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Universidade de São Paulo

Biblioteca Digital da Produção Intelectual - BDPI
Departamento de Medicina Veterinária Prevenção e Saúde Animal Artigos e Materiais de Revistas Científicas - ICB/BMP
- FMVZ/VPS

2016

Geographical distribution of Amblyomma
cajennense (sensu lato) ticks (Parasitiformes:
Ixodidae) in Brazil, with description of the
nymph of A. cajennense (sensu stricto)
Parasites & Vectors. 2016 Mar 31;9(1):186
http://www.producao.usp.br/handle/BDPI/50013
Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

cajennense (s. sculptum]. Richard de C. Prof. Dias1. 1787) was considered to represent a single tick species in the New World. cajennense (s. cajennense (s. cajennense (s. sculptum. cajennense (s. Campos9.s. Luís M. Guilloux1. Parasites & Vectors (2016) 9:186 DOI 10.br 1 Departamento de Medicina Veterinária Preventiva e Saúde Animal.) ticks were considered to be males of either A. Distribution.s. ITS2.054 A. 6.s.). . Darci M. cajennense (s. cajennense (s. Artur K. A. provide a link to the Creative Commons license. distribution.s.Martins et al.l. Benatti1.0/) applies to the data made available in this article.512 adult ticks were examined and identified as 312 A. Keywords: Amblyomma cajennense. Martins1. and indicate if changes were made. cajennense species complex or A.) in Brazil was determined by morphological examination of all lots of A. 443 A. cajennense (s.l. Arlei Marcili1. sculptum in sympatry. 1888.org/licenses/by/4. cajennense (sensu lato) (s. While these species have distinct distribution areas in the country. cajennense (s. cajennense (s.7. Results: From the tick collections. and were identified as 249 A.) and A.l. Cássio R. Conclusion: Our results confirmed that A. and reproduction in any medium. they are found in sympatry in some transition areas.) is currently represented in Brazil by only two species. Barbieri1.) and Amblyomma sculptum Berlese. A.) and A. Ramirez11. Morphological description of the nymphal stage of A. Orlando Marques de Paiva 87. São Paulo 05508-270.s.s. and by collecting new material during three field expeditions in the possible transition areas between the distribution ranges of A. The Creative Commons Public Domain Dedication waiver (http://creativecommons. since in Brazil A. sculptum and 5. Peterka4. Aline G. Faculdade de Medicina Veterinária e Zootecnia. While the A. cajennense (s.) in two large tick collections of Brazil.s.3. Phylogenetic analysis inferred from the ITS2 rRNA gene was used to validate morphological results. the etiological agent of Brazilian spotted fever. Alessandra Scofield8. The nymph of A. Costa1. sculptum. Amblyomma sculptum. Brazil Full list of author information is available at the end of the article © 2016 Martins et al. Pacheco5. At least 23 localities contained the presence of both A. which permits unrestricted use. Francisco B. Nymph * Correspondence: labruna@usp. DNA sequences of the ITS2 gene of 50 ticks from 30 localities confirmed the results of the morphological analyses. cajennense (sensu stricto) Thiago F. Open Access This article is distributed under the terms of the Creative Commons Attribution 4. and 1. Methods: The distribution of the A.s. cajennense (s.s. cajennense (s. Nunes6. Universidade de São Paulo. Terassini2. Barros-Battesti11 and Marcelo B.).l.) [these A. M.) or A. their geographical distribution is poorly known. Labruna1* Abstract Background: Until recently. A.) is morphologically very similar to A. Flávio A. a total 12. Recent studies have split this taxon into six species.) and A.) is provided based on laboratory-reared specimens.). sculptum.252 A.) is currently represented by two species in Brazil.l. cajennense (s. cajennense (s. unless otherwise stated. Pablo H. Av. sculptum is the most important vector of the bacterium Rickettsia rickettsii.l. Horta10.0 International License (http://creativecommons. Hector R.) has important implications to public health. L. The current distribution of A. A total of 1. Diego G.l.0/). Amblyomma cajennense (Fabricius. sculptum. provided you give appropriate credit to the original author(s) and the source.l. A. Camargo2.1186/s13071-016-1460-2 RESEARCH Open Access Geographical distribution of Amblyomma cajennense (sensu lato) ticks (Parasitiformes: Ixodidae) in Brazil.948 A.org/publicdomain/zero/1. Mauricio C. with description of the nymph of A. cajennense (sensu stricto) (s. Ricardo A. Amália R.746 ticks from 77 localities were collected during field expeditions.

For this purpose. cajennense species complex or A. Tocantins.s. during January 2012.s. 1844 (from Texas to western Ecuador).s.l. 5]. and the “Coleção Nacional de Carrapatos” (CNC) of the University of São Paulo. 4] and molecular analyses [5].Martins et al.) and A.) in Brazil.br). V-shaped in the former and Ushaped in the latter [2]. in order to confirm their taxonomic identification. These states were considered to represent the eastern transition area of the Fig. and Goiás. one of A. In addition. Parasites & Vectors (2016) 9:186 Background Until recently. sculptum. the A. There have been four previous descriptions of nymphs of A. 1888 (northern Argentina. A.s. Brazil. cajennense (sensu stricto) and A.) (restricted to the Amazonian region). 1). cajennense (s. Brazil. Bolivia and Paraguay).l.) [6. sculptum (see Nava et al.l.) and A.) in two large tick collections of Brazil were revised. split this taxon into six valid species. Females were identified as A. [2]). cajennense (s. cajennense (s.e. namely A.): two of A.s. cajennense (s.l. cajennense (s. the taxon Amblyomma cajennense (Fabricius. based on the genital aperture morphology (Fig. cajennense (s. cajennense (s. cajennense (s. sculptum in Brazil. a specimen from Pirassununga. Indeed. 1787) was considered to represent a single tick species occurring in southern United States. 2014 (inter-Andean valley of Peru). and Amblyomma patinoi Labruna.s. 2014 (dry areas of northern Argentina. In each tick collection.l.) or A. southeastern Brazil. São Paulo. cajennense (sensu lato) (s. the only species of A. Nava & Cáceres. sculptum. a specimen from Governador Jorge Teixeira. where these two species might overlap. it is possible that their nymphs were A. all lots of A. Amblyomma interandinum Beati. included areas of the states of Maranhão. i.) and A. cajennense (s. which can be morphologically separated only by examination of the genital aperture of females [2]. However.gov. it is not known if there is any geographical overlap between A.ibge. Paraguay. a “V” shaped in Amblyomma cajennense (sensu stricto).) is provided for the first time. Caribbean and all countries of South America with the exception of Chile and Uruguay [1].s. cajennense (s. Rondônia. the description of the nymph of A. cajennense (s. mixtum. São Paulo. New material was acquired during three field expeditions in the possible transition areas between the distribution ranges of A. Bolivia. 2014 (Eastern Andes of Colombia) [2. Brazil). Central America. cajennense (s.l.) From each lot. Mexico. [7] refers to A. Nava & Beati. With this new classification. molecular analyses were performed on representative tick specimens from different regions.) and A.) known to occur in North America [2]. cajennense by Martins et al. and one of A. the known geographical distribution of these species is still incomplete. While Cooley & Kohls [6] did not mention the specific origin of the described nymphs. A recent morphological study [2]. Brazil. In addition. the geographical coordinates of the municipality was retrieved from the “Instituto Brasileiro de Geografia e Estatística” (IBGE) website (www. Amblyomma mixtum Koch. 7]. Brazil . [2]. 1 Genital aperture of adult females. Amblyomma tonelliae Nava. Beati & Labruna. Pará.l. Methods Examination of ticks deposited at tick collections The following tick collections were assessed: the “Acari Colletion of the Instituto Butantan” (IBSP). cajennense (s. a typical area for the occurrence of A. One expedition. sculptum [8]. sculptum [2]. Finally. Amblyomma sculptum Berlese. males were morphologically identified as A. sculptum. Field expeditions for collection of ticks Three field expeditions were performed in the possible transition areas of the distribution ranges of A. Page 2 of 14 The present study aimed to determine the geographical distribution of A. São Paulo. as stated by Nava et al. Due to the lack of discriminating characters to separate males of A. a morphological description of the nymphal stage of A.) is currently represented by two species in Brazil. supported by biological [3. sculptum. since the described ticks were from the state of São Paulo.) were examined morphologically under a stereomicroscope. all lots containing adults of A. tonelliae [8]. sculptum. cajennense sensu stricto (s.l. b “U” shaped in Amblyomma sculptum.

JN866905). 62°38′W).Martins et al. we used one to three specimens from each given location. Nymphal description. and (iv) remaining natural vegetation cover (forest remains). All ticks were taken to the laboratory for taxonomic identification as stated above. Foster City.8S rRNA 3′-end. a tick colony of A.) [11.2 [19] with four independent Markov chain runs for 5. JN866902. which were considered to represent the western transition area of two species in Brazil. . KA.8S and 28S flanking regions. 1772) (AF469611) and Amblyomma americanum (Linnaeus.000. and were manually adjusted using GeneDoc. the ITS2 sequences of Amblyomma aureolatum (Pallas. were killed in hot water (70–80 °C) and immediately preserved in 70 % alcohol until further processing for description.l. plus the coordinates of the ticks collected during the three field expeditions of the present study. 15–30 day-old. since species identification through morphological examination can be achieved only for females. cajennense (s. cajennense (s.mma. sculptum from Minas Gerais. and/or for collecting questing ticks on the vegetation by dragging and visual search methods [13]. interandinum from Peru (JN866900. Cleveland. based on optical microscopy. cajennense (s. and 28S rRNA 5′-end). Brazil (JN866846). which are presented in the consensus tree. and the remaining trees were used to calculate Bayesian posterior probabilities. cajennense (s. Every time A. excluding the rRNA 5. version 2. Parasites & Vectors (2016) 9:186 two species. The first 25 % of the trees represented “burn-in”.) and A.6. road-killed tapirs (Tapirus terrestris Linnaeus.) from French Guiana (JN866864) [2.01 [17]. from different locations of Brazil. cajennense (s. we used the geographical coordinates of all lots of these two species in the IBSP and CNC tick collections. were aligned with GenBank sequences for A. A.s. OH. Description of the nymph of A. JN866897). sculptum [10]. sampling a tree every 100th generation.s. Bayesian analysis was performed using MrBayes v3. as implemented in PAUP version 4.) has been reported. in areas that could represent the central transition area of the two species in Brazil. and tested by a PCR assay targeting the entire tick ITS2 rRNA gene. Finally. 5] using Clustal X [16]. a third expedition. 1758) and anteaters (Myrmecophaga tridactyla Linnaeus.gov.gov. followed Martins et al. two main hosts for A. 12]) for tick infestations. random stepwise addition starting trees (with random addition sequences) and TBR branch swapping. Tick locations were plotted on four different maps: (i) geopolitical. sculptum in Brazil. A second expedition. São Paulo. started with adult ticks collected on the vegetation at Governador Jorge Teixeira municipality (10°31′S. (iii) climate.) in Brazil [11. Thereafter.0 (ESRI). Map sources used in the present study were obtained from the “Instituto Brasileiro de Geografia e Estatística” (IBGE) website (www. and Argentina (JN866835).000 metropolis-coupled MCMC generations. included areas of the states of Rondônia and Mato Grosso.l. The three expeditions were carried out during January or February because previous studies indicated that these months correspond in Brazil to the highest abundance of the adult stage of A. and A. Olathe. cajennense (s. tonelliae from Argentina (JN866895.) [9] or A.100 bp) were purified with ExoSap (USB.s. with primers corresponding to the flanking regions (5.ibge. Each expedition consisted of driving a car through regular roads and stopping at regular distances (usually every ≈ 50 Km) for examination of horses and pigs (main domestic hosts for A. JN866896. we processed male and female specimens taken from the CNC tick collection. From the material collected in the field expeditions. model GPS V.l. 12] were occasionally examined for tick infestations. 1758).s.) was established in the laboratory. The ITS2 alignment was used to construct a phylogenetic tree using maximum parsimony.l. Unfed F1 nymphs. cajennense (s. In addition. CA.) In July 2014. Page 3 of 14 Phylogenetic analysis The ITS2 sequences generated in the present study.0b10 [18] with 500 bootstrap replicates. also during January 2012. For construction of distribution maps of A.1. and the geographic coordinates were recorded with the use of a Global Position System apparatus (GPS Garmin. A. during February 2013. Brazil. (ii) biomes. in order to have representative specimens from nearly all Brazilian states where A. JN866882). Brazil (JN866842.br) and the “Ministério do Meio Ambiente”(MMA) website (www.s. cajennense (s. patinoi from Colombia (JN866881. 1758) (AF291874) were added as outgroup. USA) with the same primers used for PCR. USA). A. as previously described [15]. cajennense (s.br). model ABI 3500 Genetic Analyser. In addition. A. state of Rondônia. we prioritised male specimens from locations where no female specimen was available. Each tick specimen was individually submitted to DNA extraction by the guanidine thiocyanate method [14]. mixtum from Costa Rica (JN866853) and Mexico (JN866886). JN866843). Distribution maps Molecular analyses For molecular analyses. included the state of Mato Grosso. USA) and sequenced in an ABI automated sequencer (Applied Biosystems/Thermo Fisher Scientific. The distribution maps were constructed with the use of ArcGIS version 10. they were collected in plastic vials containing absolute ethanol.) ticks were found. All PCR products of the expected size (≈1.

the most common hosts were Equus caballus Linnaeus. 5. Nymphs were prepared for scanning electron microscopy (SEM) following previously described techniques [20] and SEM micrographs were taken using a HITACHI TM3000 Scanning Electron Microscope. sculptum sequences from GenBank. 1758 (5 lots. cajennense (s.s. Measurements were taken from 10 specimens using the programme Image-Pro Plus 5. cajennense (s. sculptum.s.l. 5. 3).Martins et al.s. cajennense (s. sculptum (haplotypes B-H) generated in the present study have been submitted to GenBank under the accession numbers KU169881-KU169888. Panthera onca (Linnaeus. the most common hosts were H. 5. sculptum were derived from these expeditions (Fig. cajennense (s.3 %). 184 for only A.) (only male specimens) were used to construct distribution maps according to geopolitical regions (Fig. sculptum by molecular methods. whereas A.8 %) (Additional file 1: Table S1).604 females of A. 21. as they also represented distinct haplotypes in the phylogenetic tree (Fig.970 males of A.s.7 %). 443 females of A.1 for analysis of images and morphometry. cajennense (s. lupus familiaris (21 lots. 1758 (6 lots. terrestris (46 lots.) from French Guyana (JN866864). A. 10. In addition to these transition areas. and Mato Grosso and Rondônia in western Brazil. A. as they yielded seven distinct ITS2 haplotypes.) For each lot examined. and 978 males of A.): A.) (haplotype A) and A. 5) and natural vegetation cover (Fig. Phylogenetic analyses of these ITS2 sequences with corresponding sequences from GenBank formed six main clades.) with host data (total: at least 16 different host species).l. All measurements are in micrometres and are given as the range followed by the mean in parentheses. mixtum. tridactyla (26 lots. host. A.s. sculptum. 18.). which was 100 % identical to the GenBank ITS2 sequence of A. each corresponding to a species of A. At least 23 localities contained the presence of both A.s. T.054 males of A. cajennense (s.).s. 3). cajennense (s. and 36 for A. cajennense (s. M. sculptum presented polymorphism. and C. patinoi (Fig. cajennense (s. From a total of 55 lots of A. cajennense (s. cajennense (s. terrestris (10 lots. cajennense (s. From a total of 438 lots of A. A. 4).3 %). followed by Canis lupus familiaris Linnaeus.l.s. sculptum and 1. since almost all localities with sympatric occurrence of A.s. fitted to an Olympus SZX stereoscope microscope. Distribution maps A total of 58 localities with the occurrence of only A. The three field expeditions encompassed transition areas of the two species in Brazil.) from 14 different localities. sculptum from 16 localities (Table 2). cajennense (s. 5. caballus (54 lots. 1758 (3 lots. cajennense (s. sculptum with host data (total: at least 48 different host species). 4.s. cajennense (s.s. Molecular and phylogenetic analyses DNA sequences of the ITS2 gene were generated for 24 A. These were morphologically identified as 249 females of A. 4) was from an area of tropical climate.) and A. M. Therefore. A. 9.9 %).s. 12. inferred by the presence of females of both species co-infesting the same individual hosts. date.) was restricted to areas within the Amazon biome. 2). 2). cajennense (s. Ticks collected during field expeditions A total of 1. instead of . as stated in Table 2. 1758) (23 lots. 4). biomes (Fig.s. and locality data (including geographical coordinates) are listed in Additional file 1: Table S1).). tridactyla (4 lots. and 26 A. sculptum in the Caatinga biome (Fig. cajennense (s. 1758 (12 lots. This polymorphism was extended to the A. and Atlantic forest biomes.5 %). cajennense (s. 6).l.1 %). sculptum. 7.5 %). Some of the above male specimens were identified to A.746 ticks from 77 localities were collected during the three field expeditions. sculptum. a total of 545 lots of adult ticks A. 31 for both A. cajennense (s. tonelliae. sapiens (55 lots.) Detailed information on the ticks collected at Page 4 of 14 each locality are listed in Table 1. Pantanal. 1766) (47 lots. 10. Conversely. cajennense (s. These nymphs were deposited in the CNC (accession number CNC-2913). These were identified as 67 females of A. followed by Hydrochoerus hydrochaeris (Linnaeus.) sequences.3 %).) Similarly. Ethical approval This work has been approved by the Ethic Committee in the Use of Animals of the Faculty of Veterinary Medicine of the University of Sao Paulo (project number 2660/2012). 10.) or A.) and A. these transition areas correspond primarily to the states of Maranhão and Tocantins in eastern Brazil. Mato Grosso in central Brazil. Parasites & Vectors (2016) 9:186 [7]. Results Examination of ticks deposited at tick collections A total of 335 lots of adult ticks A. 12. These were identified as 245 females of A.9 %). cajennense (s.). Bos taurus Linnaeus. and Homo sapiens Linnaeus. internadinum and A.) were examined from the IBSP tick collection. Interestingly.l. There was no polymorphism among the A.s. since all 24 tick specimens shared the same haplotype.) and A.s.6 %) and E. the 26 A. sculptum in sympatry. cajennense (s. These transition areas correspond to the geographic boundaries of the Amazon and Cerrado biomes (Fig.) were examined at the CNC tick collection. the single record of A. 648 females of A. The haplotypes of A. sculptum was found in the Cerrado.). climate (Fig. sculptum and 4.2 %).8 %) and T.l.l.

48°55.657′.s. 48°20. 49°07.503′ 9 19 E. caballus S.(s. 47°27.193′ 3 E. Parasites & Vectors (2016) 9:186 Page 5 of 14 Table 1 Data for the ticks Amblyomma cajennense (sensu stricto) [A.c. 45°23.s. 48°29. 48°27. Rosalândia Tocantins 10°34. 47°42.583′.480′ 1st 1 7 E. G. 48°45. 48°35. Tocantins Tocantins 10°07. Araguaia Pará 6°06. B. 48°43. 59°47.s.436′.843′.570′. caballus Santa Inês Maranhão 3°52.058′. Number of ticks A. caballus Barrolândia Tocantins 9°44. caballus Porto Franco Maranhão 6°20. caballus Luzinópolis Tocantins 6°10. caballus Nazaré Tocantins 6°23. caballus Tocantinópolis Tocantins 6°15. 45°16.691′ 1st 2 E.873′ 4 1 E.l.061′ 1st 3 3 6 E. 48°30.974′. 48°42.766′ 1st 3 E.177′. R. 46°46.(s. W) 1 Equus caballus Cach.105′ 1st 1 10 12 E.806′ 1st 4 74 141 E. caballus Par.594′. scrofa B.947′ 1st 1 1 1 E. 48°32. Nazaré Tocantins 11°02. 48°38.666′. 48°55. caballus Santa Inês Maranhão 3°41.618′.940′.235′ 4 E. caballus Barrolândia Tocantins 9°51. 49°05. caballus Imperatriz Maranhão 5°33.427′ 1st 1 4 E. caballus S. G. 48°03.919′ 6 32 Sus scrofa Fort.681′.446′ 1st 1 1 2 Free-living Bras.) and Amblyomma cajennense (sensu lato) [A.183′. caballus Araguanã Tocantins 6°51.018′ E. 49°10. caballus S. 47°33.(s. caballus Cariri Tocantins Tocantins 11°53. caballus Talismã Tocantins 12°47.006′ 18 17 E.c.116′ 1st 3 1 E.153′ 1st 1st 2 1st 1st 5 1st 1st 8 1st 1st 1 1st 1 1st 7 4 45 E. Tocantins Tocantins 5°58.741′ 1st 1 3 6 1st 1 E.764′ 1st 1st 7 1st 2 2 E.111′. 47°27.687′.470′ 1st 2 35 27 E.592′. 48°32.798′ 1st 1 2 1 E.) A. caballus Crix. 48°37.451′ 1st 1st 5 18 44 E. 45°32.700′. Jesus Selvas Maranhão 4°31.336′.003′. Tocantins Tocantins 11°12. caballus Brej. caballus Bequimão Maranhão 2°27.Martins et al.359′.s.c. Tocantins Tocantins 8°29. caballus Guaraí Tocantins 8°58. caballus Nova Olinda Tocantins 7°47.(s. caballus Pinheiro Maranhão 2°33. caballus Santa Helena Maranhão 2°15.l. 45°24.020′ 1st 1 6 7 E.321′ 10 E.142′. ♀♀ ♀♀ 1st Host Municipality State Geographical coordinates (S. caballus S.255′ 1st 7 10 E. Nazaré Tocantins 10°59. caballus Gurupi Tocantins 11°49.493′ 1st 3 E.783′.)] collected during three field expeditions in Brazila Exp.757′ 1st 1st 9 .664′ 5 27 Myrmecophaga tridactyla Miranorte Tocantins 9°24.737′ 1st 1 1 24 E.054′ 1st 1 2 3 E. 48°34. caballus Araguatins Tocantins 5°45. 48°54.002′. 47°51. 47°52. do Piriá Pará 1°50.039′ 1 19 E.346′. 44°47.998′ 1st 2 10 E.c.801′ 6 11 E.910′.416′ 18 S. Amblyomma sculptum (A.520′.301′ 7 E.784′.888′.955′ 9 11 E. 47°24. 49°09.518′. caballus Brej. caballus Xambioá Tocantins 6°24. Tocantins Tocantins 10°54.986′ 1st 12 25 E. caballus Alvorada Tocantins 12°23.660′.) ♂♂ 1st 21 59 E. Tabocão Tocantins 9°07. 48°33. 48°52.672′ 1st 2 8 7 E.863′. 49°07.404′.278′.841′.253′ 14 16 E. caballus Araguanã Tocantins 6°35. caballus Tocantinópolis Tocantins 6°20. Araguaia Pará 5°59. caballus Porto Nacional Tocantins 10°29.032′.582′.209′.035′. caballus Figueirópolis Tocantins 12°13.575′. 48°18. caballus N. caballus Gurupi Tocantins 11°33.502′.)]. 48°29. caballus Araguaína Tocantins 7°19. 49°01.452′ A.

caballus Sinop Mato Grosso 11°52.(s.276′. Trindade Mato Grosso 15°07.607′ 1 E. 59°52.668′. Trindade Mato Grosso 14°58.490′.634′. caballus Pont. caballus Pont.487′ 3rd 8 9 E. 60°28.000′ 137 Tapirus terrestris Sinop Mato Grosso 13°45.526′.: Field expedition a 1st expedition: considered to represent sampling in the eastern transition area of A. 55°42. 59°42. 55°36. Lacerda Mato Grosso 15°20. Amblyomma sculptum (A.775′ 2 2 E. caballus V.s.580′ 3rd 12 11 E.487′ 3rd 3rd 11 3rd 68 Total 249 443 1.654′ 1 Free-living Comodoro Mato Grosso 13°05. caballus S.122′ 1st 7 18 E.735′ E.660′. 59°57.022′. Luzia do Tide 4 2 E. 59°28.708′.094′ 12 7 E. 49°06.516′.)] collected during three field expeditions in Brazila (Continued) 1st 7 5 E. Parasites & Vectors (2016) 9:186 Page 6 of 14 Table 1 Data for the ticks Amblyomma cajennense (sensu stricto) [A. caballus Porangatu Goiás 12°52.292′. caballus Conq.Martins et al.634′ 2nd 32 7 Free-living Comodoro Mato Grosso 13°56.656′. 56°13. S.109′ 2nd 5 11 E.374′. caballus Porangatu Goiás 13°15. caballus Rosário Oeste Mato Grosso 14°48. Trindade Mato Grosso 14°55.408′.393′. 56°39. B. 49°02.216′ 2nd 11 11 Free-living V. 60°59.466′.418′ E. 59°45. caballus Comodoro Mato Grosso 13°56.509′ 3 Free-living Comodoro Mato Grosso 13°00. caballus Rosário Oeste Mato Grosso 14°48. S.058′. Goiás Goiás 13°39.s. 45°57. 56°25. caballus Porangatu Goiás 12°53. 56°19.) and Amblyomma cajennense (sensu lato) [A.271′. caballus Nova Lacerda Mato Grosso 14°10.232′.056′ 2nd 1 1 Free-living Nova Lacerda Mato Grosso 14°07. 59°59. Lacerda Mato Grosso 14°53. caballus Estrela Norte Goiás 13°49. 49°08. 49°06. B. Médici Rondônia 11°09. d’Oeste Mato Grosso 14°46.209′. 60°01. 2nd expedition: considered to represent sampling in the western transition area of the two tick species in Brazil.c.905′. 59°45. B. S.774′ 2nd 4 6 E.868′ 1 E.218′ 1 7 E.754′. Ter. 55°52. caballus Jangada Mato Grosso 15°48.550′ 2nd 2nd 4 2nd 3 2nd 3 3 2nd 1 1 E.009′ 2nd 31 31 Free-living V.685′.359′ 1st 5 5 E. caballus Pimenta Bueno Rondônia 11°52.777′ E. Trindade Mato Grosso 15°00. B. S.(s.095′ Free-living Vilhena Rondônia 12°43.052′ 2nd 5 2nd 2 2nd 2 2nd 1 4 2nd 5 6 E. S.634′ 2nd 2 2nd 2nd 4 2nd 1 49 Free-living Comodoro Mato Grosso 13°19. 59°20.868′.216′. 59°51.l. caballus Diamantino Mato Grosso 14°33. caballus Lucas Rio Verde Mato Grosso 13°45.390′ 1st 6 21 E. caballus S.364′.316′ 7 E. 61°54.)].829′ 3rd 10 34 3rd 2 3rd 1 3 E. 49°01.525′ 3rd 8 3 E.309′ 1st 1st 1 Maranhão 4°04. caballus Pont.354′ 3 E.417′ Free-living Vilhena Rondônia 12°29. 56°25.277′.576′. caballus V. 59°16.276′.c. 3rd expedition: considered to represent the central transition area of the two species in Brazil .472′ 26 E. 59°34.545′. caballus Presid.560′.328′. B. cajennense (s.) and A.722′. Lacerda Mato Grosso 15°09. 49°08. 59°23. caballus Nobres Mato Grosso 14°43.412′.153′. Trindade Mato Grosso 15°05. sculptum.118′ 1st 1 4 E. caballus Sinop Mato Grosso 11°55. 55°52.376′.278′ 21 E.500′. caballus Porangatu Goiás 13°24.054 Exp. 59°42. caballus V. 59°53.s. 60°15. 59°57.

sculptum ♂ E.s.s. sculptum ♂ E.s.) ♀ Free-living Mar/2002 Vilhena Rondônia 12°44′. sculptum ♂ Sus scrofa Aug/2010 Corumbá M. 45°23′ 1 A A. Grosso do Sul 19°0′. cajennense (s. 42°34′ 2 E A. 49°6′ 1 E A. caballus Aug/2012 Campo Formoso Bahia 10°30′.l. 46°2′ 2 E. 4). sculptum was found in areas with or without natural vegetation cover (Fig. 60°8′ 3 A A. caballus Jan/2011 Itabira Minas Gerais 19°37′.s. caballus May/2012 Alvorada do Sul Paraná 22°46′. 47°46′ 1 A A. sculptum ♂ E. cajennense (s.) ♂ E. sculptum or A. 59°28′ 1 A a A. 45°24′ 2 A A. A.s. In addition. 49°11′ 2 A A. sculptum ♂ E.) ♂ Free-living Jan/2012 Comodoro Mato Grosso 13°5′. 47°19′ 2 E A. cajennense (s. 5).) ♂ E. sculptum ♂ Equus asinus Jan/2011 José de Freitas Piauí 4°45′. The distribution of A. cajennense (s. caballus Jan/2012 Santa Inês Maranhão 3°41′.) ♂ E. 40°19′ 1 E A. 5).s.s. cajennense (s. 59°47′ 2 A A.s. The same applies to A. cajennense (s. caballus Jan/2012 Pontes e Lacerda Mato Grosso 15°9′. W) No. the Pampa biome (southernmost Brazil) was the only biome where A.Martins et al. cajennense (s. 63°34′ 2 A A. sculptum ♂ E. Guaporé Rondônia 12°3′.s. 6). cajennense (s. cajennense (s. sculptum ♂ E. Capim Pará 1°40′.) ♂ E. caballus Feb/2013 Sinop Mato Grosso 11°52′.) ♂ E. cajennense (s.) ♂ E. Parasites & Vectors (2016) 9:186 Page 7 of 14 Table 2 Data for the ticks Amblyomma cajennense (sensu stricto) (s. sculptum in the Atlantic forest biome. whereas A. sculptum ♂ Free-living Jun/2011 Tocantinópolis Tocantins 6°19′. sculptum ♂ Free-living Oct/2011 Americana São Paulo 22°44′. .) was generally restricted to areas where the natural vegetation cover was absent. caballus Jun/2011 Cumari Goiás 18°15′. There were very few records of A. 51°13′ 1 E Total a 50 This tick was collected as an engorged nymph. sculptum ♀ E. 47°24′ 2 D A.l. 59°53′ 1 A A. the occurrence of A. On the other hand. cajennense (s. cajennense (s.s. cajennense (s.) ♂ E.) ♂ Bubalus bubalis Sep/2011 S. caballus Apr/2011 Confresa Mato Grosso 10°38′. sculptum ♂ E. 55°36′ 1 A A. cajennense (s.s. caballus Jan/2012 Porangatu Goiás 12°52′. caballus Jan/2012 Bequimão Maranhão 2°27′. caballus Jan/2012 Pimenta Bueno Rondônia 11°52′. caballus Jan/2012 Pinheiro Maranhão 2°33′. cajennense (s.s. Dom. 40°13′ 1 E A. sculptum ♂ E. 60°59′ 2 B.s. sculptum ♂ E. caballus Jan/2012 S. 43°13′ 2 E A. sculptum was almost restricted to areas with tropical climate (which generally coincides with Cerrado and Atlantic forest biomes) (Fig. of specimens Haplotype code A. 43°42′ 2 E A. sculptum ♂ E. Ger. cajennense (s. 47°25′ 3 E A. cajennense (s. Franc. 43°21′ 2 A A. caballus Jul/2011 Poconé Mato Grosso 16°15′.) ♂ E. sculptum ♂ E. 47°27′ 1 G A. F A.) ♀ Canis familiaris Aug/2011 Cristalândia Tocantins 10°36′.s. caballus Dec/2008 Pirassununga São Paulo 21°59′.) was absent in Brazil (Fig.s. caballus Jul/2011 Pinheiros Espírito Santo 18°24′. 51°34′ 2 A A. cajennense (s.) ♂ Equus caballus Jan/2012 Cachoeira do Piriá Pará 1°50′. 56°37′ 1 H A. caballus Oct/2011 S. Araguaia Pará 6°6′.) and Amblyomma sculptum from Brazil used for molecular and phylogenetic analyses in the present study Tick species Gender Host Date Locality Municipality State Geographical Coordinates (S. caballus Jan/2012 Chapadinha Maranhão 3°44′. and molted to the adult stage in the laboratory the dominant semiarid climate of this biome (Fig. caballus May/2011 Seropédica Rio de Janeiro 22°44′.) was almost restricted to areas with equatorial climate (which generally coincides with Amazon biome). caballus Jul/2011 Balsas Maranhão 7°31′. 44°47′ 2 A A. In the Amazon biome. 57°39′ 2 E A.) from areas with subtropical climate in southern Brazil.) ♀ E. caballus Jan/2012 Imperatriz Maranhão 5°33′.s. 48°35′ 2 A A. C A. in the Cerrado and Pantanal biomes.s. 48°9′ 1 E A.

large and deep punctations evenly distributed. Numbers in brackets are GenBank accession numbers Description of the nymph of A.156). without auriculae. ITS2 sequences for Amblyomma americanum and A.265 (1.146–1. (Fig.280–1. 192–257 (218) wide. Hypostome rounded apically.s. sculptum. . article I with vestigial ventral prolongation.] Idiosoma. Eyes not orbited at lateral scutal angles at level of scutal midlength.040–1. 256–317 (276) long.444). without cornua. Each genotype is labelled as: Municipality name. Cervical grooves deep in scutal anterior third. as shown in Table 2. aureolatum were used as the outgroup.214). Coxa I with 2 pointed spurs separated by space equal or greater than breadth of external spur base. followed by rugose shallow depression in scutal median third.Martins et al. 7). article III 86–116 (98) long.579 (1. Numbers at nodes are support values derived from bootstrap (1. cajennense (sensu stricto). Length from apices of scapula to posterior body margin: 1. Scutum 681–784 (730) long. Trochanters without spur. Palpi length 280–345 (307). cajennense (s. dentition 2/2 with 7–8 teeth per row.000 replicates for MP/posterior probabilities for Bayesian inference analysis). Basis capituli rectangular. with an evident dorsal prolongation. external spur about 2× longer than internal. 835–944 (887) wide. and 26 genotypes of A.) [Based on 10 specimens. indicated in blue.257 (1. 291–364 (316) long. number of the tick specimen. Spiracular plate triangular with rounded angles. breadth/length ratio 1. article II 165–208 (182) long. coxae II–IV with small triangular spur. and haplotype code. inornate. including 24 genotypes of A. Length from palpal apices to dorsal posterior margin 373–473 (414). breadth 317–353 (331). posterior margin slightly concave. Gnathosoma. maximum breadth 1. Legs. posterior margin convex ventrally. length of toothed portion 164–204 (178). with 11 festoons without tubercles. 2 Bayesian inference phylogenetic tree with Maximum parsimony (MP) support values of the ITS2 rDNA sequences generated in the present study for the ticks Amblyomma cajennense (sensu lato). indicated in red. Parasites & Vectors (2016) 9:186 Page 8 of 14 Fig. outline oval.

Remark: The nymph of A. Discussion In the present study.) vs > 1. On the other hand.3 in A.s.) and A.) occurs in Brazil. This geographic distribution could be related to particular abiotic conditions required by A. If any other species of A. it might have a much more restricted distribution.s.l.s. examination of all lots of A. 72–99 (86) wide.. sculptum].s.) . the first expedition in green. cajennense (s.3 in A. cajennense (s. 6. cajennense (s. sculptum.l. cajennense (s. the second in brown and the third in orange tarsus I 431–509 (450) long.Martins et al. This result was corroborated by molecular analyses inferred from the ITS2 ribosomal gene. it is noteworthy that an extensive study in the state of Rondônia (Brazilian western Amazon) made multiple samplings throughout the state.). Amblyomma cajennense (s. As previously reported [2. The above statements are corroborated by our results shown in Fig. tonelliae by using the same criteria recently proposed to separate A.) occurs generally in the Amazon biome. sculptum and A. tonelliae from A. cajennense (s. cajennense (s.e. cajennense was generally absent from areas with dense Amazon forest [21]. i. We chose this molecular marker because a recent study revealed that the ITS2 gene fragment is suitable for evolutionary studies on A. A. plus collection of new material during three field expeditions. however. suggesting that this tick might not be adapted to extremely humid rainforests.s. cajennense (s. recently described [7].) is morphologically very similar to the nymph of A. cajennense (s. almost all records were from the edge of this biome. Dashed line ellipses include the localities that were sampled during three field expeditions for tick collections. 3 Geopolitical map of Brazil. i. A. showing the localities of Amblyomma cajennense (sensu stricto). Parasites & Vectors (2016) 9:186 Page 9 of 14 Fig. cajennense (s.) are within areas with degraded forest cover. Comparing the two descriptions. this species was not found at any area where natural vegetation cover remains in the Amazon biome. this tick is not found in thick of the rain forest [5]. which generally means the inner part of the forest. tarsus IV 320–372 (337) long.l. the absence of A.s. 99–115 (109) wide. 5]. among the so called “Seasonally Dry Tropical Forest” (SDTF) part of the Amazon biome. cajennense (sensu lato) identified in the present study. and concluded that A. sculptum. cajennense [5]. based on scutum surface and the length of the anterolateral seta on coxa I [8]. cajennense (s. since our analyses encompassed areas throughout the Brazilian land. our study confirms that A. where all records of A.s.s. confirmed that A.e.) from two large tick collections of Brazil.) is currently represented in Brazil by only two species. the only concrete difference is the scutal width/length ratio [< 1.) can be separated from A. While the absence of records from areas deeper inside the Amazon biome could be related to poor sampling in these less accessible areas. sculptum.

sculptum and A. 5). i. namely. sculptum (reported as A. Caatinga. the same cannot be applied to the Caatinga biome. where suitable hosts (e. 4). on the other hand. 3) is possibly the main limiting factor.g. Interestingly. This distribution coincides with the tropical climate. Interestingly. 22]. sculptum. which retains < 10 % of its original natural vegetation cover [24]. In fact. 2) coincides with the subtropical climate (Fig. sculptum was abundant in an open. the Atlantic forest biome is indeed the most degraded Brazilian biome.Martins et al. our single record of A. Atlantic forest. A. This assumption is corroborated by previous modelling studies [1. 4 Map of Brazil showing the localities of the ticks Amblyomma cajennense (sensu stricto). sculptum in the Atlantic forest biome were from areas where the natural vegetation cover has been degraded (Fig. the absence of A. where the semiarid climate (Figs. sculptum seems to be very adapted to the Cerrado biome. Pampa) that compose the Brazilian land deeper in the Cerrado biome (Brazilian savannah) could be related to unsuitable abiotic conditions at the opposite extreme. While A. the much drier seasonally drought period. The authors suggested that the distribution of A. this tick was not found at any area where natural vegetation cover remains in the Atlantic forest biome. sculptum has increased as a result of destruction of the Atlantic rainforest over the last few centuries. according to the six major biomes (Amazon. 2. sculptum in Brazil generally encompassed entire Cerrado and Pantanal biomes and great part of the Atlantic forest biome (Fig. sculptum in the Caatinga biome was from the state of Bahia (Figs. where A. Parasites & Vectors (2016) 9:186 Page 10 of 14 Fig.e. sculptum in southern Brazil (Fig. cajennense) was never found inside the dense forest. typical for the tropical climate that prevails in the Cerrado (Figs. 2 and 5. suggesting that the cooler temperatures during autumn/ winter in this region might be the limiting factor for the establishment of A. 2. 3). 3). Cerrado. 4). Pantanal. cajennense (sensu lato) identified in the present study. This condition can be clearly observed in Figs. It seems likely that abiotic factors of these degraded areas of the Atlantic forest . in an area that represents a narrow “invagination” of the tropical climate into the Caatinga (Fig. tapirs) were abundant. 4). degraded area within the Reserve [23]. 3. A. This result is corroborated by an extensive field study in a large Atlantic forest Reserve in Southern São Paulo. The distribution of A. It is noteworthy that nearly all records of A.

Because Rondônia is one of the Brazilian states with the highest deforestation and forest degradation indices during the last two decades [25]. we were able to find three transition areas of the distribution of A. this condition might have facilitated the expansion of A. sculptum during a large. This scenario has extreme medical relevance. Nearly one decade later. cajennense (s. A potential example of this condition is the state of Rondônia. Continuing degradation of the Amazon forest.Martins et al. 2). where these tick species were sympatric (Fig. with replacement of the original forest cover by Cerrado-like vegetation cover may favour the expansion of A. sculptum into the state. Tropical. sculptum infesting horses in Pimenta Bueno municipality (corresponds to the single record of A. 2). cajennense (s. or between the equatorial and tropical climate.s. sculptum in Rondônia in Fig. sculptum is . where Labruna et al. where A.l.s. sculptum and A. as shown in Fig. 5. sculptum prevails on either natural or degraded areas. 5 Map of Brazil showing the localities of the ticks Amblyomma cajennense (sensu stricto).) and A. Because these transition areas corresponded to the boundaries between the Amazon and Cerrado biome. This index is an indirect quantification of abiotic factors. and Subtropical) that occur in the Brazilian land “simulate” the conditions of the Cerrado biome. These results are in agreement with a recent environmental modelling study suggesting that A. extensive tick survey throughout the state during 2000–2005. Parasites & Vectors (2016) 9:186 Page 11 of 14 Fig. This statement is corroborated by a previous study with the use of satellite imagery and the distribution of A. which showed that both the Cerrado and the degraded Atlantic forest areas of Brazil presented similar normalised derived vegetation index (NVDI). In the present study. A. sculptum in Brazil. we were able to find an established population of A. according to the four major climates (Equatorial.) [1].) and A. we can infer that these transition areas represent extreme abiotic conditions for the distribution of the two species. cajennense (s. [21] did not find any A. in 2012 (during our second field expedition). since in Brazil A. sculptum into these areas. Semi-arid. cajennense (sensu lato) identified in the present study. sculptum may overlap in parts of their range due to some similarities in abiotic variables [22].

sculptum. Scale-bars: a. 60 μm. a Dorsal gnathosoma (capitulum). d. and A. c Scutum. cajennense (sensu lato) identified in the present study.Martins et al. according to natural vegetation cover. 100 μm. Parasites & Vectors (2016) 9:186 Page 12 of 14 Fig. b. 6 Map of Brazil showing the localities of the ticks Amblyomma cajennense (sensu stricto). d Coxae I–IV. 200 μm . b Ventral gnathosoma. represented in green Fig. 7 Amblyomma cajennense (sensu stricto) nymph. A. c.

) and A. Rondônia 78916-450. 2014. 1936. Brazil. Mastropaolo M. 8Curso de Medicina Veterinária. Brazil. Hence. Nava S. s/n. Beati L. sp. Universidade Federal da Integração Latino-Americana. Labruna MB. Mem Inst Oswaldo Cruz. Enéas de Siqueira Neto 340. RAD and AGAG constructed the distribution maps. Km 12. R. sp. Ann Trop Med Parasitol. (PDF 1091 kb) Competing interests The authors declare that they have no competing interests. Reassessment of the taxonomic status of Amblyomma cajennense (Fabricius. RCP. Cabrera RR. Rickettsial infection in animals and Brazilian spotted fever endemicity. Guglielmone AA. sculptum is indeed the most common human-biting tick in Brazil [28]. Parasites & Vectors (2016) 9:186 Page 13 of 14 the most important vector of the bacterium Rickettsia rickettsii. sculptum. Brazil.) that were examined in the tick collections “Acari Colletion of the Instituto Butantan” (IBSP). 8. Universidade de São Paulo. Brazil. Emerg Infect Dis. Fernando Corrêa da Costa 2367. and revised the manuscript. LMAC. 2001. R. Cuiabá. Taxonomic key to nymphs of the genus Amblyomma (Acari: Ixodidae) in Argentina. Exp Appl Acarol. Cooley RA. FBC. Nava S. 2009. 1944. Minas Gerais 36570-000.) has only been found infected by Rickettsia amblyommii. Av. Onofrio VC. Brazil. Faculdade de Agronomia. BMC Evol Biol. Szabó MP. Kasai N. Ferreira F. Av. Bloco A.30:77–111. especially because A. Labruna MB. Brazil. 10Universidade Federal do Vale do São Francisco. DMB-B. rickettsii. São Paulo 05503-900. and MBL conducted the field studies. Conclusions Received: 11 January 2016 Accepted: 15 March 2016 Results of the present study confirmed that A. 2011. 1374. 7Universidade de Santo Amaro. 1787) with the description of three new species. sapiens was the most frequent host species for A. Labruna MB. Schumaker TTS. Av. the most lethal spotted fever of the world [26]. CRLP. Universidade Federal de Viçosa. Mangold AJ.Projeto de Irrigação Senador Nilo Coelho. 2002. since in Brazil A. Horta MC. a much less or nonpathogenic rickettsial agent [27]. 2011. Porto Velho. s/n. Barros-Battesti DM. TFM. cajennense (s. with description and redescription of the nymphal stage of four Amblyomma species. et al.5:753–70. Barros-Battesti D. Soares HS.95:1016–8.98:283–92. Brazil. Universidade de São Paulo. AM. . Labruna MB. Brazil. Cafrune MM. the etiological agent of the Brazilian spotted fever.1:194–6. Terassini FA. The current distribution of A. On the other hand. Project 141945/2010-9. A.5: 252–76. Guglielmone AA. The genus Amblyomma (Ixodidae) in the United States.97:1–14. 1888 (Ixodida: Ixodidae).l. Ixodidas brasileiros e de alguns paizes limitrophes. Setor Comercial Sul. Project 2014/14464-1). the etiological agent of the most lethal spotted fever of the world. Guglielmone AA. Beati L. Brazil. Petrolina. 11. 2014. sculptum is the most important vector of the bacterium R. Risk factors to tick infestations and their occurrence on horses in the state of São Paulo Brazil. Labruna MB. Vital Brasil 1500. Vet Parasitol. Rodovia BR 407. cajennense (s. Camargo LM. Gennari SM. São Paulo. 9Deparatamento de Veterinária. 1787) (Acari: Ixodidae). sp.s. Faculdade de Medicina Veterinária. Ferreira F. cajennense (s. Vet Parasitol. and MBL performed the molecular and phylogenetic analyses. Alexandre Guimarães Areal 1927. AS. 6. Vianna MCB. Brazil.1:75–99. Brasília. Mangold AJ. Gennari SM. 5Departamento de Ciências Básicas e Produção Animal. 2010. Soares RM. ARMB. Guglielmone AA. 4Coordenação Geral do Programa Nacional de Controle da Malária. J Parasitol. FAT. 9. Mato Grosso 78060-900. Labruna MB. Vidotto O. the Brazilian spotted fever. Notes on population dynamics of Amblyomma ticks (Acari: Ixodidae) in Brazil. Galvão MAM. PHN and MBL described the nymph.). Quadra 04. Mangold AJ. 13. and Amblyomma cajennense sensu lato (s. Foz do Iguaçu. Cáceres AG. expansion of A. 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