A c a F B a 1 b P c C a A R R A A K A C M R 1 r s t b g s r A h 1 Ticks and Tick-borne Diseases 7 (2016) 842–848 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ourna l ho me pa ge: www.elsev ier .com/ locate / t tbd is mblyomma mixtum Koch, 1844 (Acari: Ixodidae): First record onfirmation in Colombia using morphological and molecular nalyses redy A. Rivera-Páeza,c, Marcelo B. Labrunab, Thiago F. Martinsb, runo Rodrigues Sampieri a, Maria I. Camargo-Mathiasa,∗ Departamento de Biologia, Instituto de Biociências, UNESP—Universidade Estadual Paulista, Avenida 24-A, 1515, Bairro Bela Vista, SP, Rio Claro CEP 3506-900, Brazil Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo-USP, Av. rof. Orlando Marques de Paiva, 87, CEP 05508-000, Cidade Universitária, São Paulo, SP, Brazil Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Calle 65 No. 26-10 Apartado Aéreo 275 Manizales, aldas, Colombia r t i c l e i n f o rticle history: eceived 1 December 2015 eceived in revised form 8 March 2016 ccepted 31 March 2016 vailable online 1 April 2016 eywords: mblyomma cajennense omplex olecular markers ickettsia rickettsii a b s t r a c t Up to some years ago, the taxon Amblyomma cajennense represented a single tick species in the New World, from southern United States to northern Argentina. Recent studies, based on genetic, reproductive and morphological data reorganized this taxon into a complex of the following 6 valid species: A. cajen- nense sensu stricto, Amblyomma mixtum, Amblyomma sculptum, Amblyomma interandinum, Amblyomma tonelliae, and Amblyomma patinoi. According to this classification, the A. cajennense complex is currently represented in Colombia by only one species, A. patinoi. Because the Colombian land is surrounded by confirmed records of A. mixtum in Panama and Ecuador, and by A. cajennense s.s. in Venezuela and the Brazilian Amazon, it is possible that these two species could also occur in Colombia. This study aimed to determine the occurrence of ticks of the A. cajennense complex in the Orinoquía region of Colombia. A total of 246 adult ticks of the Amblyomma genus were collected in three sampled regions: 71 females and 110 males in Arauca (Arauca Department), 27 females and 20 males in Nunchía (Casanare Department), and 10 females and 8 males in Yopal (Casanare Department). Based on morphological and molecular analyses, these ticks were identified as A. mixtum. Molecular analyses consisted of DNA sequences of two molec- ular markers, the nuclear second internal transcribed spacer (ITS2) and the mitochondrial cytochrome c oxidase subunit I gene (COI). The presence of A. mixtum in Colombia is of medical relevance, since this species is incriminated as a vector of Rickettsia rickettsii in Central America. © 2016 Published by Elsevier GmbH. . Introduction Up to some years ago, the taxon Amblyomma cajennense rep- esented a single tick species distributed in all tropical and ubtropical areas of the New World, from southern United States o northern Argentina (Estrada-Peña et al., 2004). Recent studies, ased on genetic, reproductive and morphological data reor- anized this taxon into a complex of the following 6 valid pecies: A. cajennense sensu stricto (restricted to the Amazonian egion), Amblyomma mixtum (from Texas to western Ecuador), mblyomma sculptum (northern Argentina, Bolivia, Paraguay, ∗ Corresponding author. E-mail address: micm@rc.unesp.br (M.I. Camargo-Mathias). ttp://dx.doi.org/10.1016/j.ttbdis.2016.03.020 877-959X/© 2016 Published by Elsevier GmbH. Brazil), Amblyomma interandinum (inter-Andean valley of Peru), Amblyomma tonelliae (dry areas of northern Argentina, Bolivia and Paraguay), and Amblyomma patinoi (Eastern Cordillera of Colombia) (Beati et al., 2013; Nava et al., 2014). According to this classifica- tion, the A. cajennense species complex is currently represented in Colombia by only one specie, A. patinoi (Nava et al., 2014). However, our current knowledge on the distribution of these species is prob- ably incomplete, and examination of new field-collected material is required for a better definition of species boundaries (Nava et al., 2014). Moreover, A. cajennense s.l. constitutes the most important human-biting ticks of South America (Guglielmone et al., 2006), and at least three species of this species complex, namely A. sculp- tum, A. mixtum, and A. patinoi, are incriminated as important vectors of the bacterium Rickettsia rickettsii, the agent of the deadly Rocky Mountain spotted fever (Krawczak et al., 2014; Labruna et al., 2014; dx.doi.org/10.1016/j.ttbdis.2016.03.020 http://www.sciencedirect.com/science/journal/1877959X http://www.elsevier.com/locate/ttbdis http://crossmark.crossref.org/dialog/?doi=10.1016/j.ttbdis.2016.03.020&domain=pdf mailto:micm@rc.unesp.br dx.doi.org/10.1016/j.ttbdis.2016.03.020 Tick-b F a l s b f 2 k e n E t t i E A c i l a c 2 t f ( a a s ( 7 S i 7 7 l o N o e s F.A. Rivera-Páez et al. / Ticks and accini-Martínez et al., 2015). Therefore, a precise knowledge of the ctual distribution of these species is of highly public relevance. The six species of the A. cajennense species complex are morpho- ogically very similar, making their morphological discrimination ometimes very difficult. The combination of morphological, distri- utional, and molecular information may sometimes be necessary or the correct determination of problematic specimens (Nava et al., 014). While A. patinoi is the only member of this complex, precisely nown to occur in Colombia (Nava et al., 2014; Faccini-Martínez t al., 2015), there have been multiple previous records of A. cajen- ense s.l. from different parts of Colombia (López-Valencia 1989; strada-Peña et al., 2004; Miranda et al., 2011). Unfortunately, hese specimens are not available for morphological reexamina- ion or molecular analysis. In addition, because the Colombian land s surrounded by confirmed records of A. mixtum in Panama and cuador, and by A. cajennense s.s. in Venezuela and the Brazilian mazon (Nava et al., 2014), it is possible that these two species ould also occur in Colombia. In view of the above and considering the medical and veterinary mportance of A. cajennense s.l. in Latin America, associated to the ack of studies on this complex in the Colombian territory, this study imed to determine the occurrence of ticks of the A. cajennense omplex in the Orinoquía region of Colombia. . Material and methods Ticks were collected directly from horses (Equus caballus), cat- le (Bos taurus), and a capybara (Hydrochoerus hydrochaeris) rom different geographical sites of the Orinoquía region Eastern Plains) of Colombia. The ecosystems of the region re tropical savanna with gallery forests and wetlands long the rivers. Ticks were collected from the following pecific sites: Department of Arauca, Arauca municipality 06◦55′43′’N, 70◦27′36”W/06◦02′0”N, 69◦25′0”W/06◦56′24”N, 0◦32′0”W/07◦1′48”N, 70◦43′39”W/07◦3′55”N, 70◦44′2”W) in eptember 2014; Department of Casanare, Nunchía municipal- ty (5◦21”1”N, 72◦4′53”W/5◦21′13”N, 72◦5′50”W/5◦21′40”N, 2◦6′7”W) and Yopal municipality (5◦19′27”N, 2◦24′31”W/5◦25′26”N, 72◦14′17”W) in February 2015. Col- ected ticks were submitted to taxonomic identification based n their external morphology following Jones et al. (1972) and ava et al. (2014) through light microscopy (Leica M205C stere- microscope). In addition, 2 male and 2 female specimens from ach municipality (Arauca, Yopal and Nunchía) were prepared for canning electron microscopy (SEM) (Hitachi Scanning Electron Fig. 1. Dorsal view of Amblyomma mixtum. (A) Male; (B) Female. (e) adjace orne Diseases 7 (2016) 842–848 843 Microscope, model TM3000) following techniques described by Corwin et al. (1979). After morphological identification, 2 male and 2 female spec- imens of each Department were individually processed for molecular analyses. For this purpose, DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) following manufacturerı́s protocol, and tested by two PCR protocols, one targeting the ribosomal second internal transcribed spacer (ITS2) region, and the second one targeting the mitochondrial cytochrome c oxi- dase subunit I gene (COI). For the ITS2 PCR, we used primers ITS2 (F) 5′-CCATCGATGTGAAYTGCAGGACA-3′ (Zahler et al., 1995) and MCLN (R) 5′-GTGAATTCTATGCTTAAATTCAGGGGGT-3′ (Mclain et al., 1995), which correspond to the 5.8S and 28S regions, respec- tively, thus amplifying a DNA fragment that contains the complete sequence of the ITS2 of the rDNA, which has ≈1000-bp in ticks of the genus Amblyomma (Marrelli et al., 2007). For the COI PCR, we used primers LCO1490 (F) 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO2198 (R) 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′, which amplify a ≈700-bp fragment (Folmer et al., 1994). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System Kit (Promega), according to the manufacturer’s instructions, and sent to Macrogen Advancing Through Genomics (South Korea) for DNA sequencing. DNA sequences were submitted to phylogenetic analyses. For this purpose, the quality analysis of the DNA sequences was per- formed with the Geneious Trial v8.14 software (Drummond et al., 2009). Sequence alignments were conducted with the ClustalW software (Thompson et al., 1997), included in the Mega 6 software (Tamura et al., 2013). Species identification and confirmation con- ducted through similarity estimation between sequences obtained from specimens collected in Colombia, in addition to representa- tive sequences of six species of the A. cajennense complex derived from GenBank (Beati et al., 2013; Nava et al., 2014). Regarding the COI gene, the studied sequences had their similarities esti- mated through public sequences from GenBank and Barcode of Life Data Systems (BOLD−), registered as A. cajennense s.l. Variation of DNA sequences was estimated using the Kimura 2 parameter-K2 P (Kimura, 1980). A tree was created for o method (Neighbor-Joining–NJ) with 1000 replications in the boot- strap test. The K2 P parameter was selected as the genetic distance model in the Mega 6 software (Tamura et al., 2013). The localities of the ticks collected in this study were plotted in a map, together with previously published records of A. patinoi, A. mixtum and A. cajennense s.s. by Nava et al. (2014), using the Geographic Coordinate System (WGS 1984, Datum: DWGS 1984) and the ESRI® ArcMap. nt enamelled stripe, (s) postero-median spot, (sm) marginal groove. http://www.barcodinglife.com http://www.barcodinglife.com http://www.barcodinglife.com 844 F.A. Rivera-Páez et al. / Ticks and Tick-borne Diseases 7 (2016) 842–848 F ntral v p medi 3 l A 8 fi v u c i i fl s e m a b ig. 2. Scanning Electron Microscopy (SEM) A. mixtum male. (A) dorsal view; (B) ve rolongation, (h) hypostome, (pal) palps, (e) adjacent enamelled stripe, (s) postero- . Results A total of 246 adult ticks of the genus Amblyomma were col- ected in three sampled regions: 71 females and 110 males in rauca, 27 females and 20 males in Nunchía, and 10 females and males in Yopal. Initially, all ticks were morphologically identi- ed as A. cajennense s.l. (Figs. 1–3). According to Nava et al. (2014), ery few external morphological characters could be consistently sed to separate species of the A. cajennense complex. One of these haracters are the female genital opening, which is “V” shaped n A. cajennense s.s., A. tonelliae and A. interandinum, “U” shaped n A. sculptum and A. mixtum, and with short and bulging lateral aps in A. patinoi. All female specimens of the present study pre- ented an “U” shaped genital opening (Fig. 3D). According to Nava t al. (2014), morphological separation of males A. sculptum from A. ixtum are easily differentiable by the ornamentation and punctu- tions of the scutum, and in this case, geographical location should e applied, since the former species seems to be restricted to parts iew; (C) ventral basis capitulum; (D) coxae I–IV. (es) coxal spur, (sp) palpal ventral an spot. of South America south to the Amazon basin, whereas the later species occurs in regions north of the Amazon basin, from northern South America to southern Texas. Because the Orinoquía region of Colombia is located northern to the Amazon, we supposed that our specimens could be A. mixtum. Attempts to conclusive taxonomic identification were per- formed through molecular analyses. In this case, fragments of the ITS2 gene were generated for 4 tick specimens of each Depart- ment. These sequences were aligned (832-bp) with representative sequences of the six species of the A. cajennense complex, in addition to A. americanum (outgroup), derived from GenBank. Phylogenetic analysis indicated that the Colombian specimens cor- respond to A. mixtum (Fig. 4). Similarly, fragments of the COI gene were generated from 4 tick specimens of each Department. These sequences were aligned (616-bp) with six sequences from GenBank and Barcode of Life Data Systems (BOLD) of A. cajennense s.l. and one of A. americanum (outgroup). The Colombian specimens F.A. Rivera-Páez et al. / Ticks and Tick-borne Diseases 7 (2016) 842–848 845 F ) vent c , (pal c t s C M a a 8 b s o 4 m o f ig. 3. Scanning Electron Microscopy (SEM) A. mixtum female. (A) dorsal view; (B oxal spur, (sp) palpal ventral prolongation, (fe) festoons, (h) hypostome, (n) notum lustered with sequences from Panama (Fig. 5), which correspond o the geographic area of A. mixtum according to Nava et al. (2014). The intraspecific genetic distances between the ITS2 rDNA equences of A. mixtum collected in Arauca and Casanare, in olombia, and the sequences from GenBank of A. mixtum from exico, Costa Rica, and Texas revealed 0.5% maximum difference nd 0.0% minimum. On the other hand, interspecific differences mong the six A. cajennense complex-species ranged from 0.9 to .3% (Table 1). Regarding the COI sequences, the minimal difference etween the Colombian species occurred with an A. cajennense s.l. equence from Panama (Table 2), an area with known occurrence f A. mixtum, according to Nava et al. (2014). . Discussion Morphological and molecular analyses of field-collected speci- ens of A. cajennense s.l. in the present study clearly confirm the ccurrence of A. mixtum in Colombia for the first time. Genetic dif- erences between the ITS2 and COI sequences of Colombian and ral view; (C) notum; (D) genital aperture. (ag) genital aperture, (esc) scutum, (es) ) palps, (se) setae. A. mixtum sequences from GenBank are in agreement with Beati et al. (2013), who performed an extensive genetic analysis of all six species of the A. cajennense complex. Because there have been previous records of A. mixtum in two neighboring countries of Colombia (Panama and Ecuador), ours findings are also corrobo- rated by geographical data, especially because there is apparently no great eco-regional differences between the Orinoquía region of Colombia and other known A. mixtum areas (Estrada-Peña et al., 2014). On the other hand, Estrada-Peña et al. (2014) suggested that the Orinoquía region could be an area of sympatry or parapatry between A. mixtum and A. cajennense s.s. While we did not find any A. cajennense s.s. in the present study, we are aware that our convenient tick sample is not representative for the region (Fig. 6). Therefore, further studies are needed to better evaluate the possi- ble occurrence of more species of the A. cajennense complex in the Orinoquía region of Colombia. The presence of A. mixtum in Colombia is of medical relevance, since this specie is incriminated as the vector of R. rickettsii in Cen- tral America (Labruna et al., 2014). On the other hand, there has 846 F.A. Rivera-Páez et al. / Ticks and Tick-borne Diseases 7 (2016) 842–848 Fig. 4. Neighbor-joining tree using the sequences of the ITS2 rDNA gene. Fig. 5. Neighbor-joining tree using the sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI). Table 1 Kimura 2 parameter (K2P) distances (in percentage) for the second internal transcribed space (ITS2) sequences of species of the Amblyomma cajennense complex, including the A. mixtum sequences generated from ticks collected in Colombia in the present study. Tick species A. mixtum A. mixtum (Colombia) A. sculptum A. patinoi A. cajennense A. tonelliae A. interandinum A. americanum A. mixtum 0.1–0.3 A. mixtum (Colombia) 0.0–0.5 0.0–0.4 A. sculptum 1.4–1.8 1.4–1.8 0.1–0.4 A. patinoi 3.5–3.8 3.5–3.8 3.8–3.9 – A. cajennense 3.4–3.6 3.4–3.6 3.6–3.8 0.9 – 7.7– 8.0– 9.7 b a t e A. tonelliae 6.5–6.9 6.6–7.2 6.6–7.0 A.interandinum 6.8–7.2 6.8–7.2 7.0–7.3 A. americanum 9.6–9.9 9.6–9.9 10.0–10.1 een several records A. mixtum infection by Rickettsia amblyommii, possible non-human pathogen or an agent of a much milder infec- ious disease (Bermúdez et al., 2009; Hun et al., 2011; Novakova t al., 2015). Previous studies in Panama and Brazil have reported 8.0 7.9−8.2 0.3 8.2 8.2–8.3 3.4–3.8 0.1 10.4 10.7–11.0 10.4–10.6 – that natural R. rickettsii-infection rates in A. cajennense s.l. ticks are usually very low (usually ≤1%) (Sangioni et al., 2005; Krawczak et al., 2014). On the other hand, reported rates for R. amblyommii- infected A. cajennense s.l. ticks are commonly >25% (Bermúdez et al., F.A. Rivera-Páez et al. / Ticks and Tick-borne Diseases 7 (2016) 842–848 847 Table 2 Kimura 2 parameter (K2P) distances (in percentage) for the mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of species of the Amblyomma cajennense complex, including the A. mixtum sequences generated from ticks collected in Colombia in the present study. Tick species A. mixtum (Colombia) A. sculptumBrazil A. cajennense. s.l. (Panamá) A. cajennense s.l. Brazil A. americanum A. mixtum (Colombia) 0.2−1.0 A. sculptum Brazil 15.6–16.0 – A. cajennense s.l. (Panamá) 3.2–3.8 16.2 0.0 A. cajennense s.l. (Brazil) 14.5–14.7 4.5 14.3 0.0 A. americanum 18.4–18.9 18.2 20.2 17.8 – F patino 2 s c t 2 t r A C n P R B B B C ig. 6. Distribution of Amblyomma mixtum; Amblyomma cajennense s.s.; Amblyomma 009; Labruna et al., 2004; Soares et al., 2015). Because laboratory tudies have suggested that previous infection by R. amblyommii ould prevent a severe disease during a subsequent infection by he highly pathogenic R. rickettsii (Blanton et al., 2014; Rivas et al., 015), it is possible that previous human contact with A. mix- um ticks could decrease potentially fatal spotted fevers, yet to be eported from the study area. cknowledgements AUIP–Asociación Universitaria Iberoamericana de Postgrado. NPq–Conselho Nacional de Desenvolvimento Científico e Tec- ológico. 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record confirmation in Colombia using morphological and molecular ana... 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgements References