First study on the helminth community structure of the neotropical marsupial Metachirus myosuros (Didelphimorphia, Didelphidae).

Metachirus myosuros is a marsupial species widely distributed in South America. Despite this, there is a lack of knowledge about its helminth parasites and helminth community structure. The aims of this study were to describe the species composition and determine the parasitological parameters of helminth communities of M. myosuros in preserved areas of the Atlantic Forest, Igrapiúna, Bahia state, northeastern Brazil. Parasites were searched from 19 specimens of this marsupial (18 were infected with at least one species), counted and identified. Ten species of helminth parasites were obtained: 7 nematodes, 2 platyhelminths and 1 acanthocephalan. The most abundant species were Aspidodera raillieti, Cruzia tentaculata, Physaloptera mirandai and Viannaia conspicua (Nematoda). These species were also the only dominant ones in the component community. Male hosts had higher prevalence of P. mirandai and greater abundance of V. conspicua. We observed a relationship between host body size and helminth abundance in both male and female hosts, and between host body size and helminth species richness in female hosts. This was the first study to analyze the helminth fauna and helminth community structure of M. myosuros. This was the first report of occurrences of A. raillieti and Didelphonema longispiculata in M. myosuros.


Introduction
Parasitism plays a key role in the ecosystems because parasites can interfere in processes such as competition, migration, speciation and host reproduction, and can also affect biodiversity (Combes, 2001;Hudson, 2005). Moreover, biotic factors (such as the host gender, age, home range and species richness) and abiotic factors (such as seasonality) may alter the spatial and temporal distribution of a parasite species, thus influencing their abundance and prevalence rates (Combes, 2001).
Metachirus myosuros may act as a wild reservoir for parasites and may contribute to the transmission of certain zoonoses. It has been reported to be naturally infected by Trypanosoma cruzi Chagas, 1909, the etiological agent of Chagas disease (Marcili et al., 2009), and by Leishmania amazonensis Lainson & Shaw, 1972, the etiological agent of American cutaneous leishmaniasis (Basano & Camargo, 2004) in Brazil.
In relation to helminths, all studies published on this host have been reports of species occurrence or species descriptions (Travassos, 1913;Travassos, 1920;Travassos, 1922;Freitas, 1937;Lent & Freitas, 1937;Proença, 1937;Sarmiento, 1954;Vicente, 1966;Travassos et al., 1969;Grisi & Castro, 1974;Gomes, 1979a, b;Noronha et al., 2002;Thatcher, 2006;Tantaleán et al., 2010;Barros, 2015;Lopes-Torres et al., 2019). No studies on the helminth fauna or its community structure in this marsupial have so far been published. The aims of the present study were to describe the species composition and analyze the helminth community structure of M. myosuros at infracommunity level (within an individual host) and component community level (set of the infracommunities of a given host population), in preserved areas of the Atlantic Forest in Igrapiúna, southern Bahia, Brazil. We also tested whether host gender and age were determinant factors for parasitism in this marsupial.

Study area
This study formed part of a comprehensive survey project on the biodiversity of the Atlantic Forest fauna and its parasites. It was conducted in preserved areas of the Pratigi Environmental Protection Area (13° 51' S; 39° 16' W) in the municipality of Igrapiúna, southern Bahia. The landscape of this area is formed by valleys and plains within the landholding of the Juliana Valley United Farms (Fazendas Reunidas Vale do Juliana). This comprises a set of agroforestry systems (including areas of rubber, cocoa, clove and peach palm production) and areas of dense ombrophilous forest that form patches linked by ecological corridors (OCT, 2019). The climate of this region is classified as wet equatorial (Af), according to the Köppen climate classification. The type Af is characterized by high temperatures, with high humidity and few seasonal variations, without any dry season (Alvares et al., 2013).

