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Introduction
Ticks are obligate hematophagous ectoparasites and important vectors of agents that cause disease in wild and domestic animals, and humans worldwide (PAROLA & RAOULT, 2001; COLWELL et al., 2011). The family Argasidae, known as soft ticks, comprises more than 200 species worldwide with 91 representatives in the Neotropics (NAVA et al., 2010; GUGLIELMONE et al., 2010; BARROS-BATTESTI et al., 2013, 2015; VENZAL et al., 2015; MUÑOZ-LEAL et al., 2016a; LABRUNA et al., 2016). While in Brazil Argasidae is represented by 24 species belonging to Antricola, Argas, Nothoaspis, and Ornithodoros genera, at least 19 species belong to the genus Ornithodoros: Ornithodoros brasiliensis Aragão, 1923; Ornithodoros capensis Neumann, 1901; Ornithodoros fonsecai (Labruna & Venzal, 2009); Ornithodoros guaporensis Nava, Venzal & Labruna, 2013; Ornithodoros hasei (Schulze, 1935); Ornithodoros jul Schulze, 1940; Ornithodoros kohlsi (Guglielmone & Keirans, 2002); Ornithodoros marinkellei Kohls, Clifford & Jones, 1969; Ornithodoros mimon Kohls, Clifford & Jones, 1969; Ornithodoros nattereri Warburton, 1927; Ornithodoros rondoniensis (Labruna, Terrassini, Camargo, Brandão, Ribeiro & Estrada-Peña, 2008); Ornithodoros rostratus Aragão, 1911; Ornithodoros rudis Karsh, 1880; Ornithodoros setosus Kohls, Clifford & Jones, 1969; Ornithodoros stageri Cooley & Kohls, 1941; Ornithodoros talaje (Guérin-Méneville, 1849); Ornithodoros faccinii Barros-Battesti, Landulfo & Luz, 2015; Ornithodoros cavernicolous Dantas-Torres, Venzal & Labruna, 2012 and Ornithodoros rietcorreai Labruna, Nava & Venzal, 2016 (GUGLIELMONE et al., 2010; DANTAS-TORRES et al., 2012; BARROS-BATTESTI et al., 2013, 2015; WOLF et al., 2016; LABRUNA et al., 2016). Of this diversity, nine species (47%) have been associated with bats or bat-frequented caves (LABRUNA & VENZAL, 2009; LABRUNA et al., 2011; DANTAS-TORRES et al., 2012; BARROS-BATTESTI et al., 2013; MARTINS et al., 2014; MUÑOZ-LEAL et al., 2016b).
The term “Caatinga” refers to the typical xeromorphic vegetation of the semiarid ecosystems in northeastern Brazil that covers an area of approximately 734,478 Km2 (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.
Materials and Methods
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 (05o15’S, 41o00’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).
Results
A total of 347 bats belonging to six families and 23 species were captured: Phyllostomidae (n=265; 16 species), Mormoopidae (n=56; 2 species), Noctilionidae (n=2; 1 species), Natalidae (n=2; 1 species), Molossidae (n=9; 1 species), Vespertilionidae (n=13; 2 species). The species Carollia perspicillata (Linnaeus, 1758) (n=66), Artibeus planirostris Spix, 1823 (n=54) and Pteronotus parnellii Gray, 1843 (n=43) were the most abundant (SILVA et al., 2015).
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).
Table 1 Ticks collected on phyllostomid bats in the Private Reserve of the Natural Patrimony (RPPN) Serra das Almas, Ceará state, northeastern Brazil. NEB: number of examined bats; NIB: number of infested bats; L: larvae.
| Bat species | NEB/ NIB | bat habitat* | Number of. ticks | Overall prevalence (%) | MI# | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| N. amazoniensis | O. cavernicolous | O. hasei | O. marinkellei | O. fonsecai | Antricola sp. | |||||
| PHYLLOSTOMIDAE | ||||||||||
| Artibeus planirostris | 54/22 | OC | 2L | 296L | 40.7 | 13.5 | ||||
| Carollia perspicillata | 66/10 | OC | 28L | 7L | 15.1 | 3.5 | ||||
| Trachops cirrhosus | 18/3 | OC | 1L | 10L | 1L | 16.6 | 4.0 | |||
| Anoura geoffroyi | 3/2 | EC | 4L | 66.7 | 2.0 | |||||
| Phyllostomus discolor | 4/2 | OC | 22L | 50 | 11 | |||||
| Tonatia sp. | 6/2 | OC | 4L | 33.3 | 4.0 | |||||
| Tonatia bidens | 4/1 | OC | 4L | 25 | 4.0 | |||||
| Tonatia saurophila | 14/1 | NC | 10L | 7.1 | 10 | |||||
| Glossophaga soricina | 17/1 | OC | 3L | 5.9 | 3.0 | |||||
| Sturnira lilium | 6/1 | OC | 1L | 16.7 | 1.0 | |||||
| Lophostoma brasiliense | 12/1 | NC | 6L | 8.3 | 6.0 | |||||
| MORMOOPIDAE | ||||||||||
| Pteronotus gymnonotus | 13/2 | EC | 10L | 15.3 | 5.0 | |||||
| Pteronotus parnelli | 43/1 | EC | 1L | 2.3 | 1.0 | |||||
| Total | 260/49 | 1 | 74 | 315 | 10 | 8 | 1 | 18.8 | 8.3 | |
*Cave use classification according to Guimarães & Ferreira (2014): essentially cave dweller (EC); opportunistic cave dweller (OC); not cave dweller (NC);
#mean intensity of tick infestation.
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). One fully engorged larva of Nothoaspis amazoniensis Nava, Venzal & Labruna 2010 was identified by the following combination of characters: dorsal plate elongate and triangular; dorsum with 12-13 setae pairs (5-6 anterolateral, 3 central, and 4 posterolateral); venter with 10 pairs of setae; posteromedian setae absent; 3 pairs of sternal setae; 1 pair of post-coxal setae; 5 pairs of circumanal setae; 1 pair of ventral posteromarginal setae and basis capituli pentagonal, following Nava et al. (2010) (Figure 2).
Figure 1 Micrographs of Ornithodoros fonsecai, Ornithodoros hasei, Ornithodoros marinkellei and Antricola sp. larvae. Ornithodoros fonsecai: dorsal idiosoma (A), dorsal plate (B), and hypostome (C). Ornithodoros hasei: dorsal idiosoma (D), dorsal plate (E), and hypostome (F). Ornithodoros marinkellei: cornua-like projection (black arrows) in basis capitulum (G) and dorsal plate (H). Antricola sp.: poscoxal setae (I). Abbreviations: Al, anterolateral seta; C, central seta; Pl, posterolateral seta; Pc, postcoxal seta.
Figure 2 Micrographs of Nothoaspis amazoniensis and Ornithodoros cavernicolous larvae collected in the present study and paratype of N. amazoniensis (NAVA et al., 2010). Nothoaspis amazoniensis: dorsal idiosoma (J), dorsal plate of the collected specimen (K), dorsal plate of paratype. (L), triangular spurs (white arrows) in coxa I (M), auricula-like projections (white arrows) in posterior margin of basis capitulum (N). Ornithodoros cavernicolous: dorsal idiosoma (O), tarsus I (P), and dorsal plate (Q). Abbreviations: Al, anterolateral seta; C, central seta; Pl, posterolateral setae; B, basiventral seta.
Four hundred and seven larvae of the genus Ornithodoros were classified in the following four species: O. cavernicolous (n = 74), O. fonsecai (n = 8), O. hasei (n = 315), and O. marinkellei (n = 10) (Table 1). The morphological characters for the identification of larvae of these species are as follows: 1) O. cavernicolous: dorsal plate with a sub-equilateral triangular shape, dorsum provided with 7 anterolateral, three central and 5 posterolateral pairs of setae, and three pairs of basal setae on tarsus I (DANTAS-TORRES et al., 2012); 2) O. fonsecai: dorsal plate elongate, pyriform, broadest posteriorly, dorsum with 13–14 pairs of setae, with 10-11 dorsolateral pairs and 3 central pairs; hypostome long, pointed apically with dental formula 3/3 in the anterior half, 2/2 posteriorly almost to base; dentition: file 1 with 17 to 18 denticles, 2 with 15 to 17, and 3 with 9 to 10 (LABRUNA & VENZAL, 2009); 3) O. hasei: 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.
Table 2 Mitochondrial 16S rDNA sequences for the analyzed ticks, and their closest similarities in GenBank.
| Tick species | GenBank - accession number | Number of larvae submitted to PCR | Host | Closest similarity in GenBank for the mitochondrial 16S rDNA sequence |
|---|---|---|---|---|
| O. cavernicolous | KX781697 | 13 | C. perspicillata, P. discolor | 100% (428-pb) O. cavernicolous, Ceará State (JF714964) |
| O. marinkellei | KX781700 | 4 | P. gymnonotus | 99% (423/426-pb) O. marinkellei, Brazilian Amazon (HM582439) |
| O. hasei | KX781698 | 2 | A. planirostris | 99% (425/427-pb) O. hasei, Mato Grosso do Sul State (KT894588) |
| O. fonsecai | KX781699 | 1 | C. perspicillata | 96% (406/425-pb) O. fonsecai, Mato Grosso do Sul State (KC769597) |
| Total | 20 |
Ornithodoros hasei was the most abundant tick followed by O. cavernicolous. O. fonsecai was collected only in C. perspicillata and Trachops cirrhosus, and O. marinkellei only in Pteronotus gymnonotus. Less frequent N. amazoniensis and Antricola sp. larvae were only collected in T. cirrhosus and P. parnelli, respectively. The occurrence of co-infestations was observed for N. amazoniensis, O. cavernicolous and O. fonsecai parasitizing T. cirrhosus; O. cavernicolous and O. hasei parasitizing A. planirostris, and O. cavernicolous and O. fonsecai parasitizing C. perspicillata. Detailed data is presented in Table 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.
Seventeen slide-mounted ticks were deposited at the CNC tick collection under accession numbers CNC-3291, CNC-3353 (O. cavernicolous), CNC-3358 (O. hasei), CNC-3363 (O. fonsecai), CNC-3365 (O. marinkellei) and CNC-3367 (Antricola sp.), and 340 ethanol preserved specimens CNC-3292 (N. amazoniensis), CNC-3354, CNC-3355, CNC-3356, CNC-3357 (O. cavernicolous), CNC-3359, CNC-3360, CNC-3361, CNC-3362 (O. hasei), CNC-3364 (O. fonsecai), and CNC-3366 (O. marinkellei).
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). To date only Desmodus rotundus (E. Geoffroy, 1810), Anoura caudifer (E. Geoffroy, 1818), and C. perspicillata were reported as hosts of this tick species; therefore, the present study adds seven host species: A. planirostris, Phyllostomus discolor, T. cirrhosus, Tonatia bidens (Spix, 1823), Glossophaga soricina (Pallas, 1766), Sturnira lilium (E. Geoffroy, 1810) and A. geoffroyi.
In contrast to O. hasei and O. cavernicolous, the other two Ornithodoros species collected in this study, O. fonsecai and O. marinkellei, presented low prevalence. From a biogeographic point of view, O. fonsecai was exclusively known from a sole cave in the state of Mato Grosso do Sul within the Cerrado biome, and by parasitizing cave-dweller bats such as Peropteryx macrotis (Wagner, 1843) and D. rotundus (E. Geoffroy, 1810) (LABRUNA & 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 were also collected from the already known host P. gymnonotus (LABRUNA et al., 2011); however, at the Caatinga biome. Similarly to A. planirostris for O. hasei, Pteronotus bats might play an essential role in the biological cycle of O. marinkellei, as emphasized in literature (KOHLS et al., 1969; JONES & CLIFFORD, 1972; VENZAL et al., 2006).
Less abundant collected ticks corresponded to one larva of N. amazoniensis and one of Antricola sp.. Previous records of N. amazoniensis were documented only from the Amazonian biome and exclusively from the bat P. parnellii (NAVA et al., 2010). Here we report for the first time this tick on T. cirrhosus and in the Caatinga biome. Both, P. parnelli and T. cirrhosus have overlapping distributional ranges and use caves as shelter in the Amazonian, Savannah and Caatinga biomes (BREDT et al., 1999; GUIMARÃES & FERREIRA, 2014). In this sense, the occurrence of N. amazoniensis on either T. cirrhosus or P. parnellii might be explained by the sharing of caves as roosting places. As both bat species are also distributed in the Savannah, further prospections might find this tick species also in this biome.
All three Antricola species that occurs in Brazil, Antricola guglielmonei Estrada-Peña, Barros-Battesti & Venzal 2004, Antricola delacruzi Estrada-Peña, Barros-Battesti & Venzal 2004, and Antricola inexpectata Estrada-Peña, Barros-Battesti & Venzal 2004 were described from mature specimens collected in caves from the Sergipe and Ceará states (ESTRADA-PEÑA et al., 2004), located in a transition area between the Atlantic rainforest and the Caatinga biomes. Additional reports included the collection of adults from caves in the Rondônia state within the Amazonian biome (LABRUNA et al., 2008). Although a detailed description of the larvae from both species has not been published, morphological characters of Antricola larvae presented by Cooley & Kohls (1944), and posteriorly by Kohls et al. (1965), coincide with the general morphology of the larva found parasitizing P. parnelli in the present study. This host-parasite association is not unexpected since both Antricola and Pteronotus bats live in caves (ESTRADA-PEÑA et al., 2004; GUIMARÃES & FERREIRA, 2014; LABRUNA et al., 2011), and because Antricola larvae have been reported on Pteronotus bats in the Amazon biome (LABRUNA et al., 2011).
This study provides new geographical ranges for five Brazilian bat-associated soft tick species, expanding the distribution of N. amazoniensis, O. marinkellei and O. fonsecai to the Caatinga biome.
