A total of six Amblyomma tapirellum ticks were found for the first time in rainforest from the lowland to the southern Pacific of Costa Rica. Tick identification was carried out by morphology and afterward confirmed by molecular analysis, using polymerase chain reaction (PCR) and DNA barcoding. Further studies are required to determine the distribution of A. tapirellum and this species' potential as a vector of bacterial agents to humans and wild hosts.
Ticks are a group of hematophagous mites that parasitize all classes of terrestrial vertebrates (Labruna et al. 2005). They cause serious problems to animal husbandry and human health. To date, most studies of Neotropical ticks have been conducted in rural and urban environments, although most species can be found on wildlife. Consequently, species living in natural environments are little known (Szabó et al. 2003). This is particularly true in Central America, where only a few studies have reported the diversity of ticks in wild environments, with the result that our knowledge of this region's tick fauna is limited.
Amblyomma tapirellum Dunn, 1933 was first described from Panama and afterward reported from Belize, Nicaragua and Colombia (Dunn 1933, Fairchild et al. 1966, Varma, 1973). Adults of this species are considered parasites of ungulates, although they have been found parasitizing other mammals, such as Carnivora, Chiroptera, Pilosa, and Rodentia (Fairchild et al. 1966, Bermúdez et al. 2010, García et al. 2014, Bermúdez et al. 2015). In addition, there are reports of humans parasitized by adults of A. tapirellum (Fairchild et al. 1966, Bermúdez et al. 2012). In the case of the immature stages, only wild carnivores have been confirmed as hosts to date (Bermúdez et al. 2015), although other groups of mammals would appear to be suitable hosts. Many aspects of the biology and distribution of A. tapirellum are unknown. In this paper, we provide additional information concerning the identification and distribution of A. tapirellum and report its presence in the tropical rainforest of Costa Rica.
Materials and methods
From 2011 to 2012, we examined ticks deposited in the collections of Universidad Nacional de Costa Rica (UNA-CR) and Instituto Nacional de Biodiversidad de Costa Rica (INBio). Collected ticks were preserved in 70% alcohol, pending their morphological identification and molecular analysis. All specimens were identified using the taxonomic keys of Fairchild et al. (1966). Subsequently, to confirm their identity as A. tapirellum, specimens were examined for characteristics described by Dunn (1933), and compared with specimens deposited in the “Dr. Eustorgio Méndez” Zoological Collection of the Gorgas Memorial Institute for Health Studies, including one female of the Dunn's Collections which is labeled as T-l. Voucher specimens were deposited in UNA-CR and INBio.
Material examined from UNA-CR (Fig. 1): 1 ♂ Costa Rica, Puntarenas, Golfito, Golfo Dulce, Parque Nacional Corcovado, sendero Río Claro. 10 m. January 6, 2012. V.M. Montenegro, R. Quesada. Collected by blanket dragging in secondary forest. 8°28′54.4″N, 83°35′31.3″ W. 1♀ Costa Rica, Puntarenas, Golfito, Golfo Dulce, Parque Nacional Corcovado, Sendero Sirena-Cruce-Guanacaste, 20 m. August 4, 2012. V.M. Montenegro, R. Quesada. Collected by blanket dragging in secondary forest. 8°29′08.37″ N, 83°35′32.94″ W. 1♀ Costa Rica, Puntarenas, Golfito, Golfo Dulce, Parque Nacional Corcovado, sendero Espaveles, 40 m. May 3, 2012. V.M. Montenegro, R. Quesada. Collected by blanket dragging in primary forest. 8°28′55,76″ N, 83°35′18.05″ W. 1♀ Costa Rica, Puntarenas, Golfito, Golfo Dulce, Parque Nacional Corcovado, Sendero Culebra, 10 m. May 4 2012. V.M. Montenegro, R. Quesada. Collected by blanket dragging, in primary forest. 8°29′13.35″ N, 83°34′51.84″W.
Revised material from INBIO (Fig. 1): 1 ♀ Costa Rica, Puntarenas, Golfito, Golfo Dulce, Parque Nacional Corcovado, Est. (Estación) Agujas, 300 m. January 12, 2012. J. A. Azofeifa, D. Azofeifa. Collected on bromeliads. 8°32′11.79″ N, -83°25′31.8″ W #103426. 1 ♂ Costa Rica, Puntarenas, Humedal Sierpe, 100 m (Fig. 1). January 5, 2011. Collected on vegetation. 8°51′57.8″ N, -84°11′43.6″ W.
In order to confirm the morphological identification, DNA from four specimens Corcovado National Park (UNA-CR) was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. These samples were amplified by polymerase chain reaction (PCR) with the primers LCO1490 5′ -GGTCAACAAATCATAAAGATATTGG-3′ and HC02198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ described by Folmer et al. (1994), to obtain a 658-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Conditions used for this reaction were reported by Hebert et al. (2003). Amplified fragments were sent for purification and sequencing to Macrogen (Seoul, Korea). Sequences were edited using the Biological Sequence Alignment Editor (BioEdit version 7.2.5) (Hall, 1999), aligned with the Clustal W algorithm (Thompson et al. 1994), and compared with sequences of the NCBI (National Center for Biotechnology Information) database using the BLASTn algorithm (Altschul et al. 1990). Subsequently, a phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis software (MEGA version 5) (Tamura et al. 2011) by the Neighbor-Joining method (Saitou & Nei, 1987), and corrected with p-distance, yielding a substitution model to analyze evolutionary divergence between species (Nei & Kumar, 2000; Srivathsan & Meier, 2012). COI gene sequences from other Amblyomma species, available in GenBank, were included in the analysis: Amblyomma auricularium (KF200137), Amblyomma calcaratum (KF200144), Amblyomma dissimile (KF200168), Amblyomma geayi (KF200159), Amblyomma longirostre (KF200095), Amblyomma nodosum (KF200131), Amblyomma oblongoguttatum (KF200165), Amblyomma ovale (KF200158), Amblyomma pecarium (KF200153), Amblyomma sabanerae (KF200152), A. tapirellum (KF200171 and KF370891), Amblyomma varium (KF200157) and Ixodes affinis (KF200161) as an outgroup.
Results and discussion
The morphological identification (Fig. 2) was consistent with A. tapirellum for all specimens examined. The partial region of the COI gene of the four UNA-CR tick samples was sequenced and included in GenBank (accession numbers: KP247501, KP247502, KP247503, KP247504); they shared between 99.6% and 100% of nucleotide identity, 99.8% to 100% when compared with sequences of A. tapirellum from Panama (GenBank accession numbers KF200120 and KF370891). All A. tapirellum sequences were grouped into the same cluster (Fig. 3). The species A. oblongoguttatum (accession number KF200165) shared the greatest sequence similarity (84.7% to 84.9%) with the A. tapirellum group.
This is the first report of A. tapirellum from Costa Rica and increases to 24 the number of Amblyomma species known from this country (Álvarez et al. 2005). However, the presence of A. tapirellum was not unexpected, since it was previously reported in Nicaragua (Fairchild et al. 1966) and has been described from Chiriquí, Panama (Fairchild et al. 1966), a city relatively close to the Costa Rican province of Puntarenas. In Costa Rica, Amblyomma mixtum and A. oblongoguttatum share similar patterns of scutal ornamentation with A. tapirellum, but they can be differentiated by characters discussed in Dunn (1933) and Fairchild et al. (1966). Ecologically, there are also differences between these species. Amblyomma mixtum is associated with open habitats and chiefly parasitizes cows and horses (Alvarez et al. 2000, Alvarez & Bonilla, 2007), while A. oblongoguttatum seems to inhabit both open environments and wooded areas and is not specific to any host group (Voltzit, 2007, Dolz, 2014). In contrast, A. tapirellum is usually found in less disturbed environments and the tapir (Tapirus bairdii Gill. 1865) is its main host (Fairchild et al. 1966).
In Costa Rica, studies of tick ecology have been largely carried out in urban and rural areas, so there is little data of the ecology of ticks associated with wildlife (Alvarez et al. 2005, Troyo et al. 2012, Jiménez et al. 2013, Dolz et al. 2013, Troyo et al. 2014). It is possible that additional research in forests will confirm the presence of other tick species in Costa Rica.
Four specimens of A. tapirellum were found in the forests of Corcovado National Park. Various potential hosts, such as peccaries and tapirs, have been reported in this park, which probably means that A. tapirellum is established there (Almeida et al. 2009, Altrichter & Almeida, 2009). However, collared peccaries (Tayassu tajacu, Linnaeus, 1978), white-lipped peccaries (Tayassu pecari Link, 1795), and Baird's tapir (Tapirus bairdii Gill, 1865) are all suffering illegal hunting pressure, and their numbers have declined in several parts of Corcovado National Park (Carrillo & Sáenz, 2011; Bustamante et al. 2013). For this reason, ecological studies are needed to determine which vertebrates are crucial to the life cycle of A. tapirellum, especially its immature stages.
Finally, records of A. tapirellum feeding on humans (Fairchild et al. 1966, Bermúdez et al. 2012) could represent a risk to people, who frequent the trails in Corcovado National Park, if populations of the principal natural hosts decrease because of poaching. We recommend further research on the ecology of this tick, especially investigations of pathogens that can be transmitted by A. tapirellum and that may affect humans and wild animals.
This study was supported in part by Fondo Especial para el Financiamiento de la Educación Superior, Consejo Nacional de Rectores (FEES-CONARE) and Vicerrectoría de Investigación, Universidad Nacional, Costa Rica.