Introduction
Nematodes of the genus Cosmocerca Diesing, Reference Diesing1861 (Ascaridomorpha: Cosmocercoidea) are cosmopolitan parasites found in the digestive tract of several amphibians (see Campião et al., Reference Campião, Morais, Dias, Aguiar, Toledo, Tavares and Silva2014; Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015; Sou et al., Reference Sou, Sow and Nandi2018a, Reference Sou, Sow and Nandib; Chen et al., Reference Chen, Zhang, Feng and Li2020; Ni et al., Reference Ni, Barton, Chen and Li2020; Harnoster et al., Reference Harnoster, du Preez and Svitin2022; Machado et al., Reference Machado, Oliveira, Benício, Araújo and Ávila2022) and some lizards (Bursey & Goldberg, Reference Bursey and Goldberg2004; Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015; Ávila & Silva, Reference Ávila and Silva2019). To date, 37 species of Cosmocerca have been reported worldwide (Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015; Sou et al., Reference Sou, Sow and Nandi2018a; Ávila & Silva, Reference Ávila and Silva2019; Banerjee & Sou, Reference Banerjee and Sou2020; Chen et al., Reference Chen, Zhang, Feng and Li2020; Ni et al., Reference Ni, Barton, Chen and Li2020; Harnoster et al., Reference Harnoster, du Preez and Svitin2022) and only 11 occur in the Neotropical region: Cosmocerca brasiliensis Travassos, Reference Travassos1925; Cosmocerca chilensis Lent & Freitas, Reference Lent and Freitas1948; Cosmocerca cruzi Rodrigues & Fabio 1970; Cosmocerca gymnophthalmicola Ávila & Silva, Reference Ávila and Silva2019; Cosmocerca parva Travassos Reference Travassos1925; Cosmocerca podicipinus Baker & Vaucher, 1984; Cosmocerca paraguayensis Moravec & Kaiser, Reference Moravec and Kaiser1994; Cosmocerca rara Freitas & Vicente 1966; Cosmocerca travasssosi Rodrigues & Fabio, 1970; Cosmocerca uruguayensis Lent & Freitas, Reference Lent and Freitas1948; and Cosmocerca vrcibradici Bursey & Goldberg Reference Bursey and Goldberg2004 (Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015; Ávila & Silva, Reference Ávila and Silva2019).
Despite the increasing number of studies regarding the superfamily Cosmocercoidea in the last 10 years (Ross et al., Reference Ross, Ivanova, Spiridonov, Waeyenberge, Moens, Nicol and Wilson2010; Jones et al., Reference Jones, Brown, Harris, Jones, Symondson, Bruford and Cable2011; Sato et al., Reference Sato, Hasegawa, Sekiya and Tsubouchi2015; Tran et al., Reference Tran, Sato and Luc2015; Chen et al., Reference Chen, Zhang, Gibson, Lü, Xu, Li, Ju and Li2018, Reference Chen, Zhang, Feng and Li2020, Reference Chen, Gu, Ni and Li2021a, Reference Chen, Ni, Gu, Sinsch and Lib; Maity et al., Reference Maity, Rizvi, Bursey and Chandra2019; Sümer et al., Reference Sümer, Birlik and Yildirimhan2019; Liu et al., Reference Liu, Yu, Shu, Zhao, Fang and Wu2020; Sata & Nakano, Reference Sata and Nakano2020; Harnoster et al., Reference Harnoster, du Preez and Svitin2022), the current knowledge about the molecular phylogeny of the family is still scarce (Chen et al., Reference Chen, Gu, Ni and Li2021a). To date, molecular data for only 39 species of Cosmocercoidea, of which 23 species are of the subfamily Cosmocercinae (18S, 28S, ITS, 12S, Cox1 and Cox2), are available in the GenBank database. Among these sequences, only 10 Cosmocerca spp. have been accessed: Cosmocerca ornata Dujardin, 1845; Cosmocerca simile Chen, Zhang, Feng & Li, 2021; Cosmocerca japonica Yamaguti, 1938; Cosmocerca longicauda (Linstow, 1885); Cosmocerca kalesari Rizvi, Bursey & Bhutia, 2011; Cosmocerca daly Harnoster, du Preez & Svitin, Reference Harnoster, du Preez and Svitin2022; Cosmocerca monicae Harnoster, du Preez & Svitin, Reference Harnoster, du Preez and Svitin2022; Cosmocerca makhadoensis Harnoster, du Preez & Svitin, Reference Harnoster, du Preez and Svitin2022; Cosmocerca sp.1; and Cosmocerca sp.2. Notwithstanding, none of these is from the Neotropical region.
