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Helminth Fauna of Two Species of Physalaemus (Anura: Leiuperidae) from anUndisturbed Fragment of the Atlantic Rainforest, Southeastern BrazilAuthor(s): Gislayne M. Toledo , Aline Aguiar , Reinaldo J. Silva , and Luciano A. AnjosSource: Journal of Parasitology, 99(5):919-922. 2013.Published By: American Society of ParasitologistsDOI: http://dx.doi.org/10.1645/GE-3212.1URL: http://www.bioone.org/doi/full/10.1645/GE-3212.1

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J. Parasitol., 99(5), 2013, pp. 919–922

� American Society of Parasitologists 2013

Helminth Fauna of Two Species of Physalaemus (Anura: Leiuperidae) from an Undisturbed

Fragment of the Atlantic Rainforest, Southeastern Brazil

Gislayne M. Toledo, Aline Aguiar, Reinaldo J. Silva, and Luciano A. Anjos*, UNESP – Univ Estadual Paulista, Campus de Botucatu, Instituto deBiociencias, Departamento de Parasitologia, CEP: 18618-970, Botucatu, Sao Paulo, Brazil; *UNESP – Univ Estadual Paulista, Campus de Ilha Solteira, FIES,Departamento de Biologia e Zootecnia, CEP: 15385-000, Ilha Solteira, Sao Paulo, Brazil. Correspondence should be sent to: gisatoledo@hotmail.com

ABSTRACT: Two amphibian species, Physalaemus cuvieri and Physalaemus

olfersii, from Serra do Mar State Park, which is an old-growth

environment of the Atlantic Rainforest in southeastern Brazil, were

surveyed for endoparasites. Hosts were sampled in 2 ponds; each was

colonized by only 1 Physalaemus species. The overall prevalence of

helminths was high and similar in both amphibian species. The mean

intensity of infection in P. olfersii did not differ statistically from that in P.

cuvieri. Nine helminth species were found: 2 acanthocephalans, 1 cestode,

and 6 nematodes. Parasite richness in the 2 host species was similar. The

composition of helminth fauna differed but the 2 hosts shared the most

prevalent taxon of nematode (an unidentified species of Cosmocercidae).

All helminth species exhibited an aggregated distribution pattern in the

host species. The present results demonstrate relatively low species

richness and the dominance of generalist parasite species. This study

contributes to knowledge regarding the structure and composition of the

helminth community in anurans.

Species of Physalaemus Fitzinger, 1826 are leiuperid frogs, a genus

which includes 45 species (Frost, 2013) with broad geographic distribution

in the tropical region of the Americas from Mexico to South America

(Nascimento et al., 2005). A systematic review of the genus by Nascimento

et al. (2005) using morphometric analyses, external morphology, color

patterns, and bone characteristics defines 7 groups of species. Physalaemus

cuvieri Fitzinger, 1826 and Physalaemus olfersii (Lichtenstein and

Martens, 1856) are representatives of 2 different clusters, the P. cuvieri

group and P. olfersii group, respectively. Physalaemus cuvieri is a small-

bodied species with nocturnal behavior found in open areas near lentic

aquatic environments (Dixo and Verdade, 2006). This species is widely

distributed throughout the northeastern, central western, southeastern,

and southern regions of Brazil as well as in parts of Argentina and eastern

Paraguay (Frost, 2013). Physalaemus olfersii is found near bodies of water

in open forests or at the forest ecotone (Dixo and Verdade, 2006)

throughout southeastern and southern Brazil (Nascimento et al., 2005;

Frost, 2013).

Features of the natural history and ecology of host species, such as size,

sex, diet composition, and behavior, are suggested to be important

determinants in structuring amphibian parasite communities (Tucker and

Joy, 1996; Bursey et al., 2001; Goldberg and Bursey, 2002; Bolek and

Coggins, 2003; Hamann, Gonzalez et al., 2006; Schaefer et al., 2006;

Santos and Amato, 2010). Moreover, characteristics of the preferred host

environments are considered as key factors with respect to parasite

colonization (Zelmer et al., 1999; Kehr et al., 2000; Muzzall et al., 2001;

Zelmer et al., 2004; Goater et al., 2005; Haman, Gonzalez et al., 2006).

The aim of the present study was to conduct a survey of helminth fauna

associated with 2 amphibian species, P. cuvieri and P. olfersii, at 2 sites in

the Serra do Mar State Park in southeastern Brazil. These data will

hopefully contribute to knowledge regarding the composition and

structure of the helminth community parasitizing frogs in an area with a

high degree of species diversity and endemism.

Twenty-two specimens of P. cuvieri and 21 specimens of P. olfersii were

sampled under a license from the Brazilian environmental agency IBAMA

(Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais

Renovaveis, No. 18240-1/2009) in January 2010 at the Serra do Mar

State Park, ‘‘Nucleo Santa Virgınia’’ (238240S and 458030W), which is an

undisturbed area of the Atlantic rainforest located in the state of Sao

Paulo, southeast Brazil. The amphibians were sampled from 2 temporary

ponds separated by a distance of 2 km; 1 pond was inhabited by P. olfersii

and the other by P. cuvieri.

The frogs were captured during active searches at night, transported to

the laboratory, and killed with a sodium thiopental solution. The

specimens were necropsied and the gastrointestinal tract, lungs, kidneys,

liver, urinary bladder, body cavity, and musculature of the hind limbs

were searched for the presence of helminths.

Parasites were fixed in alcohol-formaldehyde-acetic acid and preserved

in 70% ethyl alcohol. For species determination cestodes and acantho-

cephalans were stained with carmine and cleared with creosote.

Nematodes were cleared with lactophenol and examined using temporary

mounts. The systematic determination of helminths followed the

approaches proposed by Yamaguti (1959, 1961), Travassos et al. (1969),

Schmidt (1986), Vicente et al. (1990), Anderson et al. (2009), and Gibbons

(2010). Morphological and morphometric measures were obtained using

the computerized LAS V3 image analysis system (Leica Application Suite,

Leica Microsystems, Wetzlar, Germany) coupled to a DM5000B

microscope (interferential phase contrast). Helminth species were depos-

ited in the Colecao Helmintologica do Instituto de Biociencias de

Botucatu (CHIBB) of the Universidade Estadual Paulista, state of Sao

Paulo, Brazil.

Overall prevalence, mean intensity of infection, and mean abundance

were calculated following the methods provided by Bush et al. (1997).

Mean helminth species richness also was calculated. The discrepancy index

(D) was estimated following the method described by Poulin (1993). This

index has a minimum value of zero (D¼0), when all hosts harbor the same

number of parasites, whereas aggregation is considered a maximum (D¼1) when all parasites are found in a single host. The Quantitative

Parasitology 3.0 program (Rozsa et al., 2000) was used to determine this

index. Student’s t-test was employed to determine significant differences

between mean intensity of infection between the host species, as

recommended by Magurran (1988). The Statistica 7.0 program (Statsoft

Inc., 2004) was used for the statistical analysis. Nonparametric multi-

response permutation procedures (MRPP) were run on the PC-Ord 4.0

program (McCune and Mefford, 1999) to determine similarity in the

composition and abundance of parasites in the amphibian species. The

Sorensen (Bray-Curtis) distance was used as the dissimilarity measure to

determine whether the findings were more similar than would be expected

if the samples were randomly distributed in the environment. Delta (A)

equals the maximum value (A ¼ 1) when all groups have the same

composition of species, A ¼ 0 when heterogeneity within groups equals

expectation by chance, and A , 0 when heterogeneity within groups is

greater than that expected by chance (McCune and Mefford, 1999). The

purpose of this test is to determine the concentration within groups

(similar to t- and F-tests) and does not depend on the assumption of

normality but rather on the internal variability of the data (Zimmerman et

al., 1985).DOI: 10.1645/GE-3212.1

919

The overall prevalence of infection was 91% in P. cuvieri and 90% in P.

olfersii. Ninety-eight helminths were found infecting P. cuvieri with a mean

intensity of infection of 4.9 6 0.8 helminths/host. One-hundred forty-

seven helminths were collected from P. olfersii with a mean intensity of

infection of 7.7 6 1.7 helminths/host. No statistically significant difference

was observed between host species regarding mean intensity of infection

(Student’s t-test: t ¼�1.5112; df¼ 25; P ¼ 0.1437) (Table I).

Nematodes represented 99% of the total helminths in the component

community in P. cuvieri and 95.2% in P. olfersii. The component

community in P. cuvieri was composed of 5 taxa, i.e., larvae of an

unknown encysted nematode, an unidentified cosmocercid, Physaloptera

sp., Rhabdias sp. (Nematoda), and Acantocephalo saopaulensis (Acantho-

cephala). For P. olfersii the community component was also composed of

5 taxa: an unidentified cosmocercid, Ochoterenella sp., Oswaldocruzia

subauricularis (Nematoda), a cystacanth of Centhorhynchidae (Acantho-

cephala), and Cylindrotaenia americana (Cestoda). Data for the preva-

lence, mean intensity of infection, and site of infection for each helminth

species in P. cuvieri and P. olfersii are presented in Table II.

Due to the similarity of congeneric females of an unidentified

cosmocercid nematode, and the absence of males in several samples,

identification was not possible. Species determination is frequently based

on male characters such as spicule length, gubernaculum, and caudal

papillae.

Unidentified cosmocercids and larvae of Physaloptera sp. were the most

prevalent taxa in P. cuvieri whereas an unidentified cosmocercid, C.

americana, and O. subauricularis were the most prevalent taxa in P. olfersii

(Table II). The greatest mean intensity of infection in P. cuvieri was due to

larvae of an unidentified encysted nematode whereas an unidentified

cosmocercid was the most abundant in P. olfersii (Table II).

Parasites were recovered from the gastrointestinal tract, liver, lungs,

and body cavity. The gastrointestinal tract had the greatest richness and

abundance of nematodes in both host species. Unidentified cosmocercids

were found in the intestines of both P. cuvieri and P. olfersii. Rhabdias sp.

was found in the lungs of P. cuvieri alone and centhorhynchid cystacanths

were found in the liver of P. olfersii alone (Table II). Not surprisingly, the

discrepancy index revealed that all helminth species exhibited an

aggregated distribution pattern (Table II).

In P. cuvieri, in which there were 5 helminth taxa, the richness of

parasites ranged from 1 species (45% of the hosts), 2 species (45% of

hosts), and 3 helminth species (10% of hosts). Five helminth taxa were

identified in P. olfersii, with the majority of infected specimens harboring 1

helminth species (52.6%) and a slightly smaller percentage harboring 2

species (47.4%) (Fig. 1).

Mean parasite richness (Sm) in the 2 host species was similar (P. cuvieri:

Sm ¼ 1.5 6 0.2; P. olfersii: Sm ¼ 1.3 6 0.2). However, the permutation

analysis (MRPP) revealed that the helminth fauna found in the 2 host

species was different (A ¼ 0.047; P , 0.05).

The overall prevalence of parasites was high in both host species. This

high prevalence of infection may be related to the biology of the hosts,

which have a habitat that is terrestrial and cryptozoic and favors infection

by monoxenous life cycle nematodes by penetration of the skin or the

ingestion of eggs or larvae (Anderson, 2000). In addition, they use aquatic

calling sites in the breeding season which favors infection by heteroxenous

life cycle helminths, with higher exposure to cercariae or other potential

intermediate hosts (Hamann, Gonzalez et al., 2006; Hamann, Kehr et al.,

2006).

A high prevalence by cosmocercid nematodes was found in both host

species. These parasites infect their hosts either orally or by the active

penetration of larvae through the skin (Anderson, 2000).

Larvae of Physaloptera spp. are generally found in amphibians

(Goldberg et al., 1993), including Neotropical anurans (Bursey et al.,

2001; Goldberg et al., 2009). These parasites use insects and amphibians as

intermediate and definitive hosts; snakes, lizards, and birds are infected

when they consume anurans infected with larvae (Anderson, 2000). These

helminths were found in the stomach of P. cuvieri. The absence of these

parasites in P. olfersii may be associated with the lack of an appropriate

insect intermediate host in the pond sampled or with specialization on a

particular type of prey that is free of physalopterans.

Although acanthocephalans are less frequent in amphibian hosts

(Smales, 2007), 2 species of acanthocephalans were found in the present

TABLE I. Parameters of infection of helminth communities associated withPhysalaemus cuvieri and Physalaemus olfersii from Serra do Mar StatePark, ‘‘Nucleo Santa Virgınia,’’ municipality of Sao Luiz do Paraitinga,state of Sao Paulo, southeastern Brazil.

Parameters

Physalaemus

cuvieri

(n ¼ 22)

Physalaemus

olfersii

(n ¼ 21)

Overall prevalence (%) 91 90

Mean abundance 4.5 6 0.8 7.0 6 1.6

Mean intensity of infection (range) 4.9 6 0.8 (1–13) 7.7 6 1.7 (1–29)

Helminth richness 6 5

Mean richness (range) 1.5 6 0.2 (1–3) 1.3 6 0.2 (1–2)

TABLE II. Prevalence (P), mean abundance (MA), mean intensity of infection (MII), aggregation index (D), and site of infection (SI) of helminth speciesinfecting Physalaemus cuvieri and Physalaemus olfersii in Serra do Mar State Park, ‘‘Nucleo Santa Virgınia,’’ southeastern Brazil. *Site of infection: BC¼body cavity; S¼ stomach; LI ¼ large intestine; SI¼ small intestine; L¼ liver; Lu¼ lungs.

