Parasite Fauna of the Blackbelly Garter Snake (Thamnophis melanogaster) in Central Mexico

Abstract

Vertebrates function as intermediate hosts for the last larval stages of metazoan parasites and as their definitive hosts where sexual reproduction takes place. In this work, the metazoans that parasitize the snake Thamnophis melanogaster in Laguna de Cuitzeo, Michoacán, were identified, and their infection parameters were calculated to determine the factors that determine the presence of the metazoan species found. A total of 24 snakes (20 adults and 4 neonates) were collected, but only the adults affected by parasitic metazoans were detected, accounting for 54.2% of the total sample. One trematode species, one nematode species, and one acanthocephalan species were found inside the snake. The cystacanth Polymorphus brevis (37.5%) and Contracaecum sp. (25%) stand out for their high prevalence. Ochetosoma brevicaecum and the pentastomids of the genus Porocephalus presented a low prevalence. Only O. brevicaecum uses T. melanogaster as a definitive host. The life cycles of the metazoan that parasitizes T. melanogaster suggest that the snake acquires the infection through active transmission by penetration or passive transmission by ingestion.

1. Introduction

Most metazoans are obligate parasites (e.g., Platyhelminthes, Acanthocephalans, and Nematodes) with indirect and complex life cycles that include free-living stages and parasitic life stages within one or more hosts, which are closely related to climatic and aquatic environmental conditions. Larvae develop in intermediate hosts, and adults develop in definitive hosts [1]. Vertebrates play an important role in the life cycles of metazoan parasites, being intermediate hosts where the last larval stages take place and definitive hosts where sexual reproduction takes place [2].

Parasites play an important role in structuring biodiversity and provide much information about the natural history of their hosts and their environment [3,4,5]. In Mexico, the main research on metazoan parasites in vertebrates has been biased toward the fish group [6,7], so there are few studies on parasitic metazoans of reptiles, particularly snakes [8]. Reptiles constitute one of the most interesting groups of hosts because they act as very good systems for the study of host–parasite relationships because they occupy a wide variety of habitats with contrasting life-cycle styles and varying reproductive systems [9]. In Mexico, only Digeneans, Monogeneans, Cestodes, Nematodes, and Acanthocephalans have been identified, with a total of 239 species represented in 113 genera found in 118 species of reptiles (17%). Among the Mexican snake Thamnophis melanogaster (Peters, 1864), 15 species of metazoan parasites were found in 7 different localities [8] (Table S1). In three studied hydrological systems in Central Mexico, the sympatric snakes Thamnophis eques (Reuss, 1834) and T. melanogaster share nine species of metazoan parasites [10]. Thamnophis melanogaster has more metazoan species than T. eques [10,11], and the metazoan species Spiroxys susanae (Caballero, 1941), Telorchis corti (Stunkard, 1915), Proteocephalus variabilis (Brooks, 1978) and Contracaecum sp. (Railliet and Henry, 1912) presented the highest levels of prevalence and abundance. The ecology and physiology of this species of snake may explain this richness because T. melanogaster is an aquatic specialist in terms of both diet and foraging ability and lives in temperate habitats between 1158 and 2545 m in elevation [12,13,14]. It uses refuge on land within some meters of water but forages exclusively underwater, mainly on fishes, tadpoles, and leeches [12,14,15]. This snake is endemic to the Central Mexican Plateau [13,16]. This scenario raises the possibility of exposure and transmission of metazoan parasites through infection in the prey‒predator relationship. Recently, a study revealed that local environmental conditions, host phylogeny, and host diet may play important roles in structuring the parasite community of aquatic snakes [17].

The present work aims to identify the metazoans that parasitize T. melanogaster in a population in Central Mexico, Laguna de Cuitzeo, Michoacán, and calculate their infection parameters (prevalence, average intensity, and abundance). We found that the metazoan parasite T. melanogaster is characterized by its low richness and is composed of autogenic species.

2. Materials and Methods

2.1. Study Area

Lake Cuitzeo is located between coordinates 19°56′0″ N and 101°5′0″ W, with an altitude of 1840 m above sea level. It is the second largest lake in Mexico, with a basin of 3977 km² (Figure 1). It has a temperate climate with summer rains and annual rainfall of 906.2 mm and temperatures ranging from 10.2 to 27.5 °C. In recent years, its area and volume have been severely reduced because its main tributaries have been used to supply water to the city of Morelia and for agriculture. The average depth of this lake is almost 2 m [18].

2.2. Sampling and Handling

From March to July 2011, a total of 24 T. melanogaster individuals (20 adults, >330 mm snout–vent length, SVL [19]; and 4 neonates, <225 mm SVL; mean SVL 343 ± 94 mm; mean tail length 9 mm; 4.9 mean weight 27.85 ± 12.5 g) were collected from the shores of Lake Cuitzeo via hooks and herpetological forceps. The snakes were then weighed (g), and the snout–vent length and tail length (cm) were recorded. The snakes were killed by freezing them for 20 min; then, they were dissected longitudinally, and the skin was removed. The internal organs of the hosts were separated and placed in saline solution, and each organ was examined under a stereoscopic microscope for the collection of metazoans. The metazoans found were placed in saline solution to be counted in vivo. The trematodes and acanthocephalans were fixed in hot 4% formalin and finally preserved in 70% alcohol [20]. The nematodes were fixed in hot 4% saline formalin and preserved in 70% alcohol; all the material was labeled and placed in vials. The trematodes, acanthocephalans, and pentastomids were stained via Mayer’s Parscarmine technique and Delafield’s hematoxylin and then mounted in Canada balsam, after which permanent preparations were made [6,20]. Owing to the thickness of the cuticle of the nematodes, they were subjected to clearing techniques [6].

Each parasite was subjected to a morphometric study of important taxonomic characteristics, such as body length and width; attachment organs (hooks, papillae, suckers); testicles; seminal vesicles; ovaries; vitellogenic glands; and caeca. The specimens were photographed with the aid of a Motic 2000 microscope, and the photographs were processed with the Motic Images Plus 2.0 ML program to use these morphometric measurements to identify the organisms at the species level on the basis of the classification criteria [21,22,23,24,25,26], in addition to articles related to the parasite species found. Voucher samples of both metazoan and garter snakes were deposited at the Faculty of Science Invertebrate Collection at the Autonomous University of Mexico State, Mexico.

2.3. Analysis

The infection parameters utilized are those proposed by Bush et al. [27], i.e., prevalence (% infected), mean intensity of infection (number of parasites per infected snake), and abundance (average number of parasites per snake examined). To graphically rank the parasites found, Olmstead–Tukey analysis was used. On the basis of the prevalence data and the logarithm of abundance (Log + 1), a Cartesian quadrant was created, where each of the species found was graphed to occupy a quadrant [28], where Quadrant I represents frequent species (abundant and frequent); Quadrant II represents common species (not very abundant and frequent); Quadrant III represents rare species (not very abundant and infrequent); and Quadrant IV represents occasional species (abundant and infrequent).

3. Results

The metazoan fauna is composed of one species of trematode in the adult stage (Ochetosoma brevicaecum Caballero and Caballero, 1941), one species of nematode in the juvenile stage (Contracaecum sp. larvae III Moravec, Kohn and Fernandez, 1993), and one species of acanthocephalan in the larval stage (Polymorphus brevis Van Cleave, 1916). Pentastomids of Porocephalus Humboldt, 1811 were also found, which are considered within the phylum Arthropoda because of their type of sclerotization.

Ochetosoma brevicaecum specimens were found in the stomach and hindgut. The trematode measured on average 1729.4 µm long and 495.7 µm wide. Oral suckers with longitudinal diameters of 245.95 µm and transverse diameters of 238.35 µm were used. Short and thick intestinal caeca located preacetabularly with eggs around them. The eggs were 38–45 µ long and 20–22 µ wide. The acetabulum is smaller than the oral sucker located in the middle part of the body, measuring 181.5 µm by 92.7 µm on average. The excretory vesicle is “Y” shaped and located in the posterior region of the body (Figure 2). The testes were lobed and symmetrical, with efferent ducts leading into the cirrus sac, which extends to the reproductive pore. The seminal vesicle and prostate gland were inside the cirrus sac. The ovary was spherical with an ovoid seminal receptacle that opened into the ootype and was surrounded by the Mehlis gland. The uterus runs down to the posterior part of the body and then up to the genital pore. The vitellogenic glands consist of ovoid follicles, which form compact groups. The transverse vitelloducts lead into the yolk receptacle, which opens into the ootype.

Nematodes Contracaecum sp. were found encyst in the mesentery, liver, and stomach. The larval body is 7.95–25.45 µm long and 0.316–0.981 µm wide. The cuticle was transversely striated and more visible at the ends of the body. The anterior end is rounded, with a small ventral cuticular tooth 0.013–0.016 µm long. The primordia of the lips are poorly developed. Excretory pore near the cephalic tooth. Deiridia is 0.184–0.402 µm long from the anterior end, narrow esophagus 0.397–1.01 µm long, small round ventricle measuring 0.033–0.181 µm × 0.033–0.14 µm, short posterior ventricular appendage 0.397–0.574 µm, and nerve ring 0.231–0.330 µm from the anterior end. Dark intestine. The intestinal cecum is very long, extends anteriorly to near the nerve ring, and is 0.627–1.94 µm long. The length of the radius between the intestinal cecum and the ventricular appendage is 1:0.2–0.3 µm. The conical tail is 0.049–0.231 µm long (Figure 3).

The acanthocephalan Polymorphus brevis is found in the foregut, midgut, and hindgut within a thin-walled spherical cyst. Its body is short and robust, with a very marked widening in the anterior part of the trunk. The total length of the females varied between 1904.6 µm and 2427.3 µm, and their maximum width ranged from 472.6 µm to 676.2 µm. In males, the trunk measures from 2381.7 µm to 2755.3 µm in length, and its maximum width is 556.7 µm to 581.9 µm. The proboscis is cylindrical, measuring between 1176.9 µm and 1704.0 µm with a conspicuous bulbous widening in its middle part; it is armed with 18 longitudinal rows of hooks with 13 to 14 hooks in each row arranged in a quincuncial pattern, and the largest hooks are located in the widening area. The neck is robust and well differentiated from both the proboscis and the trunk, with a very marked constriction in its middle. The trunk is armed at its anterior end with small spines that are difficult to observe. The receptacle of the proboscis is saccular, and the lemnisci are short, equal in size to each other, and similar in length to the receptacle. Its reproductive apparatus is not well developed (Figure 4).

The Pentastomids Porocephalus were found in the larval or nymphal phase above the liver, lung, and mesentery. Its body is elongated and vermiform and features a chitinous cuticle with annular dilatations. The ventral face of the anterior part or presoma has a central oral orifice, which is a diagnostic characteristic of the genus Porocephalus. This oral orifice is accompanied by four orifices (a pair on each side); each internal orifice has a simple hook, and the sternal orifices have two hooks. Its genital primordia are rudimentary and have an apparent gonopore [29] (Figure 5).

Only adult T. melanogaster were affected by parasitic metazoans (54.2% of the sample). The cystacanth P. brevis (37.5%) and Contracaecum sp. (25%) stood out for their high prevalence. The prevalence rates of O. brevicaecum and the pentosporid Porocephalus were low, at 8.3% and 12.5%, respectively (Table 1).

The metazoan community in T. melanogaster was numerically dominated by acanthocephalans (1 species with 63 individuals), followed by nematodes (1 species with 21 individuals) and trematodes (1 species with 5 individuals). The Pentastomids Porocephalus were represented by only 4 snakes. These data are reflected in the Olmsted–Tukey analysis, which revealed P. brevis and Contracaecum as the most frequent species and O. brevizacum and the organism Porocephalus as rare species (Figure 6).

4. Discussion

The metazoan parasite recorded in T. melanogaster in Lake Cuitzeo, Michoacán, allows us to establish that only O. brevicaecum uses this snake as a definitive host. Likewise, the life cycles of the metazoans that parasitize T. melanogaster suggest that the hosts acquire the infection through active transmission (penetration) or passive transmission (ingestion). In the adult stage, O. brevicaecum is a parasite of freshwater snakes [30], such as T. melanogaster in Lake Cuitzeo. Its life cycle involves the stages of acquiring infection through active transmission when the eggs are ingested by the mollusk, its first intermediate host, where the miracidium larva hatches and develops into the sporocyst, which forms cercariae that emerge in the water and infect the second intermediate host, a fish or frog, where the trematode passes from the cercariae to the metacercaria [30,31] and can subsequently be ingested by an aquatic snake such as T. melanogaster.

The life cycle of Contracaecum sp. suggests that its definitive host is piscivorous birds, in which sexual reproduction takes place, and the eggs are discarded into the water through the bird’s feces; later, the larva is ingested by an invertebrate and then by a fish where the larva reaches its third stage [32]. Contracaecum sp. has been found in the fish Chirostoma jordani (Woolman, 1894) and Goodea atripinnis (Jordan, 1880), both of which inhabit Lake Cuitzeo and are prey for T. melanogaster [14,33]. Additionally, the presence of the cystacanth P. brevis as a parasite of T. melanogaster suggests that it was transmitted by the fish Chirostoma sp. and G. atripinnis, which are intermediate hosts of the larval phase [20]. The birds Egretta thula (Molina, 1782), Nycticorax nycticorax (Linnaeus, 1758), and Ardea alba (Linnaeus, 1758) are definitive hosts in the life cycle of P. brevis [20]. Therefore, it is very likely that Contracaecum sp., Larvae III and P. brevis do not reach their adult phase in T. melanogaster.

The metazoans found in this study have already been recorded by Jiménez-Ruíz, et al. [10] and other researchers (Table 1); however, we consider that the poverty of the species recorded in this study is due to the space and time in which the studies were carried out.

The high prevalence and abundance values in P. brevis and Contracaecum sp. are possibly explained by the frequent contact established by T. melanogaster with its second intermediate hosts Chirostoma sp. and G. atripinnis, as well as the interaction between the parasitic species and all the hosts involved in its life cycle. The low prevalence and abundance of O. brevicaecum could be due to the lack of its usual second intermediate host Girardinichthys multiradiatus [34] in Lake Cuitzeo. Ochetosoma brevicaecum has been reported in the Mexican frog Lithobathes montezumae (Baird, 1854) [35]. This frog is also found in Lake Cuitzeo [36], and T. melanogaster eats frogs in its juvenile state. Lithobathes montezumae, in its juvenile state, has little time for exposure to the parasite [37], so the probabilities of being infected and being able to transmit parasites are very low. On the other hand, the low prevalence and abundance of the pentastomid may be because the order Porocephalida uses mammals as intermediate hosts [38], which are rare in Lake Cuitzeo.

In this study, Contracaecum presented a very different prevalence and abundance from the work of Jiménez-Ruíz et al. [10], suggesting a variation possibly because of the apparently higher infection rate of intermediate hosts with Contracaecum juveniles. Another possibility is that the populations of its intermediate hosts have increased, resulting in a greater probability of transmission of the nematode.

The metazoan community in T. melanogaster is composed of parasites that do not show strict host specificity since in P. brevis and Contracaecum sp., their definitive hosts are piscivorous birds, and in the case of O. brevicaecum, it has already been reported in the snake Xenodon rabdocephalus (Wied-Neuwied, 1824) [39]. In addition, they are autogenic species because they complete their life cycle in hosts with aquatic habits [29]. On the other hand, the absence of parasites in the neonates of T. melanogaster could be related to the time of exposure to the parasites [36]. The adult stage has had more time to acquire parasitic infections. The ecology of T. melanogaster (aquatic specialist) and the transmission dynamics of parasitic metazoans are important in the structure of the parasite community.

5. Conclusions

The metazoan fauna of T. melanogaster in Lake Cuitzeo is composed of four species of parasites: one species of trematode, one species of nematode, one species of acanthocephalan, and one species of pentastomid. This community of metazoan parasites is characterized by its low richness and is composed of autogenic species. Contracaecum sp. and P. brevis were parasitized with greater prevalence and intensity, and P. brevis was the most common species in the community.

Author Contributions

Conceptualization, J.M. and P.S.-N.; Methodology, F.S.S.-T., J.M. and P.S.-N.; Formal Analysis, P.S.-N., J.M., C.S.V.-B. and A.S.; Investigation, F.S.S.-T. and P.S.-N. Writing—Original Draft Preparation, F.S.S.-T., J.M., C.S.V.-B., A.S. and P.S.-N.; Writing—Review and Editing, J.M., P.S.-N. and A.S. Funding Acquisition, J.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All the animals used were approved under the collection permit FAUT-0188 issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT) of the Government of Mexico. All experimental procedures were carried out following the guidelines of the Universidad Autónoma del Estado de México (UAEM), as well as the Mexican Federal Regulation for Animal Experimentation and Care (NOM-062-ZOO-2001).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank three anonymous reviewers of the work for providing their valuable comments, which greatly contributed to improving the quality of this manuscript. To Carmen Zepeda for her help in the preparation of Figure 1. Mariana and Javier Manjarrez-Zepeda provided moral support.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Table

Figure 1. Location of Lake Cuitzeo, Michoacan, México.

Figure 2. Adult trematode, Ochetosoma brevicaecum.

Figure 3. Contracaecum sp., larvae III.

Figure 4. Acanthocephalus in the larval phase, Polymorphus brevis.

Figure 5. Immature pentastomid, Porocepha.

Figure 6. Olmstead–Tukey analysis of metazoan species in Thamnophis melanogaster from Lake Cuitzeo, México.

Table 1

Infection parameters of parasitic metazoans in Thamnophis melanogaster from Lake Cuitzeo, Michoacán.

Parasite	Site of Infection	Number of Parasitized Hosts	Prevalence(%)	Mean Intensity(Number of Parasites/Fish	Abundance(Number of Parasites/Snake)
TREMÁTODA
Ochetosoma	Stomach and	2	8.3	2.5	0.2
brevicaecum	hindgut
NEMÁTODA
Contracaecum sp.	Mesentery, liverand stomach	6	25.0	3.5	0.9
ACANTOCÉPHALA
Polymorphus brevis	Foregut, midgut, and hindgut	9	37.5	7	2.6
PENTASTOMIDAE
Porocephala	Liver, lung and mesentery	3	12.5	1.3	0.2

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d17010011/s1, Table S1. Helminthological records of Thamnophis melanogaster in Mexico [8,10].

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