Wildlife species, Ixodid fauna and new host records for ticks in an Amazon forest area, Rondônia, Brazil

The objective of this work was to evaluate the diversity of ticks associated with free-living animals and to investigate new host records for ticks. Ticks were collected from animals rescued during the flood of the Jamari River in the municipality of Ariquemes, state of Rondônia, North Region of Brazil. A total of 39 animals were captured, out of which 10 were amphibians, 19 were reptiles and 10 were mammals. A total of 127 ticks of the Amblyomma genus were collected from these animals, distributed among seven species: Amblyomma dissimile , Amblyomma geayi , Amblyomma humerale , Amblyomma longirostre , Amblyomma nodosum , Amblyomma rotundatum and Amblyomma varium . In addition, one specimen of Rhipicephalus (Boophilus) microplus was collected. Among these specimens, 85 were adults and 42 were nymphs, with A. rotundatum being the most prevalent species. An Amblyomma spp. larvae was also collected from a lizard ( Uranoscodon superciliosus ), and one Amblyomma calcaratum and one Amblyomma dubitatum were recovered from the environment, thus totaling 130 ticks. Among the Ixodidae collected from different hosts, we provide the first report for the species A. rotundatum parasitizing Rhinella major , U. superciliosus, Leptophis ahaetulla, Chironius multiventris , and Mastigodryas boddaerti , as well as of A. humerale parasitizing U. superciliosus , A. geayi parasitizing Choloepus didactylus , and Rhipicephalus (B.) microplus parasitizing Alouatta puruensis .


Introduction
The Rondônia state, located in the North Region of Brazil, possesses wide reserves of natural Amazon forest, as well as many degraded areas.This state has been the focus of several tick-related studies, putting it in a prominent position since 34 (52%) of the 65 species in the country had already been catalogued here by 2014 (MARTINS et al., 2014).However, the Amblyomma sculptum Berlese, 1888 species was recently reported for the first time in Rondônia, bringing the total to 35 species in the state (MARTINS et al., 2016).Although an already large number of Ixodidae species have been reported, that number will tend to increase due to new discoveries.Notably, reports regarding Brazilian tick fauna in recent years have been gradually increasing for both the Ixodidae and Argasidae families.Brazil today has 71 tick species, with 46 belonging to the Ixodidae family and 25 belonging to the Argasidae family (MARTINS et al., 2014(MARTINS et al., , 2016;;BARROS-BATTESTI et al., 2015;KRAWCZAK et al., 2015;LABRUNA et al., 2016;WOLF et al., 2016;MUÑOZ-LEAL et al. 2017).
Many of these ectoparasites have been getting closer to and interacting more frequently with domestic animals and, consequently, human beings.Because of this interaction, current research studies have focused on Ixodidae populations as well as their interaction with hosts and the environment since these ticks have the vectorial capacity to transmit agents that can cause diseases in human beings (LABRUNA et al., 2005(LABRUNA et al., , 2010;;MARTINS et al., 2014).
Interventions in natural environments provide favorable conditions for infectious diseases due to both human interference, such as deforestation, and the constant changes made by man with the construction of roads, barrages, agriculture, and other activities (PATZ et al., 2004).Some tick species in Brazil, mainly from the Amblyomma genus, are pathogen vectors and are directly involved in the transmission of infectious agents to human beings, including spotted fever (Brazilian Spotted Fever) caused by bacteria of the genus Rickettsia (LABRUNA et al., 2011;OGRZEWALSKA & PINTER, 2016).
The objective of this study was to identify the tick fauna on wild animals captured in a degraded area and to investigate new records of tick-host relationships.

Location and period of collection
The capture of hosts was performed during the rescue of wild animals from the flood of the Jamari River after the construction of the Jamari Hydroelectric Central (PCH -Jamari) (10° 53' 7" S and 61° 57' 6" W), located in the municipality of Ariquemes, state of Rondônia, North Region of Brazil.The captures started in the beginning of November 2014 and lasted until February 2015.

Animal capture
All animals were captured and physically contained according to the methods in Rodrigues et al. (2016) before being transported to the wild animal triage center located near PCH -Jamari.The animal capture, triage, and translocation were executed by a team hired for the task and composed of field workers, biologists and veterinarians with the necessary technical abilities.During triage, each animal was identified by species, sex and weight.Afterwards, tick collections were performed.

Collection, identification and processing of ticks
Ticks were collected during the clinical examination and inspection of captured animals.The parasites were removed from hosts by holding ticks by their bodies and applying force against the host, turning the tick body 90° until the hypostome was completely removed from the skin.Afterwards, they were put in plastic receptacles with pierced covers for subsequent identification.
Later, the ticks were taken to the laboratory and identified with a stereomicroscope using dichotomous keys from Onofrio et al. (2006) for adults and Martins et al. (2010) for nymphs.For confirmation of the species, the Amblyomma humerale Koch, 1844 nymph was subjected to DNA extraction and sequencing, as this specimen had breakdowns that made identification by dichotomous key difficult.The ticks were deposited into the collection of the Tick Laboratory, Embrapa Gado de Corte, Campo Grande, Mato Grosso do Sul,35).

DNA extraction, PCR and sequencing
Tick DNA was extracted using a protocol based on guanidine isothiocyanate and phenol/chloroform extraction (SANGIONI et al., 2005 -adapted).The DNA was quantified with a spectrophotometer, and its integrity was verified by agarose gel electrophoresis at 0.8%.
PCR using a 16S rDNA primer for molecular identification of the A. humerale species was performed according to Mangold et al. (1998).
The product was analyzed on a 1.5% agarose gel, stained with ethidium bromide, visualized, and photographed under ultraviolet light exposure.
Purification of the amplified fragment was done using the PureLink kit (Invitrogen).The fragment was cloned in a pGEM  T-Easy (Promega) vector and sequenced using the Sanger method (SANGER et al., 1977) with an ABI 3130 Genetic Analyzer (Applied Biosystems  ) for species verification.
Sequencing analysis was obtained with the Geneious Pro 4.8.5 software.Sequence similarity of the obtained sequence was compared using BLAST (NCBI, 2017).
All amplicons were sequenced, and individual sequences were aligned with each other and with the corresponding Amblyomma sequences available in GenBank using the BioEdit Sequence Alignment Editor (HALL, 1999) with the CLUSTAL W program (THOMPSON et al., 1994).Pair-wise comparisons of sequences were made using MEGA version 5 (TAMURA et al., 2011).Phylogenetic relationships were then explored in terms of neighbor-joining distances (NJ).An NJ tree was generated using the Tamura-Nei model with any gaps in the pair-wise comparisons excluded, and support for the NJ topology was tested by bootstrapping over 1,000 replications.
During the field activities, the authors found one specimen of Amblyomma calcaratum Neumann, 1899 and one of Amblyomma dubitatum Neumann, 1899 in free-living stages in the environment.A total of 87 adult ticks (10 males and 77 females), 42 nymphs and one larva were collected.Among the nymphs, only one was from the A. humerale species, while the rest were A. rotundatum.Among adults, the most abundant species was A. rotundatum, with 59 specimens, followed by A. nodosum, A. geayi, A. longirostre and A. varium, with 13, five, three and three samples each, respectively.Only one specimen of A. calcaratum, one A. dissimile, one A. dubitatum, and one R. (B.) microplus were present, reaching a total of 130 specimens (Table 1).
BLAST analysis showed that the obtained A. humerale DNA sequence is close (99% identity) to the corresponding sequence of A. humerale from the state of Amazonas, North Region of Brazil (GQ891952), as well as 95% similar to the Amblyomma sabanerae Stoll, 1894 sequence from Costa Rica (KF702455), as illustrated in Figure 1.
Phylogenetic analysis indicated that the specimens collected from Amazon forest area, Rondônia, Brazil, correspond to A. humerale M male; F female; N nymph; L larva.
(Figure 1).The Amazonian specimens clustered with sequences from Amazonas, Brazil, which may correspond to the geographic area of A. humerale, and is located closed to Rondônia, sharing the Amazon rainforest.

Discussion
Amblyomma rotundatum ticks were present on all captured species of amphibians and reptiles, with reports of nymph and adult instars; however, only females were found among the adults.This is due to the characteristic fact that this species parasitizes different cold-blooded hosts and reproduces through parthenogenesis, despite the presence of a male specimen on a Tropidurus sp.lizard and another one on a B. constrictor snake (ARAGÃO, 1936;LABRUNA et al., 2005;MARTINS et al., 2014).Witter et al. (2016) reported A. rotundatum on R. marina and B. atrox in the state of Mato Grosso; however, according to Guglielmone & Nava (2010), this tick species has never been reported on R. major, U. superciliosus, L. ahaetulla, C. multiventris or M. boddaerti.Therefore, this study reports, for the first time, these new hosts for the A. rotundatum species.
This study reports, for the first time in Brazil, an A. humerale tick parasitizing a lizard of the U. superciliosus species, with the A. humerale nymph identified morphologically and then confirmed through DNA extraction and sequencing.A clade was demonstrated a posteriori, with a 100% probability, between A. humerale (Query) RO, Brazil and A. humerale (GQ891952), within the phylogenetic tree.Amblyomma sabaranae from Costa Rica, showed 99% of similarity with the A. humerale clade demonstrated in this study (Figure 1).The other clades did not demonstrate any similarity with A. humerale species.Labruna et al. (2002a) described the presence of A. humerale nymphs on Plica plica and Plica umbra lizards belonging to the Tropiduridae family, which is the same family to which U. superciliosus belongs, and Kentropyx calcarata, belonging to the Teiidae family, captured along the same river featured in this study.Labruna et al. (2002a) also described for the first time the presence of A. humerale in the state of Rondônia, as well as immature stages parasitizing a variety of small vertebrates.
Adult A. varium and A. geayi ticks found parasitizing sloths (C.hoffmanni and C. didactylus) in this study are associated primarily with the Xenarthra order, according to Marques et al. (2002) and Labruna et al. (2009).However, the first report of A. geayi parasitism on C. didactylus was made in this study, since descriptions of this tick species on sloths refer to the C. hoffmanni, Bradypus tridactylus, and Bradypus variegatus species (FAIRCHILD et al., 1966;LABRUNA et al., 2009;MARTINS et al., 2013).
Amblyomma nodosum adult ticks were found parasitizing T. tetradactyla.This tick species, when in its adult phase, prefers mammals from the Xenarthra order, which includes this host species.However, immature phases are involved in bird parasitism (GUIMARÃES et al., 2001;BARROS-BATTESTI et al., 2006).Witter et al. (2016) noted the presence of A. nodosum on free T. tetradactyla individuals.Different research studies have also reported adult A. nodosum present on T. tetradactyla (GARCIA et al., 2013;MARTINS et al., 2014).
Parasitism by A. longirostre adults is commonly associated with some Coendou species (GUIMARÃES et al., 2001), such as Coendou prehensilis, as registered in this study.Immature phases of A. longirostre are usually associated with bird parasitism (OGRZEWALSKA & PINTER, 2016).This is also the first report of an R. (B.) microplus semi-engorged female parasitizing an A. puruensis.This possibly occurred accidentally, since this female tick was attached to a young A. puruensis found separated from its mother and on the ground, where it possibly contacted R. (B.) microplus.Other Ixodidae species, especially Amblyomma sp.ticks, were reported parasitizing animals of the Alouatta genus (LABRUNA et al., 2002b;MARTINS et al., 2006;LAVINA et al., 2011;MARTINS et al., 2013MARTINS et al., , 2015)).Labruna et al. (2002b) also rescued animals from the flood caused by the construction of the Porto Primavera hydroelectric plant and reported the presence of 18 A. sculptum nymphs parasitizing three Alouatta caraya.
Other studies have reported the presence of A. aureolatum (Pallas, 1772) on Alouatta guariba specimens (MARTINS et al., 2006), A. aureolatum and A. ovale Kock, 1844 on Alouatta clamitans (LAVINA et al., 2011) and parasitism by A. parkeri Fonseca e Aragão, 1952and A. sculptum nymphs of A. guariba (MARTINS et al., 2013, 2015).As observed in the present study, which reported a young debilitated animal on the ground, other studies show that parasitism by Ixodidae of primates of the Alouatta genus are usually associated with the presence of stressful conditions for these animals, either because they are hurt, young, or due to their permanent residence on the ground caused by some physical debility (MARTINS et al., 2006;LAVINA et al., 2011;MARTINS et al., 2015).This study observed and presented Ixodidae fauna parasitizing several animal species in a region of the state of Rondônia, in the North Region of Brazil.Amblyomma rotundatum was described, for the first time, parasitizing amphibians and reptiles of the R. major, U. superciliosus, L. ahaetulla, C. multiventris and M. boddaerti species.Also unprecedented were the parasitism reports of A. humerale on U. superciliosus lizards, A. geayi on C. didactylus sloths, and R. (B.) microplus on A. puruensis monkeys.

Figure 1 .
Figure1.Neighbor-joining (NJ) tree built from Amblyomma humerale 16S rDNA sequence collected in the municipality of Ariquemes, Rondônia, Brazil.The numbers represent the identity generated from 1,000 repetitions.These were used when identity is >50%.The Ixodes scapularis Say, 1821 sequence was used as an external group (out group).

Table 1 .
Number, instars and tick species identification include of the genera Amblyomma and Rhipicephalus collected from wild animals rescued from a forest area in the municipality of Ariquemes, Rondônia, Brazil.