During a helminthological survey of Brazilian anurans in the São Paulo and Mato Grosso states, some Cosmocercinae nematodes were collected. By using light microscopy and molecular analyses of the mitochondrial DNA (cytochrome c oxidase subunit 1 – COI mtDNA), we were able to accurately characterize and determine the systematic position of those nematodes. This study reports the first molecular assessment of C. podicipinus and C. parva and describes a new species of Cosmocerca.
Materials and methods
Nematode identification and DNA extraction
Cosmocerca specimens from nine anurans’ hosts from Mato Grosso and São Paulo states were collected (table 1). The frogs were killed with 2% lidocaine hydrochloride and the internal organs were removed, dissected, and analysed under a stereomicroscope. Nematodes were collected from the intestines of the anurans and stored directly in 96% ethanol. Each nematode found was cut and the anterior part was used for molecular analysis and the posterior one was used for morphological studies. For the species identification, the morphology of the posterior end of the nematodes was compared with all published Cosmocerca spp.
Data for the anurans sampled from 2018 to 2020 in the municipalities of Mato Grosso (MT) and São Paulo (SP) states, Brazil, and surveyed for Cosmocerca species (NH = number of hosts).
The posterior end of the nematode specimens was cleared with lactophenol, mounted in temporary slides, and analysed under a microscope with a computerized image analysis system (Qwin Lite 3.1, Leica Microsystems, Wetzlar, Germany). Morphological measurements are given in micrometres and presented as range with values for holotype indicated in parentheses (only for the description of the new species).
Drawings were made using a camera lucida attached to a Leica DMLS microscope with phase contrast optics. The anterior end of 11 males and one female of the analysed worms were separated for molecular analyses.
After morphological identification, DNA extractions were performed using the anterior end of the specimens with DNeasy® Blood & Tissue Kit (QIAGEN) according to the manufacturer's protocol. The partial COI mtDNA was amplified by polymerase chain reaction (PCR) using the primers: COIF (5′-TTTTTTGGTCATCCTGAGGTTTAT-3′) and COIR (5′-ACATAATGAAAATGACTAACAAC-3′) (Lazarova et al., Reference Lazarova, Malloch, Oliveira, Hübschen and Neilson2006). The cycling conditions followed Chen et al. (Reference Chen, Zhang, Gibson, Lü, Xu, Li, Ju and Li2018).
The PCR reactions were performed using 3 μl of DNA extract, 1 μl of each primer, 7.5 μl or 8.5 μl of ultrapure water (Sigma-Aldrich, United Kingdom), and 12.5 μl Master Mix MyFiTM Mix Bioline®, with a final volume of 25 μl. PCR products were subjected to gel electrophoresis at 80 V in a 1.5% agarose gel, stained with Gel Red, and observed using an ultraviolet transilluminator. The products of interest were purified by adding 2 μl of ExoSAP-IT® (Affymetrix, Santa Clara, CA, USA) to 5 μl of PCR product according to the manufacturer's recommendations. Amplicons were sequenced using PCR primers on a 3500 Genetic Analyzer capillary sequencer (Applied Biosystems) using BigDye Terminator Cycle Sequencing Ready Reaction Kit v.3.1 (Applied Biosystems) according to the manufacturer's recommendations.
The newly generated sequences were assembled and edited using Sequencher v.5.2.4 (Gene Codes, Ann Arbor, MI, USA), and subjected to the Basic Local Alignment Search Tool algorithm available in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov) for preliminary identification.
Phylogenetic analyses
The newly generated sequences were aligned along with other Cosmocercidae sequences retrieved from the GenBank database. Falcaustra sinensis Liu, Zhang & Zhang, 2011 [MF113223] and Falcaustra sp. [MN729572] were used as the outgroup for the alignment.
The alignment was performed using the default parameters of the algorithm Muscle (Edgar, Reference Edgar2004) implemented on Geneious 7.1.3 (Kearse et al., Reference Kearse, Moir and Wilson2012). The best-fitting model of nucleotide substitution for the dataset was Hasegawa–Kishino–Yano + G + I, selected in the JModelTest software (Posada, Reference Posada2008) using the Akaike information criterion.
Phylogenetic trees were obtained using maximum likelihood (ML) and Bayesian inference (BI). The BI was performed with MrBayes 3.2 (Ronquist et al., Reference Ronquist, Teslenko and Mark2012) at the online platform CIPRES. The Markov chain Monte Carlo was run with 50,000,000 generations saving one tree every 1000 generations, with a burn-in of 25% of the trees. Only nodes with posterior probabilities greater than 90% were considered well supported. Phylogenetic analysis using ML was run in RAxML (Guindon & Gascuel, Reference Guindon and Gascuel2003) at the online platform CIPRES with 1000 bootstrap replicates. Only nodes with bootstrap values greater than 70% were considered well supported. The ML and BI trees were visualized and edited in FigTree v. 1.3.1 software (Rambaut, Reference Rambaut2009).
The COI mtDNA genetic divergence between sequences was calculated using the Kimura 2-parameter model with 1000 bootstrap replicates in MEGA 7 software (Kimura, Reference Kimura1980).
Results
Cosmocerca parva Travassos 1925
Three male specimens of C. parva were found in the large intestine of two Leptodactylus podicipinus (Cope, 1862) and one Dendropsophus nanus (Boulenger, 1889) from the São Paulo state.
Numerous somatic papillae lined up in two ventral, two subventral, two dorsal and two subdorsal rows. Caudal alae absent. Narrow lateral alae present spicules equal to 80.3–108.5 long, weakly sclerotized, gubernaculum well sclerotized, Y-shaped, 90.4–133.5 long. Cloacal papillae arranged in 5:3 + 1:7, as follows: five pairs of plectanes, each one with two complete rosettes and a relatively inconspicuous underlying sclerotized support in the precloacal region with three pairs of rosette papillae in the adcloacal region and one large unpaired papilla in the anterior lip of cloaca; and seven pairs of postclocal (six ventral pairs and one lateral) papillae. Tail 218.9 long, ending in a small spike.
Cosmocerca podicipinus Baker & Vaucher, 1984
Seven specimens (5 males, 2 females) of C. podicipinus were found in the large intestine of a Boana caiapo Pinheiro, Cintra, Valdujo, Silva, Martins, Silva & Garcia, 2018, two Leptodactylus latrans (Steffen, 1815), one Physalaemus centralis (Bokermann, 1962) and one Pseudis platensis Gallardo, 1961 from Mato Grosso state.
Numerous somatic papillae lined up in two ventral, two subventral, two dorsal and two subdorsal rows. Caudal alae absent. Narrow lateral alae present. Posterior end of body distinctly ventrally curved. Spicules small and equal, well sclerotized, with distal end pointed, 73.1–109.6 long, gubernaculum small and conical, well sclerotized, 96.2–124.5 long. Cloacal papillae arranged in 5:3 + 1:6, as follows: five pairs of plectanes, each with two complete rosettes of punctations directed perpendicular to the body surface and a relatively inconspicuous underlying sclerotized support that is not fused to other plectanes in the precloacal region; three pairs of broad and flat papillae which are commonly surrounded by a small rosette of punctations in the adcloacal region and one large unpaired papilla in the anterior lip of cloaca; and six pairs of postclocal, large, and simple papillae (distinguishable from somatic papillae). Tail 139.2–225.6 long, ending in a small spike.
Cosmocerca albopunctata n. sp. Alcantara and Silva
Small-sized, whitish nematodes. Body cylindrical, maximum width at about mid-body. Cuticle with fine transverse striations (figs 1–3). Numerous somatic papillae lined up in two ventral, two subventral, two dorsal and two subdorsal rows. Caudal alae absent. Narrow lateral alae present. Oesophagus divided into anterior pharynx, cylindrical corpus, isthmus and terminal posterior bulb with valves. Nerve ring located at about 1/2 of oesophageal length. Excretory pore posterior to oesophageal bulb. Deirids not observed. Tail conical, with pointed tip.
