Acta Herpetologica 10(2): 93-101, 2015 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-16150 Helminth parasites in the toad Rhinella major (Bufonidae) from Chaco region, Argentina Monika I. Hamann*, Cynthya E. González Centro de Ecología Aplicada del Litoral (CECOAL), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ruta 5, km 2.5, W 3400 AMD Corrientes, Argentina. *Corresponding author. E-mail: monika_hamann@yahoo.com Submitted on 2015, 27th April; revised on 2015, 12th September; accepted on 2015, 9th October Editor: Rocco Tiberti Abstract. The present study describes the diversity of helminth parasites of Rhinella major (Anura: Bufonidae) in rela- tion to their body size in 4 subhumid vs. semiarid sampling sites from the Argentine Chaco region. Helminths were found in 81% of the specimens examined (n = 85). Fifteen species (13 in subhumid and 7 in semiarid areas) of hel- minth parasites were found, and most of them were nematodes. Parasites were found in all the examined organs, with highest prevalence and intensity in the digestive tract. Parasite transmission to the toad host occurs by skin penetra- tion or oral ingestion. Maximum helminth richness ranged between 2 and 4 species per infected toad. The most abun- dant species was Aplectana hylambatis. Body size of the host was the main factor in determining parasite richness. The helminth parasite fauna was rather different in hosts from subhumid vs. semiarid sites, but the dominant (Aplectana hylambatis) and codominant species (Cylindrotaenia sp. and Rhabdias elegans) were the same. Mean species richness and mean species diversity of helminths were significantly different between the zones. These results suggest that the amount of rainfall and associated humidity affects the distribution and development of the parasite fauna of this toad. Keywords. Parasitic assembly, Helminth community, Habitat use, Rhinella major, Chaco region, Argentina. INTRODUCTION The small toad Rhinella major (Müller and Hellmich, 1936) is a member of the toad family Bufonidae, which can be found in South America. Its distribution in Argen- tina includes the provinces of Chaco, Formosa, Salta, Jujuy, Santiago del Estero, Santa Fe, and Tucumán (Frost, 2014), where it is able to withstand extremely dry con- ditions that predominate most of the year. R. major is a nocturnal species, living and feeding almost exclusively in terrestrial habitats. Its diet is neither generalised nor spe- cialised, with predominance of formicids (i.e. insectivore) (Duré et al., 2009). Only the systematics of its helminth parasites have been reported (see Campião et al., 2014), whereas the respective host-parasite interactions are still poorly understood. The diversity of helminth parasite species in amphib- ian hosts is often correlated with the habitat inhabited by the latter, e.g., terrestrial amphibian tend to host more nematode parasites with direct life cycles (Luque et al., 2005; Yoder and Coggins, 2007; Ibrahim, 2008; San- tos and Amato, 2010, 2013; Hamann et al., 2006a, 2012, 2013a), while aquatic/semiaquatic amphibian are espe- cially infected by trematode parasites, many of which have complex life cycles that require several hosts and may take place entirely within the aquatic habitat (Wetzel and Esch, 1997; McAlpine and Burt, 1998; Zelmer et al., 1999; Muzzall et al., 2001; Hamann et al., 2006b, 2013b; Campião et al., 2012). Such ecological factors may affect the likelihood of colonization by larval parasites in the intermediate hosts and among the vertebrate definitive hosts (Thieltges et al., 2008). The present study of the toad R. major was per- formed in two areas from the Argentine Chaco region. One zone is subhumid with longer humid periods, and characterized by dense forests interspersed with open 94 Monika I. Hamann, Cynthya E. González floodplain grassland, while the second zone is semiarid, with a shorter and relatively unstable humid period, and dominated by sparser, lower and less diverse forests com- bined with spiny shrubs. In this context, our aim is to describe the helminth fauna infecting R. major in habitats contrasting for their cli- matic features (semiarid vs. subhumid habitats), and com- pare the level of parasitic infection of differently sized toads. MATERIAL AND METHODS Study sites Toad populations were captured in four widely separated locations of the Chaco region of Argentina between October, 2011 and November, 2012. These locations span two different zones: subhumid zone (two localities: Concepción del Bermejo: 26°36’S, 60°57’W and Las Lomitas: 24°42’S, 60°35’W) and semi- arid zone (two localities: Taco Pozo: 25°36’S, 63°15’W and Ing- eniero Juárez: 23°54’S, 61°51’W) (Fig. 1). Compared to the semiarid sampling sites, the subhumid zone is characterized by a more dense vegetation with hardwood trees (e.g., Schinopsis balansae and Tabebuia avellanedae), sec- ondary woody vegetation (e.g., Zizyphus mistol, Acacia caven, Bumelia obtusifolia and Acanthosyris falcata), and herbaceous species comprising patches of grasses (e.g., Elionurus muticus) and terrestrial bromeliads (e.g., Aechmea distichantha and Bro- melia serra). The climate of this zone is characterized by high temperatures and dry north winds in the months of August to November, whereas rainfall is often higher from December to March (Table 1). During the rainy season rivers overflow favours the development of numerous marshes, swamps and lagoons that occupy former riverbeds and meanders. In the semiarid zone, the forest is adapted to dry condi- tions (xerophytic deciduous forest), with predominance of small- leaved deciduous and thorny species adapted to fluctuations in water availability, as well as to seasonal thermal variations. Woody vegetation (e.g., Bulnesia sarmientoi, Prosopis ruscifoli, Stetsonia coryne and Trithinax biflabellata) is sparse, and the her- baceous species are predominantly grasses (Trichloris sp., Gouin- ia sp. and Setaria argentina). The landscape is flat, gently slop- ing toward the east. The climate is characterized by low rainfall (mean anual about <700 mm), and high temperatures, at times exceeding 47 ºC since this area comprises part of the South American Heat Pole (Naumann, 2006). Table 1 presents the val- ues of precipitation and temperature at the study localities. Collection and examination of toads and parasites Samples of R. major were collected in subhumid (n = 61) and semiarid (n = 24) zones (Table 1). Toads were transport- ed alive to the laboratory and then euthanized in chloroform (CHCl3) solution. Their snout-vent length (SVL) was recorded. At necropsy, hosts were sexed and the esophagus, stomach, gut, lungs, liver, urinary bladder, kidneys, body cavity, muscu- lature, skin, and brain examined for parasites. Helminths were observed in vivo, counted, and killed in hot distilled water and preserved in 70% ethanol. For identification, the digeneans, and cestodes were stained with hydrochloric carmine, cleared in creosote, and mounted in Canada balsam. Nematodes were cleared in glycerine or lactophenol, and examined as tempo- rary mounts. Helminths were deposited in the Helmintho- logical Collection of Centro de Ecología Aplicada del Litoral (CECOAL) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Corrientes city, Corrientes, Argentina [accession numbers CECOAL, 11101902, Haematoloechus lon- giplexus; 12110819, Travtrema sp.; 11100008, Strigeidae gen. sp. 1; 12110803, Strigeidae gen. sp. 6; 12110820, Cylindrotaenia sp.; 12110817, Mesocestoides sp.; 12110816, Cosmocerca podicipinus; 12110823, Aplectana hylambatis; 11101906, Oswaldocruzia maz- zai; 12110825, Schulzia travassosi; 11101902, Rhabdias elegans; 12110813, R. pseudosphaerocephala; 12110814, Porrocaecum sp.; 11100003, Physaloptera sp.; 12110827, Ascarididae gen. sp.]. A sample of toad was deposited in the Herpetology Collection of CECOAL-CONICET, with accession number LHC, 5081. Statistical analysis Helminth fauna description. Prevalence, intensity, and abundance were calculated according to Bush et al. (1997). Hel- minth communities have been classified at the infracommunity level (all helminth populations within a single R. major), com- ponent community level (all helminth infracommunities within a population of the R. major) and parasite fauna (all the para- site species that exploit a given host across its geographic range) Fig. 1. Map showing the location of the study area and sampling sites in the subhumid (Las Lomitas and Concepción del Bermejo) and semiarid (Ingeniero Juárez and Taco Pozo) zones. 95Helminth parasites in Rhinella major (Bush et al., 1997; Poulin, 1997). The measures of community richness and diversity employed included: the total number of helminth species (richness), mean richness (the sum of hel- minth species, per individual toads, divided by the total sample size), Shannon index (H´) (Shannon and Weaver, 1949), even- ness (J’) as H´/H´ max (Pielou, 1966; Zar, 2010). The Berger- Parker index of dominance (d) was used to determine the most abundant species (Magurran, 2004). Helminth fauna in semiarid vs. subhumid habitats. Sorens- en similarity qualitative index was used for comparing the degree of similarity in helminth communities between habitats. The statistic Z was used to compare 2 proportions (prevalence) of helminth infection. A t-test was used to test for significant difference in the log10 helminth diversity (Hutcheson, 1970) between zones. The importance of each parasite in the commu- nity was examined according to the methodology outlined by Thul et al. (1985), in which helminth species are classified into four groups (dominant, codominant, subordinate and unsuc- cessful pioneer species) by taking into account their prevalence, intensity, and maturity factor (equal to 1.0 if at least 1 mature specimen of species j is found and equal to 0 otherwise), which is related to the degree of host specificity. Since the samples sizes were different in semiarid vs. subhumid habitats, we used rarefaction methods (implemented in the software EcoSim 7.7; Gotelli and Entsminger, 2004) for comparing the mean diver- sity and the mean richness of the helminth species. Rarefaction uses probability theory to derive expressions for the expectation and variance of species diversity and richness for a sample of a given size. This method “rarefies” its samples down to a com- mon abundance level and then compares species diversity and richness. The process was repeated 1,000 times to generate a mean and a variance of species diversity and richness. Wilcox- on’s V-test was used to compare differences in species richness between semiarid and subhumid habitats. Helminth fauna and R. major size. Spearman’s Rank test (rs) and Pearson’s coefficient correlation (r) were used to indi- cate the relationship between host body size and helminth spe- cies richness. All statistical analyses were performed using Xlstat 7.5 (Addinsof, 2004) and MVSP (Kovach, 1999). RESULTS General patterns Fifteen helminth species were found in the 85 R. major individuals collected in the Chaco region. The pre- dominant groups of parasites were nematodes (9 species), followed by trematodes (4 species) and cestodes (2 spe- cies). Parasites were found in all major organs, with high- est prevalence and intensity in the small intestine (ante- rior and middle portion of intestine), large intestine and stomach (Table 2). However, no parasites were found in the muscles. Eight species were found at the adult stage, and for these R. major is the definitive host; 7 species were at a larval stage and infect R. major as intermediate or paratenic hosts. Parasite transmission to the toad host occurs by skin penetration (60% of