Geographical distribution of Amblyomma cajennense (sensu lato) ticks (Parasitiformes: Ixodidae) in Brazil, with description of the nymph of A. cajennense (sensu stricto)

Background Until recently, Amblyomma cajennense (Fabricius, 1787) was considered to represent a single tick species in the New World. Recent studies have split this taxon into six species. While the A. cajennense species complex or A. cajennense (sensu lato) (s.l.) is currently represented by two species in Brazil, A. cajennense (sensu stricto) (s.s.) and Amblyomma sculptum Berlese, 1888, their geographical distribution is poorly known. Methods The distribution of the A. cajennense (s.l.) in Brazil was determined by morphological examination of all lots of A. cajennense (s.l.) in two large tick collections of Brazil, and by collecting new material during three field expeditions in the possible transition areas between the distribution ranges of A. cajennense (s.s.) and A. sculptum. Phylogenetic analysis inferred from the ITS2 rRNA gene was used to validate morphological results. Morphological description of the nymphal stage of A. cajennense (s.s.) is provided based on laboratory-reared specimens. Results From the tick collections, a total 12,512 adult ticks were examined and identified as 312 A. cajennense (s.s.), 6,252 A. sculptum and 5,948 A. cajennense (s.l.). A total of 1,746 ticks from 77 localities were collected during field expeditions, and were identified as 249 A. cajennense (s.s.), 443 A. sculptum, and 1,054 A. cajennense (s.l.) [these A. cajennense (s.l.) ticks were considered to be males of either A. cajennense (s.s.) or A. sculptum]. At least 23 localities contained the presence of both A. cajennense (s.s.) and A. sculptum in sympatry. DNA sequences of the ITS2 gene of 50 ticks from 30 localities confirmed the results of the morphological analyses. The nymph of A. cajennense (s.s.) is morphologically very similar to A. sculptum. Conclusion Our results confirmed that A. cajennense (s.l.) is currently represented in Brazil by only two species, A. cajennense (s.s.) and A. sculptum. While these species have distinct distribution areas in the country, they are found in sympatry in some transition areas. The current distribution of A. cajennense (s.l.) has important implications to public health, since in Brazil A. sculptum is the most important vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1460-2) contains supplementary material, which is available to authorized users.


Background
Until recently, the taxon Amblyomma cajennense (Fabricius, 1787) was considered to represent a single tick species occurring in southern United States, Mexico, Central America, Caribbean and all countries of South America with the exception of Chile and Uruguay [1]. A recent morphological study [2], supported by biological [3,4] and molecular analyses [5], split this taxon into six valid species, namely A. cajennense sensu stricto (s.s.) ( [2,5]. With this new classification, the A. cajennense species complex or A. cajennense (sensu lato) (s.l.) is currently represented by two species in Brazil, A. cajennense (sensu stricto) and A. sculptum, which can be morphologically separated only by examination of the genital aperture of females [2]. However, as stated by Nava et al. [2], the known geographical distribution of these species is still incomplete. In addition, it is not known if there is any geographical overlap between A. cajennense (s.s.) and A. sculptum.
There have been four previous descriptions of nymphs of A. cajennense (s.l.): two of A. cajennense (s.l.) [6,7], one of A. sculptum [8], and one of A. tonelliae [8]. Indeed, the description of the nymph of A. cajennense by Martins et al. [7] refers to A. sculptum, since the described ticks were from the state of São Paulo, southeastern Brazil, a typical area for the occurrence of A. