Morphological and molecular identification of ticks infesting Boa constrictor ( Squamata , Boidae ) in Manaus ( Central Brazilian Amazon )

The Boa constrictor is one of the world’s largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.


Introduction
The Boa constrictor is one of the largest snakes in the world and can reach a total length of over four meters.This predator with terrestrial and semi-arboreal habits consumes lizards, birds and mammals (CUNHA & NASCIMENTO, 1978;HENDERSON et al., 1995;BERNARDE, 2004;QUICK et al., 2005;PIZZATTO & MARQUES, 2007;PIZZATTO et al., 2009).Boa constrictor occur throughout Brazil and can be found in urban environments (MARTINS & OLIVEIRA, 1998;PIZZATTO & MARQUES, 2007;BERNARDE & ABE, 2006;BENTES, 2013).In fact, specimens are often caught in the city of Manaus, AM (central Brazilian Amazon), which has a population of almost two million (IBGE, 2012).In this region there are hundreds of rainforest fragments measuring 1 to 600 ha very close to urban areas.This combination of environments favors the parasitism of snakes by ticks (BENTES, 2013), which may be related to local parasitism dynamics (DAVIS et al., 2012).Despite this fact, only one earlier study has reported specimens of A. dissimile parasitizing a Boa constrictor captured in 1975 in an "INPA secondary forest" (ADIS, 1981).
The present study identifies tick species parasitizing Boa constrictor in urban areas of the city of Manaus, AM, with the aid of morphological and molecular tools.

Materials and Methods
All the ticks found attached to the body surfaces of Boa constrictor captured between September 2010 and November 2012 were collected by the Municipal Secretariat for Environment and Sustainability (SEMMAS) in urban areas in the city of Manaus, AM (3° 6' 7" S and 60° 1' 30" W).Most of the ticks were found attached to the head and the first 10 cm of the body.The ticks were removed manually using tweezers and stored in 70% alcohol for transfer to the laboratory, where males, females and nymphs were identified under a microscope.
The ticks were identified morphologically using dichotomous keys (GUIMARÃES et al., 2001;BARROS-BATTESTI et al., 2006).One hundred and one ticks were deposited in the Collection of Arthropods of Medical and Veterinary Importance at the Biological Institute of São Paulo, Brazil (accession number: 1313 AMB).
DNA was extracted from one male and two female ticks using a commercial kit (DNeasy Blood & Tissue Kit Qiagen  ), following the manufacturer's instructions.Polymerase chain reaction (PCR) was performed following the manufacturer's protocol (2X DreamTaq Green PCR Master Mix), usig the primers listed in Table 1 to amplify the mitochondrial 16S rRNA gene fragment (MANGOLD et al., 1998).PCR products were purified and subjected to a commercially available standard semi-automated dideoxy Sanger sequencing method.The resulting sequences were aligned with each other and with other Amblyomma mitochondrial 16S rRNA sequences available in the GenBank nucleotide database.

Results
The number of ticks collected from each of the 222 boa constrictors sampled ranged from 0 to 354 (mean: 28.4; standard deviation: 46.2), with a total number of 5929 ticks, of which 62% were females, 32% males and 6% nymphs.The morphological identification of the ticks collected from 27 Boa constrictor revealed the occurrence of A. dissimile Koch, 1844 on all the sampled individuals, whereas A. rotundatum Koch, 1844 was found on only two of these snakes.
The sequence of mitochondrial 16S rRNA from A. rotundatum exhibited 99.7% identity with a fragment from the same species available in GenBank under accession number EU805569, differing in only one of the 324 base pairs sequenced.The sequence of mitochondrial 16S rRNA from A. dissimile exhibited 87.1% identity with the corresponding fragment from A. rotundatum (Figure 1) and over 90% identity with the same gene fragments from A. longirostre and A. geayi (GenBank accession numbers EU805565 and EU805566, respectively).Identity with other sequences ranged from 81.3% to 87%.

Table 1 .a
Primers used for the PCR amplification and sequencing of the mitochondrial 16S rDNA sequence of A. dissimile and A. rotundatum.Modified primers described byMangold et al. (1998).Some nucleotides were replaced with inosines to allow for the amplification of unknown genomic sequences; b Relative to the mitochondrial A. cajennense genome taken as reference, publicly available in GenBank under accession number JX573118.