Description of Cystodiscus elachistocleis sp. nov. (Cnidaria: Myxosporea) parasitizing the gallbladder of Elachistocleis cesarii from Brazil, based on morphological and molecular analyses

Numerous pseudoplasmodia containing myxospores belonging to the genus Cystodiscus were found in the gallbladder of Elachistocleis cesarii from Mato Grosso State, Brazil. Herein, we describe Cystodiscus elachistocleis sp. nov., using morphological and small subunit ribosomal DNA sequences. The mature myxospores were ellipsoid to ovoid, measuring in average 10.6 (9.8–11.2) μm in length and 6.2 (5.6–6.6) μm in width. Polar capsules were pyriform and equal in size measuring in average 3.6 (2.8–4.3) μm in length and 2.6 (2.2–3.1) μm in width. Polar filaments had 2–4 coils. The myxospores had 3–5 transverse ridges. The phylogenetic analysis showed Cystodiscus elachistocleis sp. nov. as a sister species of Cystodiscus cf. immersus 1, in a subclade formed by species that parasitize the amphibians gallbladder. This is the first species of Cystodiscus described parasitizing a species of Elachistocleis and the third species of Cystodiscus described in Brazil.


Introduction
Myxozoans are cnidarians parasites with a complex life cycle (Lom & Dyková 2006). Lutz (1889) proposed the genus Cystodiscus Lutz, 1889 to accommodate Cystodiscus immersus Lutz, 1889 based on a large disc-like appearance found in the amphibious gallbladder. The genus was abandoned and classified as a synonym of Myxidium Bütschli, 1882; however, Hartigan et al. (2012), performing phylogenetic molecular analyses, reerected the genus Cystodiscus as a monophyletic group instead of the polyphyletic nature of Myxidium. They have a global distribution and are endoparasites of a great diversity of amphibian hosts (Hartigan et al. 2012a(Hartigan et al. , 2012b(Hartigan et al. , 2016. Cystodiscus spp. could be pathogenic but some hosts do not show pathological signs and release myxospores over time without any impact on the health of their hosts (Hartigan et al. 2012b). In pathogenic cases, the hosts may show inflammation in the nervous tissue leading to behavioral changes and even spontaneous death. Also, in cases of liver diseases, they can cause inflammation and hyperplasia, affecting the metamorphosis of tadpoles, as well as metabolism and immune function (Hartigan et al. 2012b). Due to its pathogenic potential, the parasite can have an important ecological impact on the conservation of amphibians (Hartigan et al. 2012c).
Microhylidae Günther, 1858 is one of the most diverse families of extant amphibians, distributed across most of the tropics (Frost 2020). Three subfamilies of microhylids are recognized: Adelastinae Peloso, Frost, Richards, Rodrigues, Donnellan, Matsui, Raxworthy, Biju, Lemmon, Lemmon & Wheeler, 2016, Gastrophryninae Fitzinger, 1843, and Otophryninae Wassersug & Pyburn, 1987(de Sá et al. 2012Peloso et al. 2014). Elachistocleis Parker, 1927 (subfamily Gastrophryninae) is a genus of microhylid frogs widespread distributed with 19 species, most of which occur east of the Andes in South America (Frost 2020;Sánchez-Nivicela et al. 2020). In Brazil, 12 species of Elachistocleis were reported (Segalla et al. 2019). Elachistocleis cesarii (Miranda-Ribeiro, 1920), an endemic species in Brazil, occurs from southeastern to central Brazil in eastern São Paulo, south-central Minas Gerais, Goiás, and Mato Grosso States (Toledo et al. 2010;Frost 2020). This species has sexual dimorphism and females are larger than males. They have a fossorial habit, spending most of the year sheltered under the ground, and have a marked pattern of seasonal activity (Toledo et al. 2010). This species is not on the International Union for Conservation of Nature (IUCN) red list of endangered animals.
During a study on the biodiversity of anuran parasites in Mato Grosso State, Brazil, pseudoplasmodia containing myxospores morphologically consistent with Cystodiscus were observed in the gallbladder of E. cesarii. The present study aims to describe this new species of Cystodiscus parasitizing E. cesarii from Brazil, based on morphological and molecular analyses, thus increasing the knowledge of Brazilian anurans parasites.

Anuran collection and morphological analysis
In February 2020, one female anuran, E. cesarii measuring 39 mm snout-vent length and weight of 5.8 g, was captured for a major project of biodiversity of parasites in amphibians (FAPESP 2018/09623-4;FAPESP 2018/00754-9). A new sampling effort was carried out at different times within the major project, but it was not successful in collecting more specimens of E. cesarii. The specimen was collected on a dirt road in the municipality of Araguaiana, Mato Grosso State, Brazil (14°35′5.29″ S, 51°41′19.15″ W). The host was deposited in the Herpetologica Collection of the Regional University of Ceará (Num. CHUFC A9761).
The anuran was killed using 50 mg/kg of sodium thiopental (Thiopentax®), a commercial anesthetic administered intracerebrally, following the guidelines of Sebben (2007) and the Animal Use Ethics Committee (IBAMA license 60640-1; CEUA-UNESP 1061). The coelom was opened via a midventral longitudinal incision and the gall bladder was examined for cysts or signs of myxosporean infection. Gallbladder contents were removed by puncturing the bladder wall and pipetting out the contents (Hartigan et al. 2016). The fresh smears containing pseudoplasmodia were evaluated with a differential interference contrast microscope (Leica DMLB 5000, Leica Microsystems, Wetzlar, Germany) at 1000 × magnification. The morphological measurements of myxospores freshly preserved followed the recommendations of Lom & Arthur (1989). Measurements of 30 myxospores were obtained and presented as mean ± standard deviation.

Molecular analysis
Two pseudoplasmodia from the gallbladder were fixed in absolute ethanol and used for the molecular analyses. The access to the genetic data was authorized by the Brazilian Ministry of Environment (Sisgen AA4666FA). The two isolated pseudoplasmodia were collected from the bile material with finetipped sterile needles. DNA isolation was carried out following the animal tissue protocol of the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The partial SSU rDNA gene was amplified with PCR using general eukaryotic and myxozoan primers (Table 1).
Amplification was performed in a Bio-Rad Mycycler (Bio-Rad Laboratories Pty Ltd., Gladesville, Australia), with initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 1 min, 55°C for 45 s, 72°C for 2 min and a final extension at 72°C for 7 min. PCR reactions were performed in 25 μl of solution containing 3 μl of extracted DNA and 1 μl of each PCR primer at 10 pmol, using PCR Ready-to-Go beads (Pure TaqTMReady-to-GoTM beads, GE Healthcare, Chicago, USA). The solution consisted of stabilizers, BSA, dATP, dCTP, dGTP, dTTP, ± 2.5 units of puReTaq DNA polymerase and reaction buffer. Each bead was reconstituted to a final volume of 25 μl. PCR products were analysed by electrophoresis on 1% agarose gel stained with GelRed and visualised under UV light. The products of the PCR reaction for the SSU rDNA gene were purified and precipitation reaction by Ethanol/EDTA/ Sodium Acetate according to the protocol suggested by the manufacturer was performed, and then sequenced with a BigDye® Terminator ver. 3.1 Cycle Sequencing Kit (Applied). Automatic sequencing by capillary electrophoresis was performed on the ABI3730xl DNA Analyzer (Applied Biosystems).
The partial sequences obtained were assembled and edited using a Sequencher TM ver. 5.2.4 (Gene Codes, Ann Arbor, MI) to obtain a consensus sequence. The newly generated partial sequences of SSU rDNA were aligned using Geneious ver. 7.1.3 (Kearse et al. 2012) with the ClustalW algorithm (Larkin et al. 2007) and default settings with related genetic sequences that appeared on Blastn search ( Table 2). The Bayesian inference (BI) and Maximum-Likelihood (ML) analyses were performed using MrBayes ver. 3.1.2 (Ronquist & Huelsenbeck 2003) Markov Chain Monte Carlo (MCMC) chains were run for 10 million generations and the log-likelihood scores were plotted. The 'burn-in' was set to 30%. PhyML 3.1 (Guindon et al. 2010) software was used to perform ML analysis, using bootstrap confidence calculated with 1000 replications and GTR+I+G evolutionary model which was chosen by jModeltest (Posada 2008) as the best model for this analysis. Phylogenetic trees were generated and edited in FigTree ver. 1.4 (Rambaut 2012). Chloromyxum trilineatum Sekyia, Rosyadi, Zhang & Sato, 2019 (LC417364) was used as an out group. The aligned sequences of myxosporean parasites were compared using a pair-wise distance (p-distance) matrix (Table 3).

