Helminth fauna of Leptodactylus syphax ( Anura : Leptodactylidae ) from Caatinga biome , northeastern Brazil

Leptodactylus syphax is distributed in central, southeastern and northeastern Brazil, eastern Bolivia and southern Paraguay, occupying open areas and rock outcrops, in rock cavities and termite burrows. We collected 21 frogs from the Caatinga region of the state of Ceará, northeastern Brazil, and 7,021 helminths were recovered from 18 of these hosts (overall prevalence = 85.7%). Six helminth taxa were recovered, as follows: Aplectana membranosa (n = 3,756); Schrankiana formosula (n = 3,176); larvae of Physaloptera sp. (n = 43); unidentified nematode larvae (n = 7); digenean metacercariae of Lophosicyadiplostomum sp. (n = 2); and cystacanths of Acanthocephala (n = 37). The similarity of helminth composition between L. syphax from the Caatinga and other species of the L. fuscus group showed that some anurans were clustered according to parasite species and others according to geographic locality. This study presents new helminth records for the Neotropical region, thus helping in understanding the pattern of species distribution, and it increases the knowledge of parasites associated with amphibians.


Introduction
Knowledge about biological diversity and its distribution is of such importance that it should be considered before any further study.Global diversity includes parasites such as helminths, which are associated with several vertebrates and can be modulated by their host and environment.Helminths can also influence host population conditions through co-evolutionary processes (POULIN, 1995(POULIN, , 1999)).Compared with what is known about the helminth fauna of some vertebrates, such as fish, birds and mammals, knowledge of helminth richness in amphibians is relatively poor (AHO, 1990).Considering the richness of anuran species in Brazil -around 1026 species (SEGALLA et al., 2014;FROST, Helminth fauna of Leptodactylus syphax 2015) -the number of helminth species is expected to be higher.In the Caatinga biome, located in northeastern Brazil, there are approximately 53 anuran species, and most of them have an unknown helminth fauna (ALBUQUERQUE et al., 2012;CAMPIÃO et al., 2014).One of these species is Leptodactylus syphax (Bokermann, 1969), which is distributed across central, southeastern and northeastern Brazil, eastern Bolivia and southern Paraguay, occupying open areas and rocky outcrops, in rock or termite cavities.This species does not seem to adapt well to anthropogenic disturbance (IUCN, 2015).
According to De Sá et al. (2014), the leptodactylid species Leptodactylus syphax was placed in the Leptodactylus fuscus group, which is composed of species of similar sizes that occupy similar niches and are phylogenetically close together.On the other hand, the species of this group have wide distribution and occupy different habitats, which can influence component communities of related parasites (GOATER & GOATER, 2001).However, there is only one record of a helminth parasite associated with L. syphax: the nematode Aplectana sp., which was reported from the state of Mato Grosso do Sul, Brazil (CAMPIÃO et al., 2014).
In the present study, the component community of helminths associated with L. syphax in the Caatinga, Brazil, is reported.In addition, the similarity of helminth community composition in other species of the L. fuscus group from different South American regions is compared in order to analyze whether clusters were formed according to geographic region or helminth species.
As described by Bush et al. (1997), we used ecological descriptors such as prevalence, mean abundance and mean intensity of infection.These descriptors were calculated in the SigmaStat 3.1 software (SYSTAT Software, Inc.).The similarity of the helminth fauna of the L. fuscus species group in which L. syphax is included was analyzed considering the geographic localities occupied by these species, and a cluster analysis was performed using the Bray-Curtis similarity index performed using the Past software (BRAY & CURTIS, 1957), scored as presence (1) or absence (0) of data on helminth species.

Similarity to helminth community composition of other species of the Leptodactylus fuscus group in different South American regions
Only eight species in the L. fuscus group have helminth records from South American countries, in Argentina, Brazil, Ecuador, Paraguay and Peru (Table 2).Among these, Argentina and Brazil have provided most of the records.Based on these records, a cluster with a cophenetic correlation coefficient of 0.81 was presented (Figure 1).Some hosts were grouped according to locality and others according to the composition of helminth species.Hosts sampled in the state of Mato Grosso do Sul (MS) shared the same clade of helminth species with similarity (0.6) shown in a cluster for L. elenae and L. syphax (1.0), with closer similarity than between L. fuscus and L. mystacinus (0.8).Another pair of hosts that showed a correlation was L. bufonius and L. mystacinus (0.6): although from different localities, both shared Oswaldocruzia proencai with L. bufonius from Paraguay (0.4).Leptodactylus fuscus and L. mystaceus showed similarity (0.6) from Rio de Janeiro.Leptodactylus fuscus and L. mystacinus; and L. fuscus and L. mystaceus, from different localities, presented the same similarity measurement (0.5).Leptodactylus syphax from Ceará was more similar to L. mystacinus from Rio de Janeiro (0.2) Three host species formed an outgroup because they did not share any helminth species (0.0).

Discussion
Although members of Leptodactylidae have been included in many studies on helminth fauna in relation to other families, the present study shows the helminth component community of L. syphax for the first time.In addition, this is the first study conducted on this host in the Brazilian Caatinga.Also, this is the first record of Lophosicyadiplostomum sp. for all anurans of the family Leptodactylidae and the first record of parasitism by all these helminths in hosts in the Caatinga, which extends their geographic distribution, except for Aplectana sp., Physaloptera larvae and Acanthocephala, which had already been recorded in Rhinella jimi and R. granulosa in the Caatinga biome (MADELAIRE, 2012).
The parasite community of L. syphax showed low richness, compared with the congeneric group.The environment conditions found in the Caatinga, which is characterized by long periods of drought, may have had an influence, through not allowing other forms of infection to become established (e.g.indirect-cycle parasites).Community richness would vary depending on the environmental conditions because of the way in which species respond to biotic factors (POULIN & KRASNOV, 2010).
The component community of helminths in L. syphax in this study comprised four species of Nematoda, one of Acanthocephala and one of Digenea, while Vicente et al. (1990) recorded Aplectana sp., which is a very common nematode in the intestines of frogs of the Leptodactylidae group (Table 2), and Schrankiana formosula, another nematode that is found only in the large intestine, as well as in L. fuscus (GOLDBERG et al., 2007).These two nematode species presented the highest prevalence, mean abundance and mean intensity of infection, which can be explained by the greater host body surface, which means they are more exposed to infective stages of parasites (POULIN, 1995).In addition to this, these nematode species of the family Cosmocercidae and Atractidae have direct life cycles and, in the case of A. membranosa, the females produce great quantities of eggs, thus resulting in higher reproductive rates and higher abundance of infective larvae in the environment, while S. formosula, according Anderson (2000) who claims that family Atractidae presents autoinfective species.There is a passive interaction between these two species, relating to limited resources and to the fact that both of them need to exploit their host in a very short time (DE JONG, 1976;IVES & MAY, 1985;LOMNICKI, 1988), referring to the reproductive mode of Cosmocercidae.
Larvae of Physaloptera sp. were recovered from the stomach of L. syphax, which can be considered to be an intermediate or paratenic host for this helminth because its diet consists mostly of insects, which harbor the infective larvae (OLSEN, 1986).Species of the L. fuscus group show ecological similarities, in that they live temporarily near puddles while at the tadpole stage, but in the adult stage they remain in terrestrial environments and receive further infection by nematodes through oral ingestion or penetration through the skin, in spite of their body length and different localities.Thus, these nematodes are characterized as having low specificity (ANDERSON, 2000).
The presence of cystacanths in cavities suggests that L. syphax can be considered to be an intermediate or paratenic host, such  that this helminth was acquired through ingestion of arthropods.Some species of Acanthocephala are found in amphibians: in most cases, the cystacanths adhered to the mesentery for transportation by an anuran from an aquatic intermediary host to an aquatic predatory bird, for example (KENNEDY, 2006).Metacercariae of Lophosicyadiplostomum were reported for the first time in a frog of the family Leptodactylidae, in the present study.They were found in the kidneys, thus corroborating previous studies in which this digenean was reported in cyst form at this same infection site.Many digeneans parasitize amphibians: for example, the L. fuscus group includes L. latinasus (HAMANN et al., 2006), which has been reported to be infected by both aquatic and terrestrial parasites.Metacercariae of Lophosicyadiplostomum aff.nephrocystis were found in the kidneys of Scinax nasicus, (HAMANN & GONZÁLEZ, 2009), Hyla nana and Lysapsus limellum (HAMANN & KEHR, 1998, 1999).Infection with this trematode may have occurred by penetration of cercariae beyond the host's cloaca, subsequently reaching the kidneys.On the other hand, considering the life cycle of Diplostomidae, gastropods could be ingested by anurans and then the larvae could reach anurans' kidneys as reported by Gonzalez & Hamann (2006).
Most hosts within the L. fuscus group have shown similarity regarding the areas sampled, but some species have been grouped according to helminth species that they share (Figure 1).The helminth fauna of L. syphax in the Caatinga was most similar to that of L. mystaceus and L. fuscus, in different regions, because they shared three nematode species.Nematodes generally do not have a specificity pattern, and therefore the ability of parasites to explore a wider range of hosts results in better use of resources and opportunities for successful biological cycles (POULIN, 2005).The host species in question belong to the same group and do not differ much regarding ecology and physiology, although the sampling points for each host have had very different characteristics, even involving different biomes (e.g.Caatinga, Atlantic Forest, high altitude as in Cuzco, or the Chaco region of Argentina).Diverse mechanisms for host infection, the low level of general environmental requirements for these helminths and low host specificity allow parasite infection even in completely different environments (SOUSA & GROSHOLZ, 1991).Specimens of L. syphax collected from the Caatinga presented a fauna mostly composed of nematodes considering the number of parasites, that were in most cases, parasites with a direct life cycle (ANDERSON, 2000) that did not require intermediate host.The requirement for an intermediate host usually occurs in environments with greater abundance of water.The Caatinga has extremely low rainfall, with a very long dry season (DUELLMAN, 1999), and consequently there are few environments available for parasites with an indirect life cycle, as opposed to environments from which congeneric species were collected.In addition, L. syphax presents a terrestrial habit and active forager, which favors infection by direct life cycle parasites, once this host remains most of its life in the soil.
Knowledge of the helminth fauna associated with vertebrates improves the data on biodiversity and increases the records of occurrences of species of parasites and their relationships with their hosts.This helps expand knowledge of the distribution patterns of these species and aids future studies on ecological host-parasite relationships.

Table 1 .
Prevalence (P%), mean intensity of infection (MII), mean abundance (MA) with standard error (SE), range of infection (Ri) and site of infection (SI) of helminths associated with Leptodactylus syphax from the Caatinga, Brazil.