Male of Cosmocerca albopunctata n. sp.: (A) body, lateral view; (B) anterior end, lateral view; (C) posterior end, ventral view; (D) posterior end, lateral view; and (E) detail of spicules and gubernaculum. B = oesophageal bulb; CL = cloaca; DP = dorsal papilla pair; EP = excretory pore; ES = oesophagus; G = gubernaculum; I = intestine; LP = lateral papilla pair; NR = nerve ring; P = plectane; R = rosette; RS = reproductive system; S = spicule; T = tail; VP = ventral papilla pair; Upos = unpaired postclocal papilla; and Upre = unpaired preclocal papilla. Scale bars: 1 mm (A); 200 μm (B–D); and 20 μm (E).
Enlarged view of the cloacal region of Cosmocerca albopunctata n. sp. showing spicules (S) and gubernaculum (G).
(A) Part of the precloacal region of the male of Cosmocerca albopunctata n. sp. showing rosettes (black arrows) and plectanes (white arrows); and (B) detail of preclocal rosette (black arrow) and plectanes (white arrow).
Male (3 mature specimens, 1 entire and two posterior ends): Body 6250 long; maximum width 380.1. Oesophagus 304.6 long, pharynx + corpus + isthmus 223.5 long; oesophageal bulb 81.1 × 57.1. Nerve ring 233.5 and excretory pore 533.3 from anterior extremity, respectively. Lateral alae extending from oesophageal bulb to cloaca region. Posterior end of body distinctly ventrally curved. Spicules small and equal, well sclerotized, with distal end pointed, 89.6–99.8 (89.6) long. Gubernaculum small and conical, well sclerotized 119.7–157.8 (155.4) long. Cloacal papillae arranged in 7 + 1:1 + 1:6, as follows: seven pairs of plectanes and one unpaired small rosette between the cloaca and the first pair of plectane in the precloacal region; plectane consisting of central papilla with two rows of 10 cuticular tubercles on underlying sclerotized segments; one large unpaired papilla in the anterior lip of anus and one large unpaired papilla in the posterior lip of anus; and six pairs of postclocal, large, and simple papillae (four ventral pairs, one lateral pair, and one dorsal pair). Tail 191.4–266.6 (253.7) long, ending in a small spike.
Female: Unknown.
Taxonomic summary
Type host: White-spotted humming frog Chiasmocleis albopunctata (Boettger, 1885) (Anura: Microhylidae, Gastrophryninae).
Type locality: Santa Júlia farm, municipality of Gavião Peixoto (21°50′43.74″S; 48°28′43.43″W), São Paulo state, Brazil.
Site of infection: Large intestine.
Level of infection: Three specimens of C. albopunctata n. sp.
Type specimens: Holotype and two paratypes (Helminthological Collection of the Oswaldo Cruz Institute (CHIOC) of Rio de Janeiro. The accession number of the holotype CHIOC number 39361a (male) and accession numbers of paratype CHIOC number 39361b-c (two male)).
Etymology: The epithet albopunctata is derived from the name of the host
of the new species.
GenBank Accession number: (accession numbers: OP153854 and OP153856).
Molecular characterization
The three newly generated COI mtDNA sequences of C. parva were 413, 426 and 428 base pairs (bp) in length, and showed no genetic divergence among them. The seven newly generated COI mtDNA sequences of C. podicipinus were 400, 414, 415, 419, 423, 424 and 426 bp in length, and ranged from 98.2%–100% of similarity among them. The two newly generated COI mtDNA sequences of C. albopunctata n. sp. were 426 and 427 bp in length and presented 99.1% of similarity between them (supplementary table 1).
The similarity matrix of COI mtDNA sequences from Cosmocerca species showed that the lowest genetic distance (pairwise distance) in C. parva was 11.4%–12.3% compared with C. podicipinus, and the greatest genetic distance was 23.8%–25.7% compared with C. albopunctata n. sp. Pairwise comparison of C. podicipinus with C. albopunctata n. sp. displayed 25.3%–28.6% of nucleotide divergence (supplementary table 1). The COI mtDNA sequences of Cosmocerca spp. are deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) (accession numbers: OP153854-OP153865).
Both phylogenetic reconstructions inferred by ML and BI methods recovered all sequences of Cosmocerca in a monophyletic clade (pp = 0.61; bootstrap: 83), with the sequences of Cosmocercoides as their sister group (pp = 1; bootstrap: 100). Cosmocerca spp. sequences were divided into two supported clades: one comprising all sequences of C. podicipinus and C. parva (pp = 1; bootstrap: 100); and another with the sequences of C. albopunctata n. sp., C. ornata, C. simile, Cosmocerca sp. (MT108305) and C. japonica (pp = 0.98; bootstrap: 87) (fig. 4). In this last clade, the sequences of C. japonica were positioned into two separate clades, with low support by both analyses.
Maximum likelihood (ML) topology based on COI mtDNA sequences of Cosmocercidea, showing the phylogenetic position of Cosmocerca spp. from Brazilian anurans. Numbers above nodes represent posterior probabilities values for Bayesian analyses followed by bootstrap values for ML analyses (posterior probabilities >0.90 and bootstrap values >70 were considered well supported). The branch length scale bar indicates the number of substitutions per site.
Discussion
Cosmocerca is characterized by the presence of two spicules, usually weakly sclerotized or rudimentary (Gibbons, Reference Gibbons2010), absence of tail alae, presence of rosette papillae and plectanes in males, and presence of somatic papillae and two prodelphic ovaries in females. The recognition of these combined characteristics allowed us to allocate our specimens as Cosmocerca.
Cosmocerca albopunctata n. sp. can be easily distinguished from its congeneric species by a combination of characters, such as the body size, length of spicules and gubernaculum, and the number and arrangements of the caudal papillae (7 + 1:1 + 1:6). In addition, in the new species, the position of the excretory pore is post-bulbar. Bursey et al. (Reference Bursey, Goldberg, Siler and Brown2015) have reported this type of arrangement in Cosmocerca longispicula Moravec and Kaiser, Reference Moravec and Kaiser1994 from the Panamanian region, C. longicauda (Linstow, 1885) from the Palaearctic region and Cosmocerca leytensis Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015 from the Oriental region. However, according to these authors, the excretory pore in the anterior ends of males and females of Cosmocerca spp. are rarely illustrated in a species description, which makes it difficult to affirm if this characteristic is considered a common feature in Cosmocerca spp.
To date, 37 species of Cosmocerca have been reported worldwide. Among these species, C. parva, C. uruguayensis, C. vrcibradici, C. commutata (Diesing, Reference Diesing1851) and C. longispicula present seven pairs of plectanes (Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015) as in C. albopunctata n. sp.
Among the above-mentioned species, C. parva was originally described from Hylodes nasus (Lichtenstein, 1823) (=Helosia nasus), from the municipality of Angra dos Reis, Rio de Janeiro state, Brazil (Travassos, Reference Travassos1925). This species has been reported from dendrobatids, leptodactylids, bufonids, hylids, microhylids, hylodids, odontophrynid, craugastorids and phyllomedusids in Argentina, Brazil, Colombia, Guiana, Paraguay and Peru (Campião et al., Reference Campião, Morais, Dias, Aguiar, Toledo, Tavares and Silva2014). Cosmocerca parva is distinguished from the new species by the presence of two to four pairs of papillae which are commonly surrounded by one or two small rosettes of punctations in the adcloacal region (Mordeglia & Digiani, Reference Mordeglia and Digiani1998), while in the C. albopunctata n. sp. they are absent.
Cosmocerca uruguayensis Lent & Freitas, Reference Lent and Freitas1948 was originally described from Odontophrynus americanus (Duméril and Bibron, 1841) (= Ceratophrys americana) in Montevideo, Uruguay (Lent & Freitas, Reference Lent and Freitas1948) and it has also been reported in Venezuela (Campião et al., Reference Campião, Morais, Dias, Aguiar, Toledo, Tavares and Silva2014). Cosmocerca uruguayensis could be easily distinguished from the new species by the absence of lateral alae, while in C. albopunctata n. sp. this characteristic is present.
Cosmocerca vrcibradici was originally described from the intestine of the lizard Cercosaura eigenmanni (Griffin, 1917) (= Prionodactylus eigenmanni) in Rondônia state, Brazil (Bursey & Goldberg, Reference Bursey and Goldberg2004). The species has already been registered in Alopoglossus angulatus Linnaeus, 1758, Alopoglossus atriventris Duellman, 1973, Norops fuscoauratus D'Orbignyi, 1837, Cercosaura oshaughnessyi (Boulenger, 1885) and Uranoscodon superciliosus Linnaeus, 1758 (Ávila & Silva, Reference Ávila and Silva2010). Cosmocerca vrcibradici differs from C. albopunctata n. sp. by possessing larger spicules (171–183 vs. 98.2–99.8) (Bursey & Goldberg, Reference Bursey and Goldberg2004).
Cosmocerca commutata was originally described from the intestine of Bufotes viridis (Laurenti, 1768) (= Bufo viridis) (Diesing, Reference Diesing1851). This species is commonly found in frogs (see Koyun et al., Reference Koyun, Birlik, Sümer and Yildirimhan2013). Cosmocerca commutata differs from C. albopunctata n. sp. by possessing larger spicules (180 vs. 98.2–99.8) and gubernaculum (186–213 vs. 119.7–157.8) (Bursey et al., Reference Bursey, Goldberg, Siler and Brown2015).
Cosmocerca longispicula was originally described from the intestine of Colostethus sp. from France (Moravec & Kaiser, Reference Moravec and Kaiser1994). Cosmocerca albopunctata n. sp. can be easily distinguished from C. longispicula by possessing smaller spicules (98.2–99.8 vs. 294–300) and larger tail compared to C. longispicula (191.4–266.6 vs. 135–144) (Moravec & Kaiser, Reference Moravec and Kaiser1994). This geographical location of C. longispucula reinforces the differentiation of these species.
The phylogenetic results based on the partial COI mtDNA sequences supported the new species, C. parva and C. podicipinus as members of Cosmocerca, thus corroborating Chen et al. (Reference Chen, Zhang, Feng and Li2020). Moreover, all sequences of each species were clustered together in supported clades, corroborating their current taxonomic assignation. Our phylogenetic analyses are also consistent with the morphological data for the analysed species. Although C. japonica is not a major focus of our study, the sequences of this species were positioned into two separate unsupported clades (fig. 4), indicating that these sequences might need further evaluation of their current taxonomic status.
The interspecific nucleotide divergence detected in COI mtDNA between C. albopunctata n. sp. and its congeners are: C. simile (23%–26.1%); C. ornata (28.8–29%); Cosmocerca sp. (MT108305) (30.2–31.7%); and C. japonica (27.1%–35.8%). Chen et al. (Reference Chen, Zhang, Feng and Li2020) observed a lower percentage of interspecific nucleotide divergence between Cosmocerca species (10.2–15.5%). Therefore, our results are following the expected divergence range previously found in the literature for the family Cosmocercidae, corroborating C. albopunctata n. sp. as a different species from its congeners.
The molecular identification of the cosmocercoid nematodes remains very limited (Chen et al., Reference Chen, Zhang, Feng and Li2020). The genetic data for Cosmocerca spp. generated here are very important for further studies of the DNA-based taxonomy, population and phylogenetics of the superfamily Cosmocercoidea (Chen et al., Reference Chen, Gu, Ni and Li2021a). Therefore, our results contribute to the knowledge about the species diversity and genetic data for Cosmocerca and serve as preliminary information on Neotropical cosmocercids, that should be accessed in future studies to help unravel phylogenetic relationships in cosmocercids.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0022149X22000517.
Financial support
We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing a research fellowship for E.P.A. (141322/2018-7), M.B.E. (140873/2017-1), CFS (140871/2017-9) and R.J.S. (309125/2017-0). To Fundação de Amparo à Pesquisa do estado de São Paulo (Fapesp) for a research fellowship to L.H.O. (2018/09623-4) and doctoral fellowship granted to L.P.A. (2018/00475-9). M.I.M. was supported by a postdoctoral scholarship from the Sao Paulo Research Foundation (FAPESP) (2017/16546-3).
Conflicts of interest
None.
Ethical standards
All applicable international, national and/or institutional guidelines for the ethical handling of animals were followed (ICMBio SISBIO #60640-1; CEUA-UNESP 1061; SISGEN).