Site of infection
Gallbladder.

Etymology
The species epithet is derived from the genus Elachistocleis of the host.

Remarks
Cystodiscus elachistocleis sp. nov. was morphometrically compared to all Cystodiscus spp. described worldwide ( Table 2). The species that most resembled C. elachistocleis sp. nov. was C. haldari (Sarkar, 1982), which showed similarity in all measurements available for comparison. However, the geographical distance (Brazil vs India) and the different type host (E. cesarii vs Hyla arborea (Linnaeus, 1758)) allow us to separate the two species. Furthermore, C. haldari does not present any comparative values such as the number of turns of the polar filament and the number of transverse ridges, which could differentiate the two species. Cystodiscus thyponius Gray (1993) also presented measures of length and width of myxospores similar to those found in C. elachistocleis sp. nov. However, differences were observed in the polar capsule width (2.6 ± 0.3 (2.2-3.1) vs 3.6 (3.3-5.2)) and the number of transverse ridges (3-5 vs 9-11).

Molecular analyses
Fragments of 1730-bp and 1916-bp of the SSU rDNA gene were generated. The fragments showed 100% similarity when aligned. The BLAST search of the sequences did not reveal a direct match with myxozoan sequences available in GenBank. The genetically closest species was C. immersus, which exhibited a similarity of 97.3%, and a difference of 24 out of 885 nucleotides (Table 3). A well-supported phylogenetic tree divided into three main groups was obtained (Fig. 3)

Discussion
Considering the myxospore morphology, the morphometric data, and SSU rDNA gene partial sequence obtained in the present study, we described C. elachistocleis sp. nov. This finding contributes to our knowledge of the biodiversity of Cystodiscus in amphibians from Brazil. To our knowledge, C. elachistocleis sp. nov. is the first record of a myxozoan species parasitizing E. cesarii and the third species of Cystodiscus described from Brazil.
Brazil is one of the most species-rich areas for amphibians globally (Segalla et al. 2017). However, amphibians are also considered the most threatened vertebrate group with many species facing extinction (Wake & Vredenburg 2008;Rebouças et al. 2021). Amphibians also are hosts of a variety of endoparasites and ectoparasites, which in some cases influence their fitness, behavior, feeding, reproduction, and fertility (Barta & Desser 1984;Lainson et al. 2003;Muñoz-Leal et al. 2017). For this study, only one specimen of E. cesarii was collected despite the extensive sampling effort performed. This could be due to the low presence of the host at the collection site or its ecology, which makes collecting it more difficult than other species. Despite this, several studies describe new myxozoans from a single infected host (Hartigan et al. 2012a;Chen et al. 2020;Mathews et al. 2021).
Regarding myxozoan parasites, there are only two species of Cystodiscus described on Brazilian anurans, C. immersus, and C. lyndoyense. However, the morphological and morphometric data differ from the species in the present study. The specificity and the host organ combined with myxospore morphology and molecular data are essential for a determination of a new myxozoan species. Following this taxonomic strategy, we described a new species of Cystodiscus, a gallbladder parasite of E. cesarii.
The phylogenetic analysis performed here supports previous work that suggests that Cystodiscus are amphibian gallbladder species (Hartigan et al. 2012a). The monophyletic subclade composed of Cystodiscus spp. was well supported. The low values of posterior probability found in some nodes within the subclade can be explained by the low number of described species that have their partial sequences of the SSU rDNA gene available. Cystodiscus cf. immersus 1 and Cystodiscus cf. immersus 2 appear with different sister species in the subclade, indicating that they are two distinct species.
The genus Cystodiscus is globally distributed and may have ecological implications outside of Brazil (Hartigan et al. 2012c). These parasites are associated with inflammation of nervous tissue and hepatic disease in several threatened or common frog species (Hartigan et al. 2012b). Although the situation of E. cesarii in the list of endangered species is ʻleast concern -LCʼ, a parasite that causes serious pathologies in these amphibians could further decrease the population of amphibians in Brazil, something that has been happening gradually (Subirá et al. 2012;Ceballos et al. 2020). Despite this, in the present study, no pathologies were observed in the gallbladder or any other host organ associated with parasitism by Cystodiscus elachistocleis sp. nov.