Nematodes of Proceratophrys ararype (Anura: Odontophrynidae), an endemic frog from the Araripe Plateau, northeastern Brazil

Parasites are an important component of the global biomass, having significant roles in several regulatory mechanisms in the ecosystem. Parasitism is one of the most common ecological interactions on the planet. Studies have shown that the helminth fauna of only 8% of amphibian species in Brazil have been studied, and this percentage is lower for the Odontophrynidae family, with only four of the 50 species kown to occur in Brazil having been investigated. Here, we present the helminth fauna of Proceratophrys ararype, an anuran endemic to the “Brejo de Altitude” Chapada do Araripe (Araripe Plateau), northeastern Brazil. The infection parameters analyzed were prevalence, mean intensity of infection and mean abundance of parasites. We used the Pearson’s linear correlation coefficient to check the correlations between the abundance of the parasites with the snout-vent length (SVL) of hosts. To verify the degree of aggregation of parasites in hosts, we used the dispersion index. Out of 40 specimens examined, 19 specimens were infected with at least one parasite. The overall prevalence was 47.5% with a mean infection intensity of 18.93 ± 10.77. The endoparasitic community associated with P. ararype consisted of six species of parasites, with Falcaustra mascula having the highest prevalence (25%). Most parasites had a uniform dispersion index in the hosts and their abundance was unrelated to host size. Here, we also present a compilation of all parasites associated with host species of the genus Proceratophrys from South America. Overall, we found 23 species of parasites associated with five host species (P. ararype, P. appendiculata, P. boiei, P. cristiceps, P. mantiqueira). Of these, two species of parasites (Oswaldocruzia mazzai and Strongyloides sp.) represent new records for the genus Proceratophrys. Our results demonstrate the lack of studies on amphibian helminth fauna and fill an important knowledge gap on the diversity of parasites of Proceratophrys ararype, an endemic frog from the Araripe Plateau, northeastern Brazil.


Introduction
Currently, 50 species of the Odontophrynidae family have been identified, with the genus Miranda-Ribeiro, 1920 making up 40 species registered in Brazil, Argentina, and Paraguay (Segalla et al. 2019, Mângia et al. 2020, Frost 2021. Species of the genus Proceratophry can be clumped into four species groups, based on the morphological similarities of adult individuals (Cruz et al. 2005, Prado & Pombal 2008, Mângia et al. 2018: P. appendiculata, P. bigibbosa, P. boiei and P. cristiceps species groups. The P. cristiceps group comprises 14 species including P. ararype Mângia, Koroiva, Nunes, Roberto, Ávila, Sant'Anna, Santana & Garda, 2018(Ávila et al. 2011, Brandão et al. 2013, Mângia et al. 2018). This species was identified from specimens collected at the slope of Chapada do Araripe, a humid forest in the municipality of Crato, state of Ceará, northeastern Brazil. The distribution of this species is very restricted and limited to the slope of Chapada do Araripe (Mângia et al. 2018), a climate exception area in the Caatinga biome (Tabarelli & Silva 2003), where local climatic conditions shape isolated systems (Vanzolini 1981, Borges-Nojosa & Caramaschi 2003. Currently, information on the associated parasitic fauna of P. ararype is lacking.
The parasites are integral components of the global biomass, and one of the most common life forms on the planet (Kuris 2008, Poulin 2014, Oliveira et al. 2019). Among these parasites, the helminth fauna associated with amphibians is rich and diverse, despite being hidden within known biodiversity (Poulin 2014, Campião et al. 2014). The study of parasitic fauna is extremely important due to the roles they have in several regulatory mechanisms within the ecosystem, including the negative effects they have on their hosts such as anaemia, anorexia, reduced survival and fertility, and competition (Vitt & Caldwell 2009, Matias et al. 2018. The study of these organisms not only contributes to our knowledge of animal diversity, but also clarifies the parasite dynamics of the host (Brooks & Hoberg 2001, Galli et al. 2001, Poulin 2014, because the richness of parasite species can be assumed as a characteristic of the host, where rates of parasite colonization vary according to biology of hosts (Poulin 2014, Campião et al. 2015. The latest checklist of helminths in South America stated that only about 8% of all amphibian species in Brazil have had their helminth fauna studied (Campião et al. 2014). Of the Proceratophrys genus, the helminth fauna of only four species has been studied: P. tupinamba Prado & Pombal, 2008(Boquimpani-Freitas et al. 2001, P. cristiceps Müller, 1883(Teles et al. 2017, Sampaio et al. 2020, P. boiei Wied-Neuwied, 1824 (Toledo et al. 2018), and P. mantiqueira Mângia, Santana, Cruz & Feio, 2014(Almeida-Santos et al. 2017. As such, studies on parasitism are of fundamental importance for the conservation of hosts, especially those with a restricted distribution, in addition to filling knowledge gaps in host-parasite interactions, since hosts, in general, are more susceptible to local extinctions (Pontes & Rocha 2011). Here we describe the composition and patterns of parasitic infection of helminths associated with P. ararype, an endemic frog from Chapada do Araripe, northeastern Brazil, and also present a compilation of all parasites associated with host species of the genus Proceratophrys from South America.
A total of 40 Proceratophrys specimens were collected using the active search method (visual and auditory) (Bernarde 2012). Specimens were kept in individualized plastic containers and later euthanized by lethal injection of Lidocaine Hydrochloride (CFMV 2013). We measured the snout-vent lengths (SVL, in mm) of hosts using a digital caliper Mitutoyo® (precision 0.01 mm). Hosts were fixed according to Calleffo (2002) and deposited in the Herpetological Collection of the Regional University of Cariri, (URCA-H 15.579-15.616), Crato municipality and in the Herpetological Collection of the Federal University of Cariri (CHERP-UFCA 01-02), Brejo Santo municipality, both in the Ceará state, Brazil.
We used the Pearson's linear correlation coefficient (r) to assess correlation between parasite abundance with host snout-vent length (SLV, in mm). The Mantel test was used to evaluate spatial autocorrelation between parasitic richness and sampled areas to verify if use the data as one or several parasite communities. The variance / mean ratio (s2/x ̅ ), also known as the dispersion index (ID), and the k parameter of the negative binomial distribution, were used to determine the degree of parasite aggregation within the hosts. The higher the s2/x ̅ ratio, and the lower the value of parameter k (closer to zero), the higher the level of aggregation (Pielou 1977).
To compile literature data on parasites associated with host species of the genus Proceratophrys from South America, we conducted a wide search in different databases (e.g., Google Academic, Scielo, Scopus) and in bibliographic reviews on the topic (e.g., Campião et al. 2014).

Ethical Standards
The authors assert that all procedures contributing to this study comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals. The study was approved by the Ethical Committee of Universidade Regional do Cariri (CEUA/URCA, process number 00260/2016.1) and Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio/SISBIO, number 66099-2).
There was no correlation between parasite general abundance and host snout-vent length (R 2 = 0.01, p = 0.43), when analyzing the most prevalent parasite species individually, we also found no significant correlation between parasite abundance and SVL: A. membranosa (R² = 0.13, p = 0.79), Physaloptera sp. (R² = 0.34, p = 0.56), and F. mascula (R² = 0.18, p = 0.61). Additionally, spatial autocorrelation was not observed between sample areas and parasitized individuals (R 2 = 0.3545, p = 0.11667). Examination of pattern dispersion revealed that most helminth species had a uniform distribution among hosts (Table 2), that is, the growth in the number of infected individuals is directly proportional to the prevalence of infection.
In our literature data compilation, we found, in general, five host species (P. ararype, P. appendiculata, P. boiei, P. cristiceps, P. mantiqueira) being parasitized by 23 helminth species. Proceratophrys cristiceps was the host species with the highest number of associated parasitic helminths (n = 10 spp.). Physaloptera sp. was the only helminth common to all studied Proceratophrys species (Table 3). In this study we identified two additional species (O. mazzai and Strongyloides sp.) ( Figure 1A, B) registered for the genus Proceratophrys.

Discussion
The genus Proceratophrys has 23 species of registered parasites (Campião et al. 2014, Almeida-Santos et al. 2017, Teles et al. 2017, Toledo et al. 2018, Sampaio et al. 2020. Like observations made in previous studies, we observed a particular parasitic community per host species, with Physaloptera sp. being the only helminth common to all studied Proceratophrys species (Table 3). This result could be due to the geographical locations of each species, which have different environmental conditions, and thereby affecting the composition and richness of biotic factors (Poulin & Krasnov 2010). On the other hand, P. cristiceps, which has a wide geographical distribution (Mângia et al. 2020), was the best studied species (Teles et al. 2017, Müller et al. 2018, Sampaio et al. 2020) and with the highest number of associated parasitic helminths (n = 10 spp.). This result emphasizes that habitat, along with the biology, life history of the host and study effort, can influence parasitic composition (Campião et al. 2015).
Species of the genus Aplectana are usually found infecting the large intestine of reptiles and amphibians, have a direct life cycle, and actively infect their hosts (Travassos 1931, Anderson 2000, Campião et al. 2014, Lins et al. 2017. This genus has been observed in four species of the Odontophrynidae family: Proceratophrys tupinamba and P. boiei, infected by A. delirae Fabio, 1971(Boquimpani-Freitas et al. 2001, Klaion et al. 2011, and P. cristiceps , Sampaio et al. 2020 and Odontophrynus americanus Duméril & Bibron, 1841, infected by A. membranosa (Lent & Freitas 1948 (Mcalpine 1997). Additionally, A. membranosa females produce large amounts of infective larvae in the environment, increasing infection rates (Lins et al. 2017). Falcaustra mascula has been shown to infect the small and large intestine of several amphibians (e.g., Campião et al. 2014, Toledo et al. 2015, Toledo et al. 2018. Little is known about its modes of transmission. Anderson (2000) suggested that the larvae reach its third development stage and then infect an intermediate invertebrate host, which in turn is ingested by amphibians. Toledo et al. (2018) found that among all host parasites F. mascula had the highest prevalence rate in Boana faber (Wied-Neuwied, 1821) (P = 9.1%), Leptodactylus latrans (Steffen, 1815) (P = 13.9%), and Rhinella icterica (Spix, 1824) (P = 33.3%). Therefore, our result for prevalence rate (P = 25%) is consistent with the literature data. One plausible explanation for the high infection rates observed for this parasite is that the intermediate host is an arthropod, which is a type of prey that is extensively consumed by anurans.
Oswaldocruzia mazzai have been shown to infect a variety of anuran hosts (e.g., Campião et al. 2014, Teles et al. 2015, Alcantara et al. 2018, Oliveira et al. 2019) but our study is the first to record a species of the genus Proceratophrys as a host of O. mazzai. The low host specificity is usual for some groups of helminths (Campião et al. 2015, Oliveira et al. 2019. The great diversity of hosts registered for O. mazzai is related to the direct life cycle, and the simple mode of transmission that can occur by ingesting eggs or larval penetration into the host's skin (Anderson 2000).
Nematodes of the Physaloptera genus have a worldwide geographical distribution and have been recorded in several classes of terrestrial vertebrates, including felines (Ogassawara 1986), rodents (Tung et al. 2009), lizards (Da Silva et al. 2008, Cabral et al. 2018) and anurans (Da Graça et al. 2017). This parasite was usual for all studied Proceratophrys species (see Table 3). In amphibians, this parasite is usually found in larval stage, making it difficult to identify at the species level, and is suggestive that these amphibians are not definitive hosts. Although there is not enough data about its life cycle, nematodes of this genus are known to use insects during their intermediate phase (Anderson 2000). Additionally, the acquisition of Physaloptera by anuran hosts occurs through the ingestion of infected insects, mainly Orthoptera (Klaion et al. 2011).