the species) or oral ingestion (40% of the species) (Table 2). Comparison of the parasite fauna in semiarid vs. subhumid habitats The relative importance of species within the parasite community was as follows: A. hylambatis was strongly characteristic of the community in both habitat types (Table 3). Seven codominant species contributed to a Table 1. Variables recorded in Rhinella major and climatic data of two zones from the Chaco region, Argentina. Subhumid zone Semiarid zone Concepción del Bermejo Summer 2012 Las Lomitas Spring 2012 Taco Pozo Spring 2011 Ingeniero Juárez Spring 2011 Collected toads Number of toads 31 30 10 14 Males/Females 15/16 26/4 8/2 7/7 Mean length (mm) 44.68 57.23 51.60 39.48 Min-max length (mm) 37-61 48-68 42-60 32-47 Precipitations (mm) 1 Mean anual 937 919 620 689 Total annual 649 737 456 *805 Total season 169 76 223 45 Temperature (°C) 2 Mean annual 20 to 22 22 to 24 20 to 22 22 to 24 1Data from Rohrmann H. (pers. comm.) and data from Gómez et al. (2013). 2Data from Naumann (2006).*Exceptional situation. 96 Monika I. Hamann, Cynthya E. González lesser degree in the helminth community, and two of them (Cylindrotaenia sp. and R. elegans) were common to the semiarid and subhumid zones. Two species were uncommon or rare in the community (e.g., H. longi- plexus). Seven that were able to enter the host but not to reach maturity contributed little to the community and are characteristic of different hosts (e.g., Physaloptera sp.). In both zones, helminths exhibited a low diversity with a less equitable distribution (Tabla 4). The most abundant species (d) was A. hylambatis. There were 5256 individuals of diverse helminth species in the sub- humid zone, while 646 individuals were found in the semiarid zone. The value of Sorensen qualitative simi- larity index was low (40%). Infection prevalence showed no significant differences between zones (Z = -0.94; df = 1; P > 0.05). Using a rarefaction method, the number of indi- viduals was combined and the values of mean richness and mean diversity for each of the zones were obtained (Table 5). Mean species richness of helminth parasites differed between the two habitats, i.e., it was greater in the subhumid zone (Wilcoxon V-test = 85; P = 0.003; n1 = 13; n2 = 13). Mean species diversity of helminth para- sites also differed between the two habitats, being higher in the semiarid zone (Wilcoxon V-test = 0; P = 0.0002; n1 = 13; n2 = 13). In both habitats, the helminth parasites were similar in a set of 4 species (A. hylambatis, Physaloptera sp., R. ele- gans and Strigeidae gen. sp. 6). Diversity of these helminth parasites showed significant difference between zones (t-test = -1.983; V = 732.19; P < 0.05); the subhumid zone (H’ = 0.030) showed greater inequality of species abun- dance compared to the semiarid zone (H’ = 0.054). Helminth species richness related to host body size Of the 85 toads examined from the two zones, infec- tion prevalence was 81%, and significant positive correla- tion was found between toad body length and helminth species richness (r = 0.43; n = 69; P < 0.0001). Toads from the subhumid zone showed high infec- tion prevalence (79%), and parasite species richness was significantly correlated with body length of the host (rs = 0.46; n = 48; P = 0.001). Toads from the semiarid zone presented even higher infection prevalence (88%), and also significant correlation between parasite species richness and body length of the host (rs = 0.59; n = 21; P < 0.01). Table 2. Helminths recorded in Rhinella major from the Chaco region, Argentina. Site of infection: SI-small intestine, LI-large intestine, PZ- pharyngeal zone, GB-gall bladder, M-mesentery, GM-gastric mucosa, LU-lung; stage (S): adult (A) and larval (L); mode of transmission of parasites (T): oral ingestion (I), skin penetration (P); prevalence: %; mean intensity: MI ± SD; total number: #; prevalence in Chaco region: CR. Helminths and site of infection S T Subhumid zone Semiarid zone CR Las Lomitas Concepción Bermejo Ingeniero Juárez Taco Pozo % MI ± SD # % MI ± SD # % MI ± SD # % MI ± SD # % Trematoda Haematoloechus longiplexus LU A I 10 1.0 1 1 Travtrema sp. M L P 3 1.0 ± 0.0 1 1 Strigeidae gen. sp.1 M L P 3 3.0 ± 0.0 3 1 Strigeidae gen. sp. 6 M L P 3 9.0 ± 0.0 9 7 1.0 ± 0.0 1 2 Cestoda Cylindrotaenia sp. SI A I 17 5.2 ± 1.5 26 70 2.1 ± 1.4 15 14 Mesocestoides sp. M L I 10 1.3 ± 0.5 4 4 Nematoda Cosmocerca podicipinus LI/ SI A P 23 6.0 ± 5.5 42 8 Aplectana hylambatis LI/SI A I/P 97 142.7 ± 77.0 4139 55 54.9 ± 55.5 934 86 17.0 ± 21.2 204 90 45.6 ± 38.2 410 79 Oswaldocruzia mazzai SI A P 10 2.0 ± 0.0 2 1 Schulzia travassosi SI/ GB A P 10 6.0 ± 4.1 18 3 23.0 ± 0.0 23 5 Rhabdias elegans LU A P 7 3.5 ± 0.5 7 30 3.0 ± 2.2 9 6 R. pseudosphaerocephala LU A P 17 1.6 ± 0.5 8 6 Porrocaecum sp. PZ L I 3 1.0 ± 0.0 1 1 Physaloptera sp. GM L I 30 4.3 ± 3.3 39 3 1.0 ± 0.0 1 21 1.30 ± 0.5 4 15 Ascarididae gen. sp. SI L I 3 1.0 ± 0.0 1 1 97Helminth parasites in Rhinella major DISCUSSION In general, the results of the present study indi- cate that the helminth fauna (both adults and larvae) of R. major is relatively rich, with a composition similar to those previously described for other bufonid toads (Rhinella fernandezae and Melanophryniscus klappen- bachi) from the Chaco region of Argentina (Hamann et Table 3. Classification of helminth species in Rhinella major from the Chaco Region, Argentina. Dominant species (I ≥ 1.0), codominant species (0.01 ≤ I < 1.0), subordinate species (0 < I < 0.01), unsuccessful pioneer species (I = 0). Combined data (C). Classification of helminth* Subhumid zone Semiarid zone Las Lomitas Conc. del Bermejo C Ingeniero Juárez Taco Pozo C Dominant species Aplectana hylambatis 99.262 99.742 99.544 99.472 96.471 98.865 Cylindrotaenia sp. - - - - 2.745 - Codominant species Cylindrotaenia sp. 0.108 - 0.055 - - 0.805 H.longiplexus - - - - 0.026 - Cosmocerca podicipinus 0.243 - 0.125 - - - Schulzia travassosi 0.045 0.144 0.070 - - - Oswaldocruzia mazzai - - - - 0.052 0.015 Rhabdias elegans - 0.088 - - 0.706 0.207 R. pseudosphaerocephala 0.033 - 0.017 - - - Subordinate species Rhabdias elegans - - 0.006 - - - H. longiplexus - - - - - 0.008 Unssucesfull pioneer species Porrocaecum sp. 0.000 - 0.000 - - - Physaloptera sp. 0.000 0.000 0.000 0.000 - 0.000 Ascarididae gen. sp. 0.000 - 0.000 - - - Travtrema sp. 0.000 - 0.000 - - - Strigeidae gen. sp. 1 - 0.000 0.000 - - - Strigeidae gen. sp. 6 0.000 - 0.000 0.000 - 0.000 Mesocestoides sp. 0.000 - 0.000 - - - *According to the methodology from Thul et al. (1985) Table 4. Summary of ecological indices of helminth communities in Rhinella major from Chaco region, Argentina. Combined data (C). Variables Subhumid zone Semiarid zone Las Lomitas Conc. del Bermejo C Ingeniero Juárez Taco Pozo C Component community Richness 11 5 13 3 5 7 Diversity (H’) 0.09 0.08 0.10 0.05 0.13 0.12 Evenness (J) 0.09 0.11 0.09 0.11 0.18 0.14 Dominance (d) 0.96 0.96 0.95 0.99 0.94 0.97 Identification of (d) Aplectana hylambatis Aplectana hylambatis Infracommunity Range richness 1 - 4 0 - 2 0 - 4 0 - 2 0 - 4 0 - 4 Mean richness 2.17 ± 0.93 1.22 ± 0.42 1.81 ± 0.91 1.33 ± 0.47 2.33 ± 0.94 1.76 ± 0.89 Mean diversity (HB) 0.06 ± 0.08 0.02 ± 0.07 0.05 ± 0.08 0.06 ± 0.10 0.13 ± 0.11 0.08 ± 0.10 Mean evenness (E) 0.17 ± 0.24 0.08 ± 0.22 0.13 ± 0.24 0.20 ± 0.33 0.39 ± 0.37 0.28 ± 0.37 98 Monika I. Hamann, Cynthya E. González al., 2013a, 2014). The helminthological fauna of R. major is dominated by nematode parasites. This pattern is also observed in other bufonid toads from temperate region (Bolek and Coggins, 2003; Yoder and Coggins, 2007) and tropical region (Bursey et al., 2001; Goldberg and Bur- sey, 2003; Luque et al., 2005; Ibrahim, 2008; Santos and Amato, 2010, 2013; Hamann et al., 2013a), in which the helminth community consisted mainly of nematodes. An exception is the study by Hamann et al. (2014) who found that nematode species show low infection levels in the toad M. klappenbachi; these results were related essentially to the thicker integument of this particular host, as well as by the presence of alkaloids in the skin glands that could avoid the parasitic attack. The species A. hylambatis was the dominant parasite in R. major. This had also been observed by Hamann et al. (2012) for Leptodactylus bufonius (Leptodactylidae) from Corrientes Province in Argentina, a frog with a more terrestrial habit, whose microhabitat preference significantly contributed to the high infection by A. hylambatis. Likewise, in other reports for Rhinella fer- nandezae (Bufonidae) from Brazil (Santos and Amato, 2010), and for the toad Amietophrynus regularis (Bufo- nidae) from Egypt (Ibrahim, 2008) the parasitic commu- nity was dominated by the genus Aplectana (Cosmocer- cidae). In particular, the fact that A. hylambatis -a nema- tode with direct life cycle- predominates in R. major could indicate a relationship with the mainly terrestrial habits of this amphibian. Infection by aquatic metacer- cariae (Travtrema sp., Strigeidae gen. sp. 1 and Strigeidae gen. sp. 6) showed low richness, reflecting the fact that R. major spends only little time in the water. Thus, the present study confirms that the parasite communities of R. major exhibit the general characteristics of a terrestri- al amphibian, coinciding that terrestrial amphibians con- tain greater proportion of nematode parasites (Bursey et al., 2001; Goldberg and Bursey, 2003; Bolek and Cog- gins, 2003; Luque et al., 2005; Yoder and Coggins, 2007I; Ibrahim, 2008; Santos and Amato, 2010, 2013; Hamann et al., 2013a). The infections recorded in R. major show that this anuran species represents the definitive host for more than 50% of the species found parasitizing them; apart from this, this toad had a minor role as paratenic and/ or intermediate host, reflected by the presence of lar- vae at different infection sites, e.g. infection by larvae of Physaloptera sp., which persist in the gastric mucosa for varying periods of time without reaching the adult stage (see Anderson, 2000), or by larvae of Mesocestoides sp. which remain encapsulated in the mesenteries (see Prud- hoe and Bray, 1982). These infections occur when toads eat invertebrates (e.g., insects and oribatid mites), and subsequently the ingested larval forms of these para- sites culminate their biological life cycles in some birds and mammals (e.g., by predation of R. major by fox; see Schalk and Morales, 2012). Many of the helminth species recorded in this toad were generalists, given that its pres- ence has been reported in amphibian species belonging to other families in our region (see Campião et al., 2014; Hamann et al., 2014). Proportionately, each zone had a similar number of dominant and codominant helminth species in the community, and the three species (A. hylambatis, Cylin- drotaenia sp. and R. elegans) shared by toads from both Table 5. Summary of main results of the rarefaction method. The number of individuals (n) was unified to obtain values of mean diversity (MD) and mean richness (MR) 1 ± SD for subhumid and semiarid zones. n Subhumid zone Semiarid zone MD ± SD MR ± SD MD ± SD MR ± SD 50 0.16 ± 0.12 2.47 ± 1.03 0.22 ± 0.12 2.78 ± 0.95 100 0.18 ± 0.09 3.70 ± 1.23 0.24 ± 0.08 3.76 ± 0.95 150 0.19 ± 0.08 4.51 ± 1.26 0.24 ± 0.07 4.39 ± 0.96 200 0.20 ± 0.06 5.27 ± 1.28 0.25 ± 0.05 4.81 ± 0.92 250 0.20 ± 0.05 5.68 ± 1.27 0.26 ± 0.04 5.28 ± 0.92 300 0.20 ± 0.05 6.21 ± 1.25 0.26 ± 0.04 5.56 ± 0.87 350 0.20 ± 0.05 6.55 ± 1.27 0.26 ± 0.03 5.81 ± 0.84 400 0.20 ± 0.04 6.90 ± 1.25 0.26 ± 0.03 6.05 ± 0.76 450 0.20 ± 0.04 7.11 ± 1.21 0.26 ± 0.03 6.28 ± 0.71 500 0.21 ± 0.04 7.39 ± 1.18 0.26 ± 0.02 6.50 ± 0.61 550 0.21 ± 0.04 7.59 ± 1.22 0.26 ± 0.02 