Noteworthy records of the ticks Ornithodoros rostratus and Amblyomma sculptum parasitizing Pteronura brasiliensis in the central-western region of Brazil, with pathogen investigation notes

Abstract A male of Pteronura brasiliensis (Carnivora: Mustelidae) was found dead on the banks of the Rio Negro, in the Pantanal wetlands of Mato Grosso do Sul state, Aquidauana municipality. Two ticks found attached to its skin were morphologically identified as a second-instar nymph of Ornithodoros rostratus (Argasidae) and a male of Amblyomma sculptum (Ixodidae). In order to complement the morphological identification, these tick specimens were subjected to DNA extraction, and tested using PCR assays to confirm the molecular identity the specimens. Also, the tick DNA samples were tested and were negative in the PCR assays for all the pathogens tested. We also examined 30 batches, consisting of 174 individuals of O. rostratus deposited in the Acari Collection of the Butantan Institute, and we found material from four Brazilian states, including one batch containing 2 males and 2 females from Aquidauana, of Mato Grosso do Sul state, collected from the soil. This was therefore the first record of O. rostratus parasitizing P. brasiliensis and the first locality record (Aquidauana). Likewise, A. sculptum is commonly found in the Pantanal and is reported here for the second time parasitizing the giant otter, which is a host little studied regarding the ectoparasites.

In addition, O. rostratus, along with Ornithodoros brasiliensis Aragão, 1923, Ornithodoros furcosus Neumann, 1908, and the recently described species Ornithodoros improvisus Muñoz-Leal & Venzal, 2023, form the neotropical group of the Pavlovskyella subgenus.Ornithodoros brasiliensis is endemic to southern Brazil, while O. furcosus occurs in the higher-altitude Andean parts of Ecuador, Colombia and Peru, and O. improvisus occurs in Chile (Muñoz-Leal et al., 2023).The Pavlovskyella species can be found in their hosts' burrows due to their habit of living buried in sand or soft soil near their principal hosts' habitats.They can also be found living in cellars, stables, and even primitive human dwellings (Clifford et al., 1964).Several Pavlovskyella species are vectors of tick-borne relapsing fever caused by Borrelia spp.within their geographical ranges (Hoogstraal, 1985).
Until the second half of the twentieth century, O. rostratus was abundant in some areas of the Brazilian states of São Paulo, Minas Gerais, Mato Grosso do Sul and Goiás (Aragão, 1936;Aragão & Fonseca, 1961).Since then, this species has not been recorded in any new areas and has only been reported by Cançado et al. (2008), in the locality of Nhecolândia, Mato Grosso do Sul, where it was parasitizing domestic and free-ranging animals, from loose soil on a farm; and by Luz et al. (2019), in the locality of Corumbá, Mato Grosso do Sul, where four nymphs were collected through dragging, and three nymphs and 35 adults from dry-ice traps.The life cycle of O. rostratus was studied by several research as Guglielmone & Hadani (1980), Venzal & Estrada-Peña (2006), Ribeiro et al. (2013) and Costa et al. (2015), and according to these authors, there are five to six nymphal instars.However, the last instar is rare (Ribeiro et al., 2013).
Even though O. rostratus and A. sculptum have distinct distribution areas in Brazil, ticks of the A. cajennense complex can be found in sympatry in some transition areas.According to Martins et al. (2016), A. cajennense s. s. generally occurs on the border of the Amazon biome and is not found in the thicker parts of the rainforest.Amblyomma sculptum is found in the Cerrado, Pantanal and degraded Atlantic Forest biomes, but only one record from the Caatinga biome (Martins et al., 2016) has been reported.
Here, we report the presence of O. rostratus and A. sculptum in a carcass of a male giant otter (Pteronura brasiliensis) for the first and second time, respectively, in the municipality of Aquidauana, state of Mato Grosso do Sul, including pathogen investigation in both species.

Occurrence Report
During a study carried out by the team responsible for the Giant Otter Conservation Fund, a male of P. brasiliensis (Gmelin) (Carnivora: Mustelidae) was found dead on the banks of the Rio Negro at the coordinates 19°34′58″ S and 56°09′44″ W, in the Pantanal region of the state of Mato Grosso do Sul, in the municipality of Aquidauana, in June 2022.Upon inspection, the animal was found to have two specimens of ticks attached to its skin.The ticks were brought to the Vector-Borne Bioagents Laboratory (VBBL), Department of Pathology, Reproduction and One Health, UNESP, Jaboticabal, SP, where they were morphologically identified as a second-instar nymph of O. rostratus (Argasidae) (through comparison with specimens from the laboratory's colony) and a male of A. sculptum (Ixodidae).
Sanger sequencing was performed at the Research Center for the Human Genome and Stem Cells, Institute of Biosciences, University of São Paulo, state of São Paulo.The sequences thus obtained were assembled using the Sequencing Analysis 5.3.1 software and were subjected to BLAST analysis to infer similarities with other tick sequences available in GenBank.Different haplotypes were visually distinguished through alignment using the CLUSTAL W algorithm implemented in Geneious R11.
We also examined all specimens of O. rostratus deposited in the Acari Collection of the Butantan Institute (IBSP).We found 30 batches of specimens that included 183 individuals (68 females, 47 males, 5 first-instar nymphs, 6 second-instar, 23 third-instar, 28 fourth-instar and 6 fifth-instar), from four Brazilian states: Mato Grosso do Sul -Aquidauana, Corumbá, Ladário, Nhecolândia and Três Lagoas; Goiás -Anápolis and Goiânia; Minas Gerais -Uberlândia; and São Paulo -Araçariguama, Avaré, Bananal, Barretos, Guaraci and Jaboticabal.The geographical distribution of this species based on material deposited in the IBSP collection and the new locality record is shown in Figure 1.The free-living ticks were trapped by CO 2 traps according to Cançado et al. (2008), and after that these alive ticks were donated by PHD Cançado to the Laboratory of Parasitology in the Instituto Butantan, São Paulo State, to start a colony in 2010.Since that, the colony of O. rostratus has been maintained in vials and packed in a biological oxygen demand (BOD) incubator at 27 ± 1º C and 90 ± 10% humidity, in the same laboratory.Also, this N2 nymph (Figure 2) had the following characteristics: idiosoma outline with oval discs and mammillae; length from anterior to posterior body margin 2679.28 mm, breadth 1537.20;legs with dorsal humps on tarsi; two strong and protuberant dorsal humps on tarsus I, one proximal (close to the tibia) and one distal (close to Haller's organ); also strong and protuberant humps on tarsi II and III; tarsus VI with very small, almost imperceptible, distal and proximal dorsal humps; all coxae with smooth surface, upper and lower borders surrounded by small mammillae that were present on supracoxal folds; preanal, transverse postanal, median postanal and dorsoventral grooves present; spiracular plate rounded; gnathosoma, including palpi and hood, visible dorsally; hypostome blunt apically, dentition 2/2.

Figure 1 .
Figure 1.Geographical distribution of Ornithodoros rostratus based on records within the IBSP collection.Symbols: red circle -IBSP data (literature records); and yellow circle -new record.