Helminth parasites of bats (Chiroptera, Phyllostomidae) in the Department of Junin, Peru and Check list of records made in Peru

The objective of this work was to determine the parasitic helminth fauna of the phylostomid bats of Junin, Peru, as well as to prepare an updated checklist of records made in Peru. Thirty bats of 6 different species belonging to the family Phyllostomidae were captured in the locality of Chanchamayo, Junin, Peru. Of these species, Artibeus lituratus Olfers, 1818, A. planirostris (Spix, 1823) and Carollia perspicillata (Linnaeus, 1758) were parasitized by trematodes and nematodes; Phyllostomus discolor (Wagner, 1843) by cestodes and nematodes; Carollia benkeithi Solari & Baker, 2006 and Glossophaga soricina (Pallas, 1766) by nematodes. A total of 175 trematode specimens, 115 nematode specimens and 8 cestode specimens were collected. All parasites found in the survey are new records for the department of Junin. The checklist developed in this study found 26 parasite species in the literature. Litomosoides brasiliensis Almeida, 1936, was the endoparasite with the largest number of hosts. The number of hosts with at least one species of parasite recorded was 22 bat species on the checklist.

The chiropterans, being a highly diversified group in relation to species and food habits, are not oblivious to helminth parasitosis, because bats usually acquire these parasites passively through their diet, with the digestive tract and body cavity being the most parasitized by helminths (Gregory et al., 1996;Cuartas-Calle & Muñoz-Arango 1999;Poulin & Morand 2004).
Parasitic helminths of Chiroptera can be associated with intrinsic factors (age, body size, gender, individual genetic aspects, immune response, foraging strategy and nutritional status) and extrinsic factors (social structure, behavior patterns, distribution areas, activity areas, migration and evolutionary history) of the hosts and their habitats (Esteban et al., 1991;Poulin & Morand, 2004;Pinheiro et al., 2013;De Albuquerque et al., (2016). Among these studies it has been observed that bats can act as definitive hosts (e.g. Vampirolepis) and intermediaries (e.g. Physocephalus L 3 ) for parasitic helminths. (Walldorf & Mehlhorn, 2014;Falconaro et al., 2018).
In relation to these investigations that have been registered in relation to parasitism in Chiroptera, only about one-third of the species of bats known in South America have been reported to have helminths; likewise, in Peru fourteen parasitic helminth species have been reported from bats (Santos & Gibson, 2015).
Studies related to the biology and ecology of Chiroptera have increased in recent decades, including studies related to the helminth parasites and their high specificity within these hosts (Hill & Smith, 1984;Dick, 2007;Díaz et al., 2011;Frank et al., 2014).
In Peru, the first studies related to parasitic helminths of Chiroptera were done by Freitas and Ibañez (1963), who carried out parasitological studies on the mouse-tailed bat Talarida brasiliensis (I. Geoffroy, 1824) (Molossidae) in the department of La Libertad (Vargas et al., 2009a).
Within this context, this research aimed to determine the parasitic helminth fauna of Phyllostomid bats from Fundo San José, Chanchamayo, Junin, Peru, as well as to prepare an updated check list of records made in Peru.
Host bats. Mist nets of 10 x 2.5 m were installed among the trees near crops, always trying to obstruct the most likely entrance that the bats could have. Monitoring of the nets was made between 22:00 h˗02:00 h at 30˗minute intervals, following the methodology of Pacheco et al. (2007). For the identification of the hosts, the taxonomic keys of Ibáñez (1981), Eisenberg & Redford (2000), Pacheco et al. (2007) and Díaz et al. (2016) were used. The bats collected were deposited in the Colección Zoológica of the Museo de Historia Natural of the Professional School of Biology, Facultad de Ciencias Naturales y Matematica, Universidad Nacional Federico Villarreal (MUFV˗ZOO), Lima, Peru. According to the IUCN Red List of Threatened Species, all species of bats collected in this study showed a state of conservation of Less Concern. The collection was authorized by Directorial Resolution N°024-2014-SERFOR-DGGSPFFS.
Endoparasite helminths. For the collection of endoparasitic helminths, the abdominal cavity, thoracic cavity, lungs, stomach, small intestine, large intestine, gonads, heart, gallbladder, liver and spleen were examined (Lamothe, 1997). Nematodes were fixed in 70% boiling ethyl alcohol, and trematodes and cestodes pre-pressed between two slides and fixed with 4% formalin for 24 h; they were then stored in 70% ethyl alcohol for transfer to the laboratory (McAllister et al., 2005). The trematodes and cestodes were stained in Semichon's acetic carmine and mounted in Canada balsam. In the identification of the nematodes alcohol-phenol was used to clarify the cuticle and observe in detail the copulatory structures of males and other important structures in both sexes (Oviedo et al., 2010).
All the specimens studied were deposited in the Colección de Helmintos parásitos e invertebrados afines of the Museo de Historia Natural (MUFV: ZOO: HPIA), Universidad Nacional Federico Villarreal, Lima, Peru and in the Museo de Historia Natural (MUSM), Universidad Nacional Mayor de San Marcos, Lima, Peru.
List of parasitic helminths of the bats of Peru. The parasite-host lists of the parasitic bat helminths of Peru presented in this work were compiled from data obtained from searches of the literature in physical libraries of the Peruvian's universities, scientific collections, and museums, and in the virtual literature in databases such as the Web of Science, Scopus, Scielo, Helminthological Abstracts, Research Gate and Google Scholar (Martín-Martín et al., 2018). The list elaborated in this study is composed of taxonomic classifications of the parasites, the hosts registered in Peru and their respective trophic guild, the site of infection, locality, i.e., the governmental department in which the parasitized host was reported, and the bibliographic reference. The abbreviations correspond to the trophic guild of the host: low-flying insectivore LFI, high-flying insectivore HFI, insectivore-piscivore IPI, omnivore-nectivorous OMN, high-flying frugivore HFF, low-flying frugivore LFF, insectivore-carnivore INC, hematophagous (HEM) and omnivore-predator OMP (Patterson et al., 1996).

Site of infection. Thoracic and abdominal cavity.
Identification. Based on 18 individuals (7 male, 11 female). Two large latero-ventral cephalic papillae half the length of the buccal capsule, cuticular wall of the buccal capsule usually with 2 rings, 1 triangular in longitudinal section, the other smaller anterior, almost always visible. Male: 4 pairs of post-cloacal papillae, almost aligned in the midline, the anterior pair regularly away from the cloacal opening. Spiral tail with 2 chitinized unequal spicules, the smaller one with triangular shape at the posterior end followed by a protrusion. Female: The vagina usually subspherical but can also be elongated, the vulva is mostly located far behind the esophagus. Tail generally straight.
Remarks. This species was redescribed by Guerrero et al. (2002), based on material collected from Peru and French Guiana, who also describe the late larval stage of this species to understand the evolution of this genus with additional and more detailed data. It was reported for the first time for Peru by Guerrero et al. (2002) in the bats Anoura caudifer (Geoffroy, 1818), C. perspicillata and Sturnira lilium Geoffroy, 1810, from the towns of Camisea -Cusco and Paquitza -Manu National Park. In this study, L. brasiliensis is registered for the first time for the department of Junin.
Seuratidae Railliet, 1906Seuratum Hall, 1916 Seuratum sp. (Fig. 3A  Identification. Based on 10 individuals. Body fusiform, maximum width at level of ventral sucker. Tegument thin, unarmed. Oral sucker subspherical, subterminal, somewhat smaller than ventral sucker. Ventral sucker usually transversely oval. Pharynx, esophagus, and intestinal ceca absent. Gonads primarily in middle third of body; occasionally ovary extends into posterior third of body. Testes usually opposite, rarely diagonal, subspherical to oval, located on either side of body approximately at level of posterior margin of ventral sucker. Cirrus sac thin-walled, oval to subspherical, anterior to and occasionally overlapping with ventral sucker. Cirrus sac contains coiled seminal vesicle and cirrus. Genital pore median at approximately third of distance between oral and ventral sucker. Ovary, usually smaller than testes, median or submedian, usually post testicular. Vitellarium consists of numerous follicles of varying shape and size, irre- gularly distributed posterior to gonads. Uterus strongly developed with numerous loops occupying posterior two thirds of body, occasionally extending slightly anterior to ventral sucker, usually overlapping gonads. Remarks. The genus Anenterotrema parasitizes exclusively neotropical bats. Currently there are six species and the last of them, Anenterotrema iannaconei, was discovered by Achatz et al. (2018) from the duodenum of Carollia brevicauda Schinz, 1821 collected from the locality of Tingo María, province of Huánuco, Peru; and was the first record of this genus in Peru. Anenterotrema iannaconei differs from the other species in having smaller eggs, lack of a transverse fold in the anterior end of the body, a much larger cirrus sac, by its relatively smaller oral suckerin comparison to ventral sucker, and the ovary is almost immediately postacetabular and anterior to almost all vitelline follicles (Achatz et al., 2018).

Site of infection. Small intestine
Identification. Based on 5 individuals. Cestodes of small size, scolex with 4 suckers without hooks or rostellum. Proglottids slightly craspedote, mature from 4 to 6, gravid from 3 to 4; each proglottid with a set of reproductive organs, the genital pores alternating irregularly. Muscular and pronounced genital atrium. Number of testicles in mature proglottids is 50 to 60, grouped bordering the posterior side of the ovary. Cirrus without spines opens at the base of the atrium. Beneath the sac of the cirrus runs through the vagina which is surrounded by a dense layer of cells. The seminal receptacle is lobed and is located behind the ovary. Ovary with 2 lobed wings. Uterus difficult to distinguish.
Remarks. This genus was created from samples collected from the Nasua nasua (Linneaeus, 1766) ring-tailed coati. Atriotaenia hastati was first described by Vaucher (1982a) from the bat Phyllostomus hastatus (Pallas, 1767), collected in Paraguay. In Peru it was recorded for the first time by Vargas et al. (2009a) for the same host collected in the Cerros de Amotape National Park, Tumbes. We registered P. discolor as a new host for A. hastati.

DISCUSSION
The diversity of Neotropical bats is 390 species in 99 genera (Solari & Martínez-Arias, 2014). The diversity of mammals reported for Peru is 467 species, making Peru the country with the third greatest diversity of species in the Americas and the fifth in the world Quintana et al., 2009). Of this diversity reported for Peru, 172 are species of the order Chiroptera, the group of mammals with the greatest diversity (Díaz et al., 2016). Bats occupy several levels in the food chain, which adds to their diversity and makes them important regulators of complex ecological processes in tropical forests (Ochoa, 1992;Reis et al., 2013;Walldorf & Mehlhorn, 2014).
In the evaluation of the bats from Junin, conducted in this study, it has been observed that 5 out of 6 species of bats evaluated were parasitized by the nematode L. brasiliensis, which registers a wide range of hosts only in South America (Santos & Gibson, 2015). The high prevalence of L. brasiliensis as observed in Table 1 is probably due to the life cycle of this nematode, which uses different hematophagous arthropods (e.g., ticks, mites, mosquitoes, batflies, bugs) as intermediate hosts, as suggested by Scott et al. (1951) and Santos & Gibson (2015). Life cycle studies have been carried out on Litomosoides species that parasitize field rodents and it has been found that species of ticks, mosquitoes and bed bugs can act as vectors for this parasite (Williams, 1948). We suggest that the success of parasites transmitted by vectors (hematophagous arthropods) is greater than that of those transmitted by intermediate hosts (eaten by the final host), because one or more intermediate hosts infect a single host individual, while a single vector has the potential to transmit the parasite to several final hosts by means of a mechanism of micropredation of the vector to the host (Wilson et al., 2017). Likewise this genus of nematode, Litomosoides, together with Litomosa Yorke & Maplestone, 1926, are the most well-known nematode genera that parasitize bats (Ramasindrazana et al., 2016). Based on the list presented in this study, only the genus Litomosoides is registered for Peru and based on the records presented by Santos & Gibson (2015) also only for South America. Guerrero & Bain (2011) considered that L. brasiliensis, as generally regarded, may represent a complex of species, and not just a single taxonomic entity.
The parasitism by Litomosoides species in A. planirostris has been reported in bats of South America, being L. chandleri Esslinger, 1973 the only species registered up to now in A. planirostris (Santos & Gibson, 2015). In our study, we reported this bat as a new host for L. brasiliensis.
