Ticks parasitizing bats (Mammalia: Chiroptera) in the Caatinga Biome, Brazil

In this paper, the authors report ticks parasitizing bats from the Serra das Almas Natural Reserve (RPPN) located in the municipality of Crateús, state of Ceará, in the semiarid Caatinga biome of northeastern Brazil. The study was carried out during nine nights in the dry season (July 2012) and 10 nights in the rainy season (February 2013). Only bats of the Phyllostomidae and Mormoopidae families were parasitized by ticks. The species Artibeus planirostris and Carolia perspicillata were the most parasitized. A total of 409 larvae were collected and classified into three genera: Antricola (n = 1), Nothoaspis (n = 1) and Ornithodoros (n = 407). Four species were morphologically identified as Nothoaspis amazoniensis, Ornithodoros cavernicolous, Ornithodoros fonsecai, Ornithodoros hasei, and Ornithodoros marinkellei. Ornithodoros hasei was the most common tick associated with bats in the current study. The present study expand the distributional ranges of at least three soft ticks into the Caatinga biome, and highlight an unexpected richness of argasid ticks inhabiting this arid ecosystem.

The term "Caatinga" refers to the typical xeromorphic vegetation of the semiarid ecosystems in northeastern Brazil that covers an area of approximately 734,478 Km 2 (MARINHO-FILHO & SAZIMA, 1998), corresponding to 11% of Brazil's territory.This region includes areas of semi-deciduous and cloud forests, and presents low rainfall and humidity regimes (COSTA et al., 2004).While ticks are associated with bats in other Brazilian biomes (LABRUNA & VENZAL 2009;LABRUNA et al., 2011;MUÑOZ-LEAL et al., 2016b), to date documented reports of ectoparasites on bats from the Caatinga Biome include only streblid flies (Diptera: Streblidae) (RIOS et al., 2008;BEZERRA et al., 2016) and spinturnicid mites (Acari: Spinturnicidae) (ALMEIDA et al., 2016).In this study we introduce the diversity of ticks parasitizing bats in the Caatinga biome, providing new records of hosts and localities for this understudied group of parasites.

Study site and capture of bats
The study was carried out during nine nights in the dry season (July 2012) and 10 nights in the rainy season (February 2013) at Serra das Almas Natural Reserve (RPPN), municipality of Crateús, state of Ceará (05 o 15'S, 41 o 00'W).This reserve covers an area of 6,146 hectares and is considered an outpost of the Caatinga Biosphere Reserve (ARAÚJO et al., 2011).
Bats were captured with five to ten mist nets (36 mm), 6 -18 m long, 2.5 m height, placed in existing trails.Sampling period extended for six hours after sunset.Mist nets were examined at intervals of 10 min, and captured bats were kept in individualized and numbered cloth bags.These bags were used only once per day to avoid ectoparasite exchange among collected hosts.Voucher bat specimens were fixed in 10% formaldehyde and preserved in 70% alcohol, as previously described (VIZOTTO & TADDEI, 1973;HANDLEY, 1988), and deposited in the Adriano Lucio Peracchi (ALP) collection, at the Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil.Identification of bat species followed the descriptions of Silva et al. (2015) and the taxonomic nomenclature of Nogueira et al. (2014).Accession numbers for bat vouchers are as follow: ALP 10157, 10132, 10138, 10150, 10165, 10167, 10168, 10184 and 10455.The capture and collection of specimens was authorized by the Chico Mendes Institute for the Conservation of Biodiversity (ICMBio) of the Brazilian government (license number 32684-1).