Helminth species

Physalaemus cuvieri (n ¼ 22) Physalaemus olfersii (n ¼ 21)

P (%) MA 6 SE

MII 6 SE

(Range) D SI* P (%) MA 6 SE

MII 6 SE

(Range) D SI*

Acanthocephala

Acanthocephalus saopaulensis 4.5 0.05 6 0.04 1 (1) 0.913 LI — — — — —

Centrorhynchidae (cystacanths) — — — — — 9.5 0.1 6 0.1 1 (1) 0.909 L

Cestoda

Cylindrotaenia americana — — — — — 14.3 0.2 6 0.2 1.7 6 0.7 (1–3) 0.848 SI

Nematoda

Nematode encysted (larvae) 13.6 0.77 6 0.53 5.7 6 2.7 (2–11) 0.89 S — — — — —

Cosmocercidae unidentified 81.8 2.7 6 0.5 3.3 6 0.5 (1–10) 0.447 SI, LI 90.5 6.3 6 1.5 6.9 6 1.6 (1–25) 0.491 SI, LI

Ochoterenella sp. — — — — — 4.8 0.05 1 (1) 0.909 BC

Oswaldocruzia subauricularis — — — — — 14.3 0.3 6 0.2 2.3 6 0.9 (1–4) 0.857 SI

Physaloptera (larvae) 40.9 0.77 6 0.25 1.9 6 0.4 (1–4) 0.667 S — — — — —

Rhabdias sp. 9.1 0.14 6 0.10 1.5 6 0.5 (1–2) 0.848 Lu — — — — —

920 THE JOURNAL OF PARASITOLOGY, VOL. 99, NO. 5, OCTOBER 2013

study, i.e., adults of Acanthocephalus saopaulensis in P. cuvieri and the

cystacanth of a centhorhynchid in P. olfersii, affirming that amphibians

can act as either definitive hosts (Smales, 2007; Pinhao et al., 2009) or

second intermediate–paratenic hosts (Smales, 2007).

Centrorhynchid cystacanths have been reported in several amphibian

species (Rodrigues, 1986; Azevedo-Ramos et al., 1998; Goldberg and

Bursey, 2003; Hamann, Gonzalez et al., 2006; Goldberg et al., 2009;

Santos and Amato, 2010) whereas A. saopaulensis has been found in a

much smaller number of amphibian hosts (Smales, 2007; Pinhao et al.,

2009).

Cylindrotaenia americana is known from a wide variety of North

American and South American amphibians (Wardle and MacLeod, 1952;

Tantalean and Garcıa, 1989; Recharte, 1995; Rego, 1995; Bursey et al.,

2001; Goldberg et al., 2002) and was found in the small intestine of P.

olfersii in the present study.

The parasite fauna was composed of generalist species, as all taxa found

in both host species also infect other amphibian species and even different

families. According to Aho (1990), generalist parasites are the major

species composing helminth communities in amphibians. For example,

cosmocercids, O. subauricularis, and Rhabdias sp. found here are also

known to infect Rhinella icterica (Spix, 1824) (Pinhao et al., 2009),

Leptodactylus podicipinus (Cope, 1862) (Campiao et al., 2009), and

Rhinella fernandezae (Gallardo, 1957) (Santos and Amato, 2010) from

other localities in the Neotropics. Hosts in same locality, even those of

different species and genera, can share helminth taxa when exposed to

similar ecological conditions (Aho, 1990).

Therefore, the present findings are in agreement with a number of

studies regarding the community structure of helminths in amphibians

with relatively low species richness (compared with other vertebrate hosts)

and the dominance by generalist species (Aho, 1990; Boquimpani-Freitas

et al., 2001; Van Sluys et al., 2006; Campiao et al., 2009; Pinhao et al.,

2009; Klaion et al., 2011). These results contribute to knowledge on the

structure and composition of the helminth community in anurans. Further

studies should be carried out in the region to gain a better understanding

of the ecological relationships between amphibian hosts and their parasite

fauna.

The authors are grateful to Carla S. Cassini for help with the

manuscript and colleagues from the Laboratorio de Parasitologia de

Animais Silvestres (Lapas) – UNESP for help with the fieldwork. G. M.

Toledo and A. Aguiar thank the Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (CNPq) for MSc. scholarships (130585/2011-4

and 130583/2011-1). L. A. Anjos thanks the Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP) for the postdoctoral grant

(2008/50417-7). R. J. Silva is grateful to FAPESP for financial support

(2008/58180-6).

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