The Strongyloides genus has a low specificity, with records for several classes, including mammals (occasionally humans), birds, reptiles, and amphibians (Little 1966, Urquhart et al. 1998. Although there is a lack of detailed biology on this parasite, it is known to have a direct or indirect life cycle, with the former being the most usual (Santos et al. 2010). The infection occurs on land through skin penetration or ingestion of infected preys (Mati & Melo 2014, Sulieman et al. 2015. Even though this nematode infects several amphibian species (Campião et al. 2014, Sulieman et al. 2015, this is the first record of the genus Strongyloides acting as a parasite for species of the Odontophrynidae family. The parasite Raillietnema spectans was initially described in the large intestine of leptodactylids and bufonids (Alcântara et al. 2018), and it has been registered for several other species: Rhinella crucifer (Wied-Neuwied, 1821), R. icterica and Leptodactylus latrans (Campião et al. 2014), Pleurodema diplolister (Peters, 1870) (Teles et al. 2015), Physalaemus albifrons (Spix, 1824), P. cicada Bokermann, 1966P. cuvieri Fitzinger, 1826(Oliveira et al. 2019, and Dermatonotus muelleri (Boettger, 1885) (Alcantara et al. 2018). This parasite is known to presents a direct life cycle and transmission that occurs via ingestion or penetration of larvae in the skin (Anderson 2000). In this study, R. spectans had the lowest prevalence, contrary to the results from Alcântara et al. (2018) and Oliveira et al. (2019). This low prevalence may be related to host phylogeny, which is reflected in the structuring of parasitic interactions (Krasnov et al. 2012), or geographical, biological, and life history effects on the host, which can influence parasitic composition (Campião et al. 2015).
Helminths registered for the genus Proceratophrys are usually found in other taxa of amphibians, and therefore can be considered generalists (Campião et al. 2014, Müller et al. 2018). Nevertheless, studies on Procetatophrys cristiceps (Teles et al. 2017, Müller et al. 2018, and P. ararype (this study), in northeastern Brazil, and on P. boiei, P. mantiqueira and P. tupinamba, in humid forests of southeastern Brazil (Boquimpani-Freitas et al. 2001, Klaion et al. 2011, Almeida-Santos et al. 2017, Toledo et al. 2018, show a greater similarity between helminth communities in locally close hosts. These results suggest that geographic distribution of the host, and the different local conditions, could influence the composition of helminth fauna. We did not find correlation between parasitism and host size. This result contrasts with those found for the genus Leptodactylus, where body size accounted for 17% of the variation in species composition, compared to the 3% accounted for the host's habitat (Campião et al. 2016a). However, our results were similar to those described by Oliveira et al. (2019) for Physalaemus species. This similarity between the results may be due to the smaller size variation, when species are analysed    (Müller, 1883) Aplectana   Table 2). One of the most usual characteristics of parasitic infections in populations of vertebrate hosts is aggregation, because these infections rarely happen or are due to the high lethality in infected hosts, which cannot survive for long periods (Von Zuben 1997). Therefore, the uniform dispersion model, predominant in the helminth species of this study, may be due to parasite mortality, a process dependent on the density and mortality of the host induced by the parasite. Additionally, regular or uniform distribution is also observed if there is strict competition between individuals, or if there is positive antagonism, causing a constant minimum distance between individuals (Odum & Barrett 2008).
Anurans have the supracommunity pattern of diversified generalist helminth parasites with low host specificity and wide distribution (Campião et al. 2014). Due to the increase in studies conducted on this topic (e.g., Campião et al. 2016a, b, Lins et al. 2017, Teles et al. 2017, Leivas et al. 2018, Alcantara et al. 2018, Oliveira et al. 2019, it is quite common to find new records of hosts containing parasites that have not previously been described for the species (e.g., Aguiar et al. 2014. In this study, we present new records on species of parasites (O. mazzai and Strongyloides sp.) for the genus Proceratophrys, increasing the total number to 21 helminths. Additionally, all helminth species found represent their first records for Procetatophrys ararype, a frog endemic to the Brejo de Altitude Chapada do Araripe, Northeastern Brazil.

Author Contributions
Wilmara Mascarenhas: Contribution to data collection; contribution to identification of parasites and amphibians; contribution to data analysis and interpretation; contribution to manuscript preparation and critical revision Cicero R. Oliveira: Contribution to data collection; contribution to identification of parasites and amphibians; contribution to data analysis and interpretation; contribution to manuscript preparation and critical revision.
Ronildo A. Benício: Contribution to data analysis and interpretation; contribution to manuscript preparation and critical revision.
Robson W. Ávila: Contribution to identification of parasites and amphibians; contribution to manuscript preparation and critical revision.
Samuel C. Ribeiro: Contribution to data collection; contribution to data analysis and interpretation; contribution to manuscript preparation and critical revision.