Collection and identification of ticks
Ticks were removed with tweezers and kept in vials containing 70-90% ethanol for taxonomic identification.In the laboratory, ticks were slide-mounted in Hoyer's medium, and photographed with an Olympus DP70 camera implemented in an Olympus BX40 optical microscope (Olympus Optical Co. Ltd., Japan) for morphological and morphometric analyses.Fully engorged specimens were measured using a stereoscope SteREO Discovery V12 (all measurements are given in mm).Ticks were identified at genus level according to Barros-Battesti et al. (2013), and species diagnoses followed Kohls et al. (1969), Jones & Clifford (1972), and original descriptions of other Neotropical Ornithodorinae (KEIRANS & CLIFFORD, 1975;NAVA et al., 2010;DANTAS-TORRES et al., 2012).Additional comparisons of immature specimens were made using part of the material deposited at the "Coleção Nacional de Carrapatos" (CNC) of the Faculty of Veterinary Medicine of the University of São Paulo, São Paulo, Brazil.
Molecular analyses were performed in order to confirm morphological identifications of ticks.For this purpose, larvae were individually submitted to DNA extraction by the guanidine isothiocyanate-phenol technique (SANGIONI et al., 2005).DNA of fully engorged larvae was extracted through a small incision in the posterior region of the idiosoma using a 23G needle.Extracted DNA was subjected to conventional polymerase chain reaction (PCR) targeting a fragment of approximately 460-bp of the mitochondrial 16S rRNA gene, as described elsewhere (MANGOLD et al., 1998).PCR products of the expected size were purified and sequenced using an ABI automated sequencer (Applied Biosystems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyzer, Foster City, California, USA) with the same primers used in the PCR.
Prevalence of tick infestation (number of infested bats/number of examined bats x 100), and tick mean intensity of infestation (total number of collected ticks/total number of infested bats) were calculated according to Bush et al. (1997).
Overall, 49 out of 260 (18.8%) bats, belonging to 12 species of the Phyllostomidae and Mormoopidae families were infested by ticks (Table 1).Of these, Anoura geoffroyi, Artibeus planirostris and C. perspicillata were the most infested species, with a prevalence of 66.7%, 40.7% and 15.1%, respectively (Table 1).A total of 409 larvae belonging to Antricola, Nothoaspis and Ornithodoros genera were collected (Table 1).The single larva of Antricola was identified to genus level by the presence of three postcoxal setae (KOHLS et al., 1965) (Figure 1   dorsal plate pyriform, 15 pairs of dorsolateral setae and 4 pairs of central setae; hypostome apically pointed with 3 rows of denticles (17-19 denticles in row I; 16-17 in row II, and 11-12 in row III); dentition formula 3/3 in the anterior two-thirds and 2/2 towards the base; hypostome arising from a triangular median extension (KOHLS et al., 1965); 4) O. marinkellei: dorsal plate long and narrow, triangular, surface smooth, posterior margin slightly concave; dorsum with 13 pairs of setae, 7 anterolateral, 3 central, and 3 posterolateral, 3 first anterolateral setae short and thick; venter with 8 pairs of setae plus pair on anal valves; posteromedian setae absent; hypostome with dental formula 2/2 throughout length, file 1 with 21 denticles and file 2 with 20-21 denticles, additional denticles around apex, apex pointed, presence of two cornua-like projections in the posterior margin of basis capitulum (LABRUNA et al., 2011) (Figures 1, 2).In addition, morphological identifications of 20 larvae were confirmed by molecular analyses.GenBank accession numbers for the obtained sequences and their respective similarities with other conspecific ticks are shown in Table 2. Attempts to amplify and generate a confident sequence of the mitochondrial 16S rRNA gene from the N. amazoniensis were unsuccessful.
Ornithodoros hasei was the most abundant tick followed by  1. Overall, the mean intensity of tick infestation was 18.8 ± 3.6 ticks per bat, ranging from 1 to 61 ticks per bat (Table 1).Ticks were found attached on the tail membrane, legs, around eyes and mouth of the bats.

Discussion
In this study, the majority of tick-infested bats constitute species associated with caves, where bats live in colonies or use the caves as opportunistic roosting places (GUIMARÃES & FERREIRA, 2014).
Ornithodoros hasei was the most common tick found in association with bats from the Caatinga area prospected in the current study, and A. planirostris corresponded to the most parasitized host.In a recent study from the Pantanal biome, Muñoz-Leal et al. (2016b) also evidenced high prevalence of O. hasei larvae on A. planirostris.As suggested by these authors, and reinforced by the data from the Caatinga biome, A. planirostris, a bat with wide distribution in Brazil, might play an important role in the biological cycle of O. hasei, contributing to the dispersion of this tick.
Ornithodoros cavernicolous, the second most common species, was collected only in cave-roosting bats.This soft tick was described from free-living specimens from caves in the Caatinga biome, and from bat-associated larvae in the Cerrado Biome of central-western Brazil (DANTAS-TORRES et al., 2012) & VENZAL, 2009).Here we report new records of this tick in the Caatinga biome, parasitizing the bats C. perspicillata and T. cirrhosus.The occurrence of O. fonsecai in these new localities expands its actual distribution towards the north of Brazil in ≈2400 km, and adds two new hosts.
In Brazil, O. marinkellei was previously reported in caves from eastern (LABRUNA et al., 2011) and western (HENRIQUE-SIMÕES et al., 2012) Amazonian biome, in association with three bat species of the genus Pteronotus (LABRUNA et al., 2011).In this way it is not surprising that in the current study larval stages of this tick

Table 2 .
Mitochondrial 16S rDNA sequences for the analyzed ticks, and their closest similarities in GenBank.