6.68 ± 0.52 600 0.21 ± 0.03 7.83 ± 1.22 0.26 ± 0.01 6.84 ± 0.39 650 0.21 ± 0.03 8.00 ± 1.22 0.26 ± 0.00 7.00 ± 0.00 99Helminth parasites in Rhinella major zones were categorized the same way (Table 3); i.e., the relative importance of these species was similar in both habitats. Nevertheless, more larval species were present in toads from the subhumid zone. This difference may be influenced in part by low water availability in the semi- arid zone given by shorter humid periods. Thus, because of this factor some invertebrates that act as intermediate hosts are not active, and therefore the development and survival of infective stages does not progress to allow completion of the parasite life cycle in this zone. In the same way, soil with high contents of sand combined with scarce humidity can also affect the survivorship and transmission of infective nematode larvae (see Pizzatto et al., 2013). On the other hand, the dominant species (A. hylam- batis) confers a relatively high level of predictability regarding the parasite communities of this toad, such that any sample of R. major from a single locality is like- ly to include this common helminth. This cosmocercid species that reaches high local prevalence also tends to have a wide geographical range (see Poulin, 2007). Final- ly, the helminthological composition showed little simi- larity between the sampling sites, and this result clearly highlights the important role of local biotic and abiotic factors in determining the proportion of infected toads in a population and the number of helminth species infecting them. Available information suggests that parasite species richness increases with host size. For instance, several authors in North America (Bolek and Coggins, 2003; Yoder and Coggings, 2007) have found a positive rela- tionship between species richness and size of Anaxyrus americanus (Bufonidae), and Ibrahim (2008) in Egypt has shown that host age of A. regularis played a sig- nificant role in determining community species rich- ness. The results of the present study also indicate that host size plays an important role in parasitic infections, in agreement with the results of previous analyses of Argentine amphibians (Hamann et al., 2010, 2013a, 2014); these results suggest that larger (i.e., older) toads have been exposed to parasites for a longer time and also expose a larger surface area for parasitic attack. In the present study, this is supported by the fact that in the subhumid zone the amphibians present larger body sizes with higher infection level. In conclusion, this study revealed that the size and habitat of R. major influence its helminth infections. In particular, our results suggest that climate (e.g. amount of rainfall and soil moisture) is an important factor affecting the distribution and development of the parasite fauna of R. major. ACKNOWLEDGMENTS We thank Ing. Rohrmann, H. of office of the Pro- vincial Water Administration (APA) of the Chaco Prov- ince, Argentina who provided information and docu- ments necessary to our work. We also thank Dr. Cuevas, M.A. of the office of the Secretary of Natural Resources, Department of Fauna and Protected Areas of the Pro- duction Ministry of Chaco Province, Argentina, and Dr. Bray, H.D of the Secretary of Natural Resources and Environmental Quality of the Production and Envi- ronment Ministry of Formosa Province, Argentina to authorize the capture of the toads for this research. Financial support was provided by the Consejo Nacional de investigaciones Científicas y Técnicas (CONICET) of Argentina, through grant PIP 11220090100345. REFERENCES Addinsoft. (2004): Xlstat for excel, version 7. 5 Addinsoft, New York. Anderson, R.C. (2000): Nematode parasites of verte- brates: their development and transmission, 2nd edn CABI International, Oxford. Bolek, M.G., Coggins, J.R. 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