Sampling methods
The marsupials were collected using Tomahawk® live traps (16 × 5 × 5 inches), which were placed on the ground along six transects of 15 points. Captures were carried out during ten consecutive nights in August 2014 and again in March 2015. All the traps were baited with a mixture of peanut butter, banana, oatmeal and bacon.
The animals were identified by external morphology, weighed, measured, had their sex registered, and euthanised for helminth recovery. The animals were euthanised as follows. The animals were anesthetised with ketamine hydrochloride (100mg/mL) associated with xylazine hydrochloride (20mg/mL) in the proportion 1:1 with 0.1mL/100g dose. When the animal was completely anesthetised, 19.1% potassium chloride was applied intracardiac, using a 2 mL/kg body weight dose. All animals were preserved by taxidermy and deposited as voucher specimens in the Mammal Collection "Alexandre Rodrigues Ferreira" of the Santa Cruz State University (CMARF-UESC). The presence of helminths was investigated in the trachea, lungs, esophagus, heart, kidneys, liver, pancreas, spleen, small intestine, large intestine, cecum, mesenteric veins, reproductive apparatus and body cavities.
The helminths recovered were washed in physiological saline solution (0.85% NaCl). Nematodes were fixed in AFA solution (93 parts 70% ethanol, 5 parts 0.4% formalin and 2 parts 100% acetic acid) and were heated to 65 °C. The flatworms of the classes Trematoda and Cestoda were compressed in cold AFA. The specimens of acanthocephalans were kept in distilled water, fixed and compressed in cold AFA for protraction of the proboscis, as described by Amato et al. (1991). The nematodes were cleared with lactophenol and were placed between a slide and coverslip for identification under an optical microscope. The trematodes, cestodes and acanthocephalans were stained using Langeron's carmine, differentiated with 0.5% hydrochloric acid, dehydrated in an increasing alcohol series, cleared in methyl salicylate and mounted in Canada balsam as permanent preparations (Amato et al., 1991).
Specimens were counted using a stereoscopic microscope and were identified under an optical microscope (Zeiss Axio Scope A1) that was coupled to an Axio Cam MRc digital camera for photomicrography. The species were identified using morphological characteristics, as described by Vicente et al. (1997) and Anderson et al. (2009) for Nematoda; Travassos et al. (1969) for Trematoda; and Gomes (1979b) for Cestoda and Acanthocephala and other species descriptions. Voucher specimens were deposited at the Helminthological Collection of the Oswaldo Cruz Institute (Appendix 1).
The animals were captured under authorization from the Brazilian Government's Chico Mendes Institute for Biodiversity and Conservation (ICMBio; license number 17131-4) and from the Ethics Committee for Animal Use (CEUA) of the Oswaldo Cruz Foundation (license number LW-39/14) in collaboration with the Federal University of Paraíba (UFPB), Federal University of Rio de Janeiro (UFRJ) and State University of Santa Cruz (UESC). Biosecurity techniques and personal safety equipment were used during all procedures involving animal handling and biological sampling (Lemos & D'Andrea, 2014).

Data analysis
The abundance, intensity and prevalence of each helminth species were calculated according to Bush et al. (1997). The mean abundance was calculated by dividing the total number of parasites by the total number of hosts. For the mean intensity, the total number of parasites was divided by the number of infected hosts. Prevalence was calculated by dividing the number of hosts infected by the total number of hosts and multiplied by 100. The sex ratio of the most abundant helminth species was tested if it differed from 1: 1, using the χ 2 contingency test.
The mean species richness was calculated as the mean number of helminth species for each infracommunity, while the species richness was the total number of helminth species recovered. The estimated species richness was calculated using the nonparametric Jackknife 2 estimator (Magurram, 2004).
The parameters of the most prevalent and abundant species were compared in relation to host gender. The abundances were compared using the nonparametric Mann-Whitney test. The prevalences were compared using the χ 2 contingency test.
We also investigated the influence of host age, based on host body size, on the total abundance and species richness for each infracommunity, using linear regression separately for each host gender. Significance of the regression coefficient (beta) was evaluated using t test. Since body size is related to age, we hypothesized that older hosts would be more parasitized than younger ones.
The importance value, I, was calculated for each of the helminth species as described by Thul et al. (1985). From this, each species was then classified in the community as dominant (I ≥ 1.0), co-dominant (0.01 ≤ I < 1.0), subordinate (I < 0.01) or an unsuccessful pioneer (I= 0).
The analyses were done in accordance with Zar (1996), using the Past software, version 3.21 (Hammer et al., 2001). The data were tested for normal distribution using the Shapiro-Wilk test. In all analyses, the significant level was taken to be 5%.

Results
A total of 19 specimens of M. myosuros were captured (8 females and 11 males). Ninety-five percent of the hosts (18) were infected by at least one helminth species. A total of 3,299 helminths were recovered. The overall helminth species richness was 10 and the estimated species richness was 14.66. The mean helminth species richness was 4, and this ranged from zero to seven.
We recovered a total of 124 adult helminths of P. mirandai, 2,371 of C. tentaculata, 128 of A. raillieti, 464 of V. conspicua and 98 of V. pusilla. The nematodes C. tentaculata and V. conspicua were the most abundant species ( Table 1). The prevalence was higher for the nematodes A. raillieti, C. tentaculata, P. mirandai and V. conspicua (Table 1). Viannaia pusilla, R. coronatus and the Trichostrongyloidea specimens were found only in male hosts ( Table 1). The nematode D. longispiculata was found in a single male host in 2014 ( Table 1).
The sex ratios of A. raillieti and P. mirandai did not significantly differ from 1:1 ( Table 2). Cruzia tentaculata, V. conspicua and V. pusilla had significantly more females than males ( Table 2).
The influence of host gender on the parameters found was investigated for the species A. raillieti, C. tentaculata, P. mirandai and V. conspicua. The nematodes P. mirandai and V. conspicua showed a statistically significant difference in prevalence in relation to host gender, with higher values for male hosts ( Table 3). The abundance of V. conspicua was significantly higher in male hosts ( Table 4).
The nematodes were dominant in the component community of M. myosuros with the exception of D. longispiculata and V. pusilla, which were co-dominant and Trichostrongyloidea specimens, which were subordinate ( Table 5). The cestodes were co-dominant and the trematodes were subordinate ( Table 5). The acanthocephalans were co-dominant (Table 5).

Discussion
Metachirus myosuros is recorded as a new host for the nematodes A. raillieti and D. longispiculata. This study also provides the first record of the species A. raillieti, C. tentaculata, D. longispiculata, V. conspicua, V. pusilla, M. bivittata, R. coronatus and O. microcephalus in the state of Bahia, Brazil. Previous studies on helminths in M. myosuros were based on new taxonomic descriptions and new records of species occurrence ( Table 6) without reports of abundance or prevalence indices. Twenty-four helminth species had previously been described infecting this marsupial (      Small intestine Freitas (1937) Igrapiúna, BA, Brazil Thatcher (2006) Present study reported by Travassos (1913), Proença (1937), Vicente (1966) and Thatcher (2006). Didelphonema longispiculata was reported by Wolfgang (1953) in Didelphis marsupialis in North America and the Lesser Antilles.
Cruzia tentaculata was reported parasitizing M. myosuros by Travassos (1922) and Thatcher (2006) in Brazil and by Tantaleán et al. (2010) in Peru. Viannaia conspicua and V. pusilla were found in this marsupial by Freitas (1937) in Angra dos Reis, state of Rio de Janeiro, Brazil, and the latter was also reported by Thatcher (2006) in Brazil. Lent & Freitas (1937) reported P. mirandai in M. myosuros in Angra dos Reis, Brazil; Barros (2015) found this species in the states of Acre and Bahia, Lopes- Torres et al. (2019) in the state of Espírito Santo, Brazil, and Tantaleán et al. (2010) in Peru.
Cruzia tentaculata, V. conspicua and V. pusilla were found in the present study to have sex ratios biased toward females, which suggests that this may be an ecological reproduction strategy in these species. A larger number of females leads to higher numbers of eggs released, thus increasing the chances of host infection. This pattern is most common in species with a polygamous mating system (Poulin, 2007), but there is no information in the literature about the mating system of these species. Nonetheless, similar results were found by Antunes (2005) In some studies, it was observed that male marsupials of other species presented behavior of a more exploratory nature: these included Marmosa (Micoureus) demerarae (Thomas, 1905) (Morais-Júnior & Chiarello, 2005 and Didelphis aurita (Wied-Neuwied, 1826) (Loretto & Vieira, 2005). Such behavior may increase the chances of parasitic infection. In addition, testosterone can decrease host immunity and this can also be correlated with higher occurrence of parasites in male hosts (Combes, 2001). This may explain the greater abundance of V. conspicua and higher prevalence of P. mirandai and V. conspicua in male hosts, as well as the occurrence of V. pusilla, R. coronatus, D. longispiculata and Trichostrongyloidea specimens only in male hosts.
The helminth abundance increased with increasing host body size, suggesting that older animals were infected with more parasites than younger ones. It can be expected that larger hosts may have a larger number of parasites as well as more parasite species (Guégan et al., 1992;Morand & Poulin, 1998;Combes, 2001). For helminth species richness, this relationship could be observed only for females.
The fact that the overall estimated richness (14.66) was higher than the observed results (10) may indicate that more species could be present in the helminth fauna if the sample size were to be increased, and that rare species may also be present in the helminth community. The presence of only two subordinate species among the ten species observed corroborates this idea. The dominance of the nematodes A. raillieti, C. tentaculata, P. mirandai and V. conspicua in the component community of M. myosuros may indicate that these species have characteristics that make them more tolerant to the external environment and to the specificities of the host-parasite interaction, in relation to the other helminths found. Except for P. mirandai, the other three species have been reported to be transmitted through egg ingestion (Jiménez et al., 2011;Taylor et al., 2017), which can facilitate transmission of the parasite. Other studies have also reported that A. raillieti and C. tentaculata are dominant species in other marsupial hosts. Quintão e Silva & Costa (1999), in the state of Minas Gerais, Brazil, and Antunes (2005) in the state of Rio Grande do Sul, Brazil, found high prevalence and high mean intensity rates for C. tentaculata and A. raillieti in D. albiventris. Jiménez et al. (2011)  This was a novel study on the helminth community structure of the marsupial M. myosuros, albeit preliminary. This study not only contributes new records of helminth species and helminth parasitological parameters, but also enables better understanding of the parasite community and parasitism in small mammals.