Is important to indicate that 2 species of nematodes have been registered in A. lituratus in South America: Bidigiticauda vivipara Chitwood, 1938and Cheiropteronema globocephala Sandground, 1929(Guerrero, 1985Durette-Desset & Vaucher, 1988). In our study only 1 individual was evaluated of A. lituratus which was found parasitized by the nematode L. brasiliensis, this being the first record of the genus Litomosoides for this host. It is likely that increasing the size of the sample to be evaluated in this species of bat in Peru, also increases the report of more species of parasites, because A. lituratus has registered 6 species of helminths in South America (Santos & Gibson, 2015).
In relation to the bat C. benkeithi, this species is relatively new, was described and separated from C. castanea and C. brevicauda by Solari & Baker (2006). Being a relatively new species, no parasitic records were found in this bat, this study is the first to provide information about the helminths associated with C. benkeithi.
In this study it was observed that nematodes had the highest prevalence of infection with 56.7% (17 of 30) of hosts parasitized. This higher prevalence of nematodes in Chiropteran hosts was observed by Cardia (2012) in a study of bat helminths from the Central-West region of the State of Sao Paulo, Brazil. Cardia (2012) indicated that 30.5% of the hosts infected by helminths were exclusively with nematodes. In our study, nematodes were the largest parasitic component found in the bats evaluated, and likewise the group with the greatest diversity of species, suggesting that nematodes can coexist and develop successfully in the presence of other helminths and that, in turn, it would not be competent for the development of other helminths.
The largest number of parasites observed was the trematode Anenterotrema iannaconei. High number of trematodes were also observed in bats from the valleys of Aburrá, Colombia (Castiblanco & Vélez, 1982). These authors sug-gest that the high number may be due to the feeding heterogeneity of the bats, which include insects, which may present immature forms of trematodes (McAllister et al., 2007). We found low prevalences of A. iannaconei, even though this factor is strongly affected by the behavior and feeding strategies of the bats (Coggins, 1988).
In our study, only 1 individual was observed parasitized with a species of cestode; this low occurrence of parasitism by tapeworm species was also noted by Vargas et al. (2009a), who recorded 2 species of cestodes from 39 bats collected in Amotape, Tumbes, Peru. In the same way, Nogueira et al. (2004) found only 1 species of cestode Vampirolepis elongatus (Rego, 1962) in the bat Platyrrhinus helleri (Peters, 1866), collected in the Amazon of Brazil.
All the parasites mentioned in the description are new records for the department of Junin, Peru. We also added Peru as the new geographic registry for a species of Seuratum. Phyllostomus discolor is also registered as a new host of the nematode Seuratum sp. and of the cestode A. hastati. Artibeus lituratus and A. planirostris are new hosts for the trematode A. iannaconei. Bats A. lituratus, A. planirostris and C. benkeithi are also new host records for the nematode L. brasiliensis.

List of parasite helminths of bats in Peru
Santos & Gibson (2015) presented a list of parasitic helminths of bats in South America. According to this list, 14 parasitic helminth species have been recorded from bats in Peru. In the present work we update this information and increase the list of parasite helminths of bats to 26 species, an increase of 185.7%.
Twenty-six species of helminths were recorded among trematodes, cestodes and nematodes, of which 21 were identified at the species level, 5 were identified at generic level ( Table 2). The nematode L. brasiliensis was recorded from 7 chiropteran hosts and is the helminth with the highest number of bat hosts. Twenty-two bat species had at least one parasitic infection, representing 4 families and 15 genera. The bats A. planirostris and P. hastatus have the highest number of parasitic records in Peru with 7 species of helminths each one, followed by C. perspicillata with 4 species, and A. lituratus, P. discolor and T. brasiliensis with 3 species each (Table 3). Among the 24 departments of Peru, ten recorded at least one study about parasites of Chiroptera, and the department of Loreto has the largest number of parasitological studies from this group of mam-

Artibeus planirostris
Intestine Loreto Vaucher (1982b) Vampirolepis mazanensis (Vaucher, 1986) Saccopteryx bilineata Intestine Loreto Vaucher (1986) Rynchonycteris naso Intestine Loreto Vaucher (1986) Vampirolepis phyllostomi (Vaucher, 1982) Phyllostomus    (Table 2). According to the trophic guild of the bat hosts, the highest percentages 18.18% (n=4) was for Low-flying frugivore (LFF) and High-flying frugivore (HFF) among the 9 categories trophics (Table 3). The information reviewed in this research was carried out until December 2018. This does not mean that from 2015 to 2018 there have been 12 more species registered for Peru; on the contrary, there was only one related study of bat helminths in Peru since the year 2008 which was carried out by Vargas et al. (2009ab). It is probable that the oversight of 185.7% of the available information, and possibly why it was not included in the list of Santos & Gibson, is related to access to other studies. This type of information is described by Oliva & Luque (2010) as "gray information", which raises the question of if "... there is a gray information not accessible in the databases and that normally corresponds to local journals, records presented at conferences, thesis of pre-post degree and / or technical reports? "(p.102). In the preparation of this updated list of bat helminths in Peru we have not included the abstracts of conferences or theses, but if we had access to information from local scientific journals that do not have a virtual version, then they were included (Martín-Martín et al., 2018).
In the elaboration of this updated list we have included information on trophic guild, i.e., the habit and feeding strategy of the hosts, because little is known about the life cycles of parasitic helminths in this group of mammals (Coggins, 1988) and we believe that additional knowledge about the feeding ecology of bats can provide important insights into the relationships between these groups, as suggested by Ubelaker (1970) and Reis et al. (2013). The diet is important to explain parasitism of helminths in bats, because many of their diet components such as arthropods, molluscs and small vertebrates are involved in the development of the helminth biological cycle, acting as intermediate or paratenic hosts (Esteban et al., 1991;McAllister et al., 2007). But not only the diet can influence the parasitism, but also the corporal size and geographical distribution of the bats, because these factors promote the colonization of new species and would be associated with a high diversity of parasites (De Albuquerque et al., 2016). It is thus that the larger bats would be expected to harbor the richer parasite assemblages, since they provide a greater spatial variety for niche diversification (Morand & Harvey, 2000;Vitone et al., 2004;Milano, 2016). This could explain why A. planirostris and P. hastatus, which have a larger body size (above 90 mm), had greater helminth richness than C. perspicillata, which has a smaller body size (below 65 mm) (Reis et al., 2013).
In the development of the review, it has been observed that several fruit or nectivorous bats have reported cestode parasitism. This could be explained by the fact that there are species such as Glossophaga soricina (Pallas, 1766), known to be nectivorous, which presents flexibility in its diet extending to insects such as beetles (Coleoptera), flies (Diptera) and moths (Lepidoptera) (Clare et al., 2014).
Also, a greater geographic range of bats could accommodate a greater diversity of parasites, because their distribution would be shared with other bats, allowing the acquisition of other parasitic species (Krasnov et al., 2004;Milano, 2016). That is, in a smaller geographic space and short periods of time, the coexistence between host species can facilitate the transfer of parasites and increase the richness of parasite species in the communities protected by the individual host species and in a larger geographic space, the host species with wide geographical distributions will overlap with those of other host species from which they can acquire new parasites on a longer time scale (Milano, 2016). It is to be expected then, that host species with broader geographical distributions have higher species richness than those with more restricted distributions (Poulin & Morand, 2004). The 6 bats surveyed in Junin, Peru have wide distribution in all South American (Reis et al., 2013).
In the developed list we have found that A. planirostris and P. hastatus are the bats with the highest records of parasitic helminths in Peru. In the case of A. planirostris, in spite of being a species with a diet based on fruits and of which it would be expected less records of helminth parasites, it turned out to have greater parasitic diversity than P. hastatus, which is an omnivore species. Although the diet of A. planirostris is predominantly of fruits (80%), some individuals of this species consume arthropods, including beetles (Coleoptera), dipterans and accidentally at the time of ingestion of the fruits, hymenoptera (ants) and mites (Hollis, 2005). Likewise, both species are known to have wider geographic distribution than other species of their respective genus (Gardner, 2007;Díaz et al., 2016), which would explain, along with the information on their feeding habits, why both species of bats are the most for parasitized by helminths.   Our results show that it is still necessary to increase the number of collections for the search of helminths in bats in Peru. Only 22 species of bats (12.79%) have at least one parasite helminth record of a total diversity of 172 species of Chiroptera in the Peruvian territory.