First record of Ornithodoros faccinii ( Acari : Argasidae ) on toads of genus Rhinella ( Anura : Bufonidae ) in Brazil Primeiro registro de Ornithodoros faccinii ( Acari : Argasidae ) em sapos do gênero Rhinella no Brasil

Although a group of soft ticks (Argasidae) associated with amphibians was recently discovered in Brazilian rainforests, parasitism by these ticks on cold-blooded animals remains less common than on mammal and bird species. In this study, we identified ticks that were collected from toads that had been caught in December 2016 and January 2017, at Itinguçú waterfall (22°54’05” S; 43°53’30” W) in the municipality of Itaguaí, state of Rio de Janeiro. Tick specimens were identified using a morphological and molecular approach. In total, twelve larvae of Ornithodoros ticks were collected from three individuals of Rhinella ornata and were identified as Ornithodoros faccinii. Our results include a longer 16S rRNA mitochondrial sequence for O. faccinii that supports its phylogenetic relatedness to Ornithodoros saraivai, and we report this tick species parasitizing Rhinella toads for the first time in Brazil.

The aims of the current study were to provide the first report of O. faccinii parasitizing anurans of the family Bufonidae in Brazil and to reassess its phylogenetic position by means of a longer partial sequence of 16S mitochondrial rRNA.

Study site and toad-catching
Ticks were collected from toads between December 2016 and January 2017, at Itinguçú waterfall in the municipality of Itaguaí, state of Rio de Janeiro, distant 30 meters (22°54'05" S; 43°53'30" W) from the type locality of O. faccinii.
Anurans were caught manually through an active search, at dusk on alternate days, over a total six days, comprising 19 hours of sampling effort.All the animals thus caught were examined for tick infestation, and they were released at the same location where they had been caught, in order to cause minimal impact on the population of these hosts.The taxonomic nomenclature for amphibians that was used followed the system of the Brazilian Society of Herpetology (SBH, 2017), and the animals were caught and manipulated in accordance with the recommendations of the Brazilian Institute for the Environment and Renewable Natural Resources -Chico Mendes Institute for Biodiversity Conservation (IBAMA-ICMBio -number 36164-1).

Collection and morphological identification of ticks
Larval stages of soft ticks were removed with tweezers, kept in vials containing RNAlater (Sigma-Aldrich) and transported to the laboratory.For morphological and morphometric analyses, three ticks were slide-mounted in Hoyer's medium, and were photographed using an Olympus DP70 camera that was coupled to an Olympus BX40 optical microscope (Olympus Optical Co. Ltd., Japan).Specimens were identified to genus level as described by Barros-Battesti et al. (2013), and species-level diagnosis was performed by comparing the morphological traits of the slide-mounted ticks with those of other Neotropical Ornithodorinae (ENDRIS et al., 1989;BARROS-BATTESTI et al., 2015;MUÑOZ-LEAL et al., 2017b).Type species of O. faccinii from the Acari collection of the Butantan Institute (IBSP 10316) were examined in order to compare the number of dorsal setae.

Molecular tools
For molecular analyses, larval DNA was extracted using the bead-beater/phenol-chloroform method (SANTOLIN et al., 2013).Subsequently, conventional PCR as described by Mangold et al. (1998) was performed using the primers 3'-CCGGTCTCAACTCAGATCAAGT-5' (forward) and 3'-GCTCAATGATTTTTTAAATTGCTGT-5' (reverse), targeting a fragment of approximately 460 bp from the mitochondrial sequence encoding 16S rRNA.Sequencing of PCR products was performed by combining the same amplification primers, purified amplicons and BigDye Ready Reaction mix (ABI Corp) in an automated genetic analyzer (model 3500; ABI Corp).The sequences were assembled using Sequencher (Version 5.3, Genecodes Corporation, CA, USA).We used the BLAST search algorithm ( ALTSCHUL et al., 1990) to determine closest gene identities.

Phylogenetic analysis
The sequences obtained for the mitochondrial 16S rRNA gene were aligned with another 60 Argasidae sequences retrieved from GenBank using Clustal X (THOMPSON et al., 1997), and were manually adjusted using the GeneDoc software.Sequences from Ixodes holocyclus Neumann, 1899 and Ixodes uriae White, 1852 were used as outgroups (accession numbers of all sequences are shown in the phylogenetic tree).The alignment was used to construct a phylogenetic tree using maximum parsimony, as implemented in PAUP version 4.0b10 (SWOFFORD 2002), with 500 bootstrap replicates, random stepwise addition to start trees (with random addition sequences) and TBR branch swapping.Bayesian analysis was performed using MrBayes v3.1.2(HUELSENBECK & RONQUIST, 2001) with four independent Markov chain runs for 1,000,000 metropolis-coupled MCMC generations, in which one tree was sampled every 100 th generation.The first 25% of the trees represented burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities.

Results
We caught 15 anurans (seven specimens of Rhinella ornata (Spix, 1824) and eight of Thoropa miliaris (Spix, 1824), and a total of 12 larvae of the genus Ornithodoros were collected from three specimens of R. ornata (20%) (Figure 1).Nine ticks were collected from the dorsal region of the hind limbs and three from the abdomen.
Three larvae were slide-mounted and were morphologically identified as O. faccinii, based on the following characters: idiosome with 11 pairs of dorsolateral setae and three pairs of central dorsal setae; seven (instead of six) pairs of dorsal anterolateral and four pairs of dorsal posterolateral setae; dorsal plate smooth, elongated, almost rectangular, with anterior and posterior margins rounded; ventral idiosome provided with seven pairs of setae (three pairs sternal, three circumanal and one postcoxal), plus one anal pair; posteromedian setae absent; hypostome pointed; and dental formula 3/3 in the anterior third, then 2/2 towards base (BARROS-BATTESTI et al., 2015) (Figure 1).Slide-mounted ticks were deposited in the "Danilo Gonçalves Saraiva" National Tick Collection (CNC) of the School of Veterinary Medicine of the University of São Paulo, São Paulo, Brazil, under accession number CNC-3514.
Morphological identifications of ticks were confirmed by means of molecular analyses, since DNA extracted from nine ticks retrieved a mitochondrial 16S rRNA consensus sequence of 428 bp that matched the shorter and sole 366-bp sequence of O. faccinii available in GenBank (KP861242), in which the nucleotides were 100% identical.The 16S sequence obtained in this study was deposited in GenBank under the accession number KY661385.
The phylogenetic relationships between this tick and other Neotropical Argasidae were inferred from alignment of a partial fragment of the mitochondrial 16S rDNA gene, including the longer sequence for O. faccinii that was obtained in the present study.This alignment showed that O. saraivai was a sister taxon clustering together within a larger clade composed of six other species of Ornithodoros (Figure 2).

Discussion
The record of O. faccinii larvae parasitizing anurans of the family Bufonidae is the first report of this in Brazil.Out of the 211 valid species of soft ticks worldwide, only representatives of the genus Ornithodoros have been recorded in association with amphibians.Indeed, early records of soft ticks on anurans in the Neotropical region documented larvae of a species of Ornithodoros that was morphologically related to the O. talaje group, and O. puertoricensis parasitizing Eleutherodactylus cooki Grant, 1932, in Puerto Rico (CAPRILES & GAUD, 1977).Much more recently, Rivas et al. (2012) reported that they had collected approximately 200 larvae of an unidentified species of Ornithodoros from a single specimen of Rhinella arenarum (Hensel, 1867) in Argentina.Additionally, larval stages of O. puertoricensis were also reported by Bermúdez et al. (2013), collected from the axillae of a specimen of Rhinella marina (Linnaeus, 1758) in Panama.Lastly, soft ticks corresponding to the recently described O. faccinii and O. saraivai were recorded as parasites of T. miliaris and Cycloramphus boraceiensis Heyer, 1983, in the states of Rio de Janeiro and São Paulo respectively (BARROS-BATTESTI et al., 2015;SÁ-HUNGARO et al., 2016;MUÑOZ-LEAL et al., 2017b).To date, these two species are the sole amphibian-associated soft ticks in Brazil.The frogs T. miliaris and C. boraceiensis are nidicolous endemic species of the Atlantic rainforest that frequent moist fissures in rocky environments near waterfalls (COCROFT & HEYER, 1988;FEIO et al., 2006;AMPHIBIAWEB, 2017).However, specimens of T. miliaris were also observed beyond its usual habitat in rocky environments near waterfalls during the fieldwork, under damp leaves in the forest understory, and often sharing the same space with specimens of R. ornata.Occurrences of both amphibian species in the same microhabitat might explain the records of O. faccinii larvae on R. ornata.In turn, it cannot be ruled out that O. faccinii might also occur not only in association with lotic water environments, but also in the moisture of the Atlantic rainforest soil, where Rhinella toads live.
Although, in the original description of O. faccinii, Barros-Battesti et al. (2015) used the same primers as in the current study in order to sequence an approximately 460-bp fragment of mitochondrial 16S rRNA, the sequence available for this species did not exceed 366 bp, and was 62 bp smaller than the sequence presented in the current study, which was composed of 428 bp.Barros-Battesti et al. (2015)    study, our phylogenetic tree showed that O. faccinii forms a monophyletic group with O. saraivai and that it clusters within a larger clade that rather excludes O. capensis and O. sawaii from the group.In this way, the present study corroborates the phylogeny of Argasidae using partial sequences of this mitochondrial gene that was presented by Muñoz-Leal et al. (2017b).
Although, in the current study, larval stages of O. faccinii collected from R. ornata presented shared identity of at least 366 bp with the sequence of 16S mitochondrial rRNA gene that was characterized in the original description of this species (BARROS-BATTESTI et al., 2015), we observed one noteworthy difference in the dorsal setae, in comparing our material with the dorsal chaetotaxy reported in the original description of this species: seven instead of six pairs of dorsal anterolateral setae.In Ornithodoros larvae, dorsolateral setae appear arranged in one anterior group, chiefly composed by seven pairs, and one posterior group with a variable number.Therefore, presence of six pairs of dorsal anterolateral setae is a morphological characteristic that rapidly separates O. faccinii from every other Neotropical species of the genus Ornithodoros.Since the larva of O. faccinii was described exclusively from field-collected material, morphological analyses on laboratory-reared larvae that had not been exposed to tegumentary attrition caused by their microhabitat or by their host would need to be performed to evaluate whether this morphological dissimilarity is a naturally polymorphic characteristic.
constructed a phylogenetic tree in which O. faccinii clustered in a clade with Ornithodoros capensis Neumann, 1901, and Ornithodoros sawaii Kitaoka & Susuki, 1973.The first species, except Antarctica, occurs in all zoogeographic regions of the world, including the coasts of the Atlantic and Pacific oceans in the Neotropical Region, while

Figure 2 .
Figure 2. Phylogenetic tree inferred from 16S rDNA gene partial sequences, including the new sequence of Ornithodoros faccinii that was generates in the current study.