Ornithodoros faccinii n. sp. (Acari: Ixodida: Argasidae) parasitizing the frog Thoropa miliaris (Amphibia: Anura: Cycloramphidae) in Brazil

Background Most argasid ticks from the Neotropical region are parasites of mammals and birds, with a few records from reptiles. Many species of the genus Ornithodoros are known only through larval descriptions, and their chaetotaxy and morphological characteristics have been used to separate the taxa. In the present study, we describe the larva and the nymph of first instar of a new species of the genus Ornithodoros that was collected from frogs of the species Thoropa miliaris. Methods Larvae of Ornithodoros were collected from frogs of the species T. miliaris at waterfalls in the state of Rio de Janeiro, southeastern Brazil. The larval and nymphal description was based on optical and scanning electron microscopy. Molecular analysis using the argasid 16S rRNA sequences available in GenBank was also conducted. Results Ornithodoros faccinii sp. n. is closely related to Ornithodoros clarki Jones & Clifford, Ornithodoros marinkellei Kohls, Clifford & Jones, Ornithodoros capensis Neumann and Ornithodoros sawaii Kitaoka & Susuki. However, the larval morphology of the new species is unique. The mitochondrial 16S rDNA partial sequence of O. faccinii generated in the present study was deposited in GenBank under the number KP861242. Conclusions The larvae collected from Thoropa miliaris are a new species, Ornithodoros faccinii n. sp. This is the first report of argasid ticks on frogs in Brazil, the second on frogs and the third on Amphibia in the Neotropical region.

In the present study, we describe the larva and nymph of first instar of a new species of the genus Ornithodoros collected from frogs of the species Thoropa miliaris (Spix, 1824) (Anura: Cycloramphidae) and determine the phylogenetic position of this species in comparison with other argasids from different regions. Adults were not described because they were not found in the host's natural environment and we were unable to obtain this stage under laboratory conditions.

Tick collection and morphological study
Larvae of Ornithodoros n. sp. were collected from frogs of the species T. miliaris. The frogs were found at the Itinguçu waterfall (22°54'07.47" S, 43°53'34.14" W), which is at the locality of Coroa Grande, municipality of Itaguaí, state of Rio de Janeiro, southeastern Brazil, in 2010 and 2012. Collection of these amphibians was authorized through the System of Authorization and Information on Biodiversity (SISBIO), in accordance with the protocol number 36164-1.
One of the frogs, which was infested with five larvae was also kept in a special vivarium by one of us (H. Luz) until the larvae dropped off. Some engorged larvae dropped off three days later and these specimens were kept in separate vials and were packed into a biological oxygen demand (BOD) incubator at 27°± 1°C and 90 ± 10% relative humidity (RH) for larval ecdysis. Two nymphs emerged after thirteen days. Eight larvae were prepared on slides (including the holotype) and were measured under a Leica DM2500 microscope coupled to the NiS-Elements BR 64-bit measurement system, v. 3.33.13, and two nymphs were measured under a Leica stereomicroscope. All the measurements were made in micrometers (μm), as the mean followed by the standard deviation and range in parentheses (the holotype measurements are in square brackets). The specimens were prepared and examined, and micrographs were produced through light and scanning electron microscopy (SEM) in the Electron Microscopy Laboratory, at the State University of São Paulo (UNESP), Rio Claro campus. Four larvae and two nymphs were examined under high and low vacuum, respectively.
The description of the new species was based on the literature relating to the taxonomy of the subfamily Ornithodorinae [5,6,15,16]. For the present study, regarding the current usage of valid tick names, we followed the nomenclature proposed in the specific literature on ticks worldwide [2].

Molecular study
DNA was extracted from two larvae using the guanidine isothiocyanate-phenol technique [17]. The extracted DNA samples were then subjected to PCR targeting a fragment of approximately 460 base pairs (bp) of the mitochondrial 16S rDNA [18]. The products were purified and sequenced using the same primers as used in the PCR. These sequences were aligned using Clustal X [19] and adjusted manually using the GeneDoc software, with sequences previously determined for other argasid species available in GenBank, and also with sequences from Ixodes holocyclus Neumann and Ixodes uriae White (Ixodidae), which were used as outgroup (the accession numbers of all the sequences are shown in the resulting phylogenetic tree). The phylogenetic tree was inferred by means of the maximum parsimony (MP) method using PAUP version 4.0b10, with 500 replicates of random addition taxa and TBR branch swapping [20]. All positions were equally weighted and Bayesian analysis was performed using MrBayes v3.1.2 with 1,000,000 replicates [21]. The first 25% of the trees represented burnin, and the remaining trees were used to calculate Bayesian posterior probability.

Ethical approval
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).

Results
We examined 50 frogs from which 15 were infested, showing prevalence of 30% and the mean intensity of infestation was 1.5 ticks.        ; number of setae on palpal articles I-IV is 0, 4 (1V, 3D), 5 (1V, 4D) and 9 tibiotarsal, respectively ( Figures 3A, C, E). There are 11 short spurs in the inner side in each trochanter, some of them are bifid (Figures 3B, D). These spurs can also be observed by optical microscopy using immersion objective.

Phylogenetic position
The phylogenetic relationships based on a partial sequence of the mitochondrial 16S rDNA gene ( Figure 5)

Type-locality
Itinguçú waterfall (22°54'07.47" S, 43°53'34.14" W), which is at the locality of Coroa Grande, municipality of Itaguaí, state of Rio de Janeiro, Brazil.   Phylogenetic tree based on the 16S rDNA ticks. The alignment was produced using Clustal X and the tree was inferred by means of the MP method with 500 replicates of random addition taxa. The species Ixodes holocyclus and Ixodes uriae were used as outgroup. The Bayesian support (posterior probability) values are derived from 1,000,000 replicates.

Type material
Holotype larva mounted on slide deposited in the Acari Collection of the Butantan Institute, under the number (IBSP 10316); 7 paratype larvae in slides, 5 paratype larvae and 2 paratype nymphs in 80% ethanol (IBSP 10317); 2 paratype larvae deposited in the National Tick Collection ( University, Statesboro (USNMENT 00862006). They all come from same host and locality.

Gene sequences
The mitochondrial 16S rDNA partial sequence of O. faccinii n. sp. generated in the present study was deposited in GenBank under the number KP861242.

Etymology
The name of the species is a tribute to Professor Dr. João Luiz Horácio Faccini of the Federal Rural University of Rio de Janeiro, who has always been devoted to studies of parasitic mites and ticks in Brazil.

General
In accordance with section 8.5 of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank with the life science identifier (LSID) zoobank.org:pub: A229A69C-9DD5-4E93-8 AC8-8DF237556CA8.

Species relationships
The larvae of O. faccinii n. sp. have morphological characteristics that indicate that this species does not belong to any subgenus previously proposed, although they present some characteristics close to the subgenus Alectorobius. The larvae of this subgenus present dorsal plate pyriform and widest posteriorly; dorsal surface with 11-18 pairs of dorsolateral setae and 3-5 pairs of central setae, usually pointed and barbed; ventral surface with 8 or 9 pairs of setae plus a posteromedial seta; basal capituli without cornua or auriculae and palpal articles without spines [15]. Nevertheless, the presence of small spurs at the lateral base of the hypostome and at the inner side of the trochanter of the palpi, as well as the dorsal plate that is smooth, elongated almost rectangular, with the anterior margin slightly rounded and narrowed and the posterior rounded and almost convex, comprise the main characteristics that morphologically distinguish O. faccinii n. sp. from the other species of Alectorobius. Although the new species is phylogenetically related to O. capensis [5], occurring in the Nearctic, Ethiopic, Oriental and Palearctic regions, and O. sawaii [22], restricted to the Japanese islands, which are both parasites of marine birds, it is morphologically distant. Few dorsal setae are also common in O. brasiliensis and O. rostratus, which in turn have dorsal plate similar to O. faccinii n. sp. [4]. But the new species is easily separated from them by the presence of long and pointed hypostomes.
The PMS (absent in most larvae of O. faccinii n. sp.) is also absent in O. marinkellei [25], in O. setosus [6], O. amblus [26], O. clarki [16], O. spheniscus [27], and O. yunkeri [28]. However, O. marinkellei presents 2 large auricula-like projections in the capitulum, which are absent in the new species [25]  The nymph of O. faccinii n. sp. has characteristics that resemble species of Alectorobius, such as integument with distinct mammillae and discs, idiosoma pointed anteriorly, legs with micromammillated cuticle and absence of dorsal humps on tarsi I-IV [15]. However, the absence of hood and cheeks, and the U-shaped capsule of Haller's organ separate the new species from most other species of the genus Ornithodoros belonging to the group Alectorobius in the Neotropical region, for which the nymphal stage has been described [29]. The U-shaped capsule aperture of Haller's organ seems to be unique to O. faccinii n. sp., as far as we are currently aware.  Kohls, 1941, also has no hood and cheeks, but the legs of this species have a smooth surface while the legs of O. faccinii n. sp. are micromammillated. An incomplete description of nymphs of O. sawaii has been presented and the authors commented that the nymphs and adults are quite similar [22]. Unfortunately, most descriptions of nymphs of the genus Ornithodoros are generally poor on detail and illustrations, and this inhibits the ability to make comparisons between species [29].
The presence of genital primordium in the N1 of O. faccinii n. sp. may indicate that this species only has one nymphal instar, because the genital primordium is indicative that the next stage could be the adult [30]. Otobius lagophilus Cooley & Kohls, 1940, and Ornithodoros peropteryx , are species of soft ticks that only have one nymphal stage and thus, these species undergo two molts in the process of reaching the adult stage [31,32]. According to southern specialists, it is possible there are more species with this behavior, especially in the Neotropical region [32]. Although it has been reported that the nymphs of O. sawaii present a smooth circular structure with very small setae ventrally, in place of the genital aperture, there was no mention of which nymphal instar was used in the description [22].
The host Thoropa miliaris is a frog that is endemic to the Atlantic Forest and is found in rocky environments near the coast in southeastern Brazil [33]. Thus, this report provides the first record of an argasid tick parasitizing Amphibia in Brazil.
The morphological and phylogenetic studies are congruent and they support O. faccinii as a new species, thereby increasing the Brazilian argasid fauna to 22 species.