronatus, the acanthocephalan Oligacanthorhynchus microcephalus and the nematodes Cruzia tentaculata, Gnathostoma turgidum, and Turgida turgida. In general, the helminth fauna of each didelphid species showed a stable taxonomic composition with respect to previously sampled sites. This situation suggests that the rate of accumulation of helminth species in the inventory of these 3 species of terrestrial marsupials in the Neotropical portion of Mexico is decreasing; however, new samplings in the Nearctic portion of this country will probably increase the richness of the helminthological inventory of this group of mammals.


Introduction
Less than 25% of the 525 species of mammals distributed in Mexico have been examined for helminth parasites (García-Prieto et al. 2012). To date, 336 nominal taxa of helminths have been recorded in mammals, 26 associated with 3 species of terrestrial opossums (Virginia opossum, Didelphis virginiana Kerr, the common opossum Didelphis marsupialis Linnaeus, and the Gray four-eyed opossum Philander opossum Linnaeus) from this country. However, the knowledge of the helminth richness associated with this host group is incomplete due to the wide distribution of these mammals in Mexico. Didelphis marsupialis occurs from Tamaulipas State and west San Luis Potosí until the Yucatán peninsula. Didelphis virginiana inhabits almost all of Mexico, except for the central Plateau and Baja California peninsula. Philander opossum occurs from south Tamaulipas State along the Gulf of Mexico coast and Chiapas State (Arcangeli-Álvarez 2010, Cervantes et al. 2010). The main objective of this work is to present new records of helminth species parasitizing these 3 species of opossums in Mexico and to compare the finding to previous records.

Materials and methods
From August 2011 to November 2013, 68 opossums (8 Didelphis sp., 40 D. virginiana, 15 D. marsupialis, and 5 P. opossum) were collected in 18 localities from 12 Mexican states (Table 1), under the collecting permit FAUT 0057 issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT), Mexico. Mammals were shot by local hunters or caught with Tomahawk traps and then killed with intraperitoneal sodium pentobarbital overdose. Opossums were dissected within the following 4 h. and all organs were examined under a stereomicroscope. Helminths were placed in Petri dishes with 0.85% saline solution. Platyhelminths and nematodes were fixed with hot 4% formalin and preserved in 70% ethanol; acanthocephalans were chilled in distilled water for 10-12 h. Once the proboscis was everted, they were preserved in 70% ethanol. Platyhelminths and acanthocephalans were stained with Mayer's paracarmin, cleared with methyl salicilate, and mounted in Canada balsam. Nematodes were cleared using Amman's lactophenol and temporarily mounted for morphological study (Lamothe-Argumedo 1997). Voucher specimens of all helminth species were deposited at Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City.

Results
A total of 12,188 helminths representing 21 taxa were identified in the 68 opossums collected from 18 localities within 12 states of Mexico ( Figure 1). Six trematode, 2 cestode, 3 acanthocephalan, and 10 nematode species were collected. Below, we present Remarks. These specimens belong to A. caudalitestis due to the position of the reproductive organs and the separation of the vitelline glands in two fields lying anterior and posterior to the ovary. Furthermore, the uterus has a zig-zag shape, occupying intercecal extension and the S-shape of the excretory vesicle, sinuous between both testes (Caballero et al. 1952).

Brachylaima didelphus Premvati & Bair, 1979 ‡
Site of infection. Intestine.  Remarks. The specific identification of this material follows Premvati and Blair (1979) and is based on the disposition of the vitellaria which extending from pharynx to posterior end.
Remarks. In accordance with Thatcher (1970) this species is characterised by having body flattened and pyriform, covered with small spines. Cirrus and cirrus sac large. Parasites in gall-bladder of marsupials.
Remarks. Rhopalias caballeroi is distinguished by the absence of oral and flanking spines, and because it has between 4 and 11 spines visible within tentacle sacs (Haverkost and Gardner 2008). ( Remarks. The diagnostic traits of this species are: flanking and oral spines present. Between 3 and 11 spines visible within tentacle sacs, which extend far beyond the posterior margin of the pharynx (Haverkost and Gardner 2008). Remarks. This species was identified by having tentacle sacs that do not extend beyond the posterior margin of the pharynx and by having only flanking spines (Haverkost and Gardner 2008 Remarks. In accordance with Cañeda-Guzmán et al. (2001), T. didelphidis is distinguished by the morphology of the scolex that is formed by 4 well separated lobes each containing 1 noncircular sucker opening laterally inside the exterolateral cavity, a large-sized body and by the shape of gravid proglottids that are inversely craspedote, among others.
Oncicola luehei (Travassos, 1917 Remarks. These specimens belong to O. luehei because the dimensions of the proboscis, the number of hooks (36), as well as its size and arrangement fits to the morphology mentioned by Machado (1950). Remarks. Our material was identified following Prestwood (1976); this species is characterised because the oral opening is surrounded by lips, the morphology and size of the spicules and the number and arrangement of bursal rays.

Family Aspidoderidae Skrjabin & Schikhobalova, 1947
Aspidodera raillieti Travassos, 1913 Site of infection. Intestine. Remarks. We identify these nematodes according to the re-description made by Adnet et al. (2009), who established the number of caudal papillae (ten pairs of button-like papillae, symmetrically ventro-laterally located), as well as the single median papilla at the anterior cloacal lip and four pairs of post-cloacal papillae, as diagnostic traits of this species.
Remarks. These specimens were identified based on the re-description of this species (Matey et al. 2001). Its diagnostic traits are: the presence of 2 spongelike areas on the inner side of each pseudolabia, and the number of caudal papillae (22).

Family Trichuridae Railliet, 1915
Trichuris didelphis Babero, 1960 Site of infection. Caecum. Remarks. Our material was identified based on the original description (Babero 1960). This species is characterised by the size of the spicule (0.47-0.6 mm), by having a spiny sheath, by the size of mature eggs (0.068 × 0.032 mm) and the posterior position of the vulva. Remarks. Our specimens were identified following Guerrero (1985). The synlophe of Viannaia viannai at mid-body has 3 ventral ridges orientated to left, short spicules (0.133-0.141 mm) and bursal ray arrangement 2-1-2 type.

Remarks.
To date, 12 species of the genus Travassostrongylus have been described, all parasitizing New World marsupials; Travassostrongylus orloffi Travassos, 1935 is the only species of this genus recorded in Mexico as parasite of Didelphis marsupialis; however, the finding of only 8 females make species identification difficult, because taxonomy of this group is based on male characteristics (Scheibel et al. 2014).

Discussion
As a result of this study, we reported 66 new locality records, 9 new host records, and added one species to the composition of the helminth fauna of the opossums in Mexico: the trematode B. didelphus parasitizing D. virginiana, which had not been recorded in this country (see García-Prieto et al. 2012). A total of 21 helminth taxa were obtained from the 3 opossums species analyzed (6 trematodes, 2 cestodes, 10 nematodes and 3 acanthocephalans), all in adult stage, with exception of the larvae of G. turgidum collected during their migration through the liver of the hosts. The richest helminth fauna among the 3 host species was recorded in D. virginiana, (parasitized by 17 species), followed by D. marsupialis (11 species) and P. opossum (8 species). The digestive tract had the highest number of helminth species (12 intestinals, 2 in gall-bladder, 2 in caeca, and 3 in stomach); only 2 of the 21 taxa, D. hayesi and Capillariinae gen. sp. were found in another site of infection (lungs). The geographic distribution of the helminth species was heterogeneous. The nematode C. tentaculata was the only species found in all localities. Other helminth species were collected from 7 (T. didelphis), 8 (R. coronatus) and 9 (V. viannai) localities; however, most taxa (12) were found in only one locality.
These data bring the number of taxa parasitizing D. virginiana, D. marsupialis, and P. opossum to 37, 21 and 20, respectively (García-Prieto et al. 2012). In this work we sampled in 9 previously unstudied localities; nevertheless, 47.2%, 52.4% and 40% of the taxa collected were reported previously from the Virginia opossum, Black-eared opossum and Gray four-eyed opossum, respectively. These species are typical of didelphids in other parts of the Americas (see Alden 1995;Corrêa Gomes et al. 2003;Haverskot and Gardner 2008;Bertoni-Ruiz et al. 2011;Richardson et al. 2014), conforming a group basically represented by the trematode R. coronatus, the acanthocephalan O. microcephalus and the nematodes C. tentaculata, G. turgidum, and T. turgida; these species have been recorded associated to any of the three opossum species in 7,10,15, 9, and 17 Mexican states, respectively. In states where the 3 host species are distributed sympatrically, O. microcephalus and R. coronatus are the species more frequently shared between them. On the other hand, the most restricted geographic areas are presented by the trematodes A. caudalitestis, B. didelphus, and P. magnacirrus, the acanthocephalan O. luehei, and the nematodes Gongylonema sp., and Travassostrongylus sp., which are present exclusively in one locality. In total, the records of this group of mammals come from 20 of the 32 states of the Mexican Republic; however, the geographic information is asymmetrical, because most of the samplings were made in the state of Veracruz (13 sites). Other states, as Campeche and Quintana Roo, have been sampled once. Moreover, most of the species that have been found parasitizing these didelphid species represent point locality records in only one study about its parasites cover states or regions, particularly Los Tuxtlas, Veracruz. However, the host's collections were made along 13 years, in different year season and with a very distinct sample size (see Cañeda-Guzmán 1997).
Considering only the 27 nominal helminth species recorded to date, the 3 host species shared 12 worm species along the sampled sites in Mexico; 8 were exclusively found in D. virginiana, and 2 are specialist to P. opossum. The cestode T. didelphidis and the acanthocephalan O. luehei are shared by the 2 species of the genus Didelphis but not by P. opossum; the Virginia opossum and the Grey four-eyed opossum shared the digenean D. proloba and the nematode A. raillieti, whereas D. marsupialis and P. opossum shared only P. magnacirrus. The helminth fauna of these hosts throughout their range is composed by one group of 20 specialist species, and by P. mexicanus, O. microcephalus, O. luehei, P. gethi, A. raillieti, D. longispiculata, and T. minuta that act as generalist species. Accidental species have not been reported in any of the samples carried out to date in Mexico. At a local scale, both phenomena had been also observed in marsupials of French Guiana (Jiménez et al. 2011;Byles et al. 2013).
The structuring factor of the helminth fauna in the three didelphid species is the diet; most of the helminth species infect these host species through ingestion of eggs, larvae or intermediate hosts. Fifteen of the 27 named helminth species have indirect patterns of transmission (T. didelphidis, B. didelphus, B. virginiana, D. proloba, A. caudalitestis, P. mexicanus, O. microcephalus, P. gethi, O. luehei, P. nickoli, G. turgidum, T. turgida, G. mexicanum, D. longispiculata, and D. hayesi), five are transmitted directly by eggs ingestion (A. raillieti, C. americana, C. tentaculata, T. didelphis, T. minuta) and for P. magnacirrus, R. baculifer, R. coronatus, R. macracanthus, R. caballeroi, V. didelphis and V. viannai, the life cycle is unknown (Table 2). This result is in agreement with the generalist lifestyles and diets of the three species of opossums (Krause and Krause 2006), that exposed them to the same parasite species; local differences in composition and abundance of helminth species could be related to local availability of parasites (or their intermediate hosts), as well as to the compatibility among host and helminth species, as has been showed by Cañeda-Guzmán (1997) and Jiménez et al. (2011).
The data obtained in this study came from 68 opossums collected from 18 localities (nine not previously sampled for helminths); however, the helminth fauna of each didelphid species showed a stable taxonomic composition with respect to previously sampled sites. Only one species of trematode not previously found in this group of hosts in the country was added to their parasitological record as results of our samples. In spite of the reduced scope of our samplings, this situation suggests that the rate of accumulation of helminth species in the inventory of the 3 species of terrestrial marsupials distributed in the Neotropical portion of Mexico included in this study is decreasing; however, new samplings in the Nearctic portion of this country will probably increase the richness of the helminthological inventory of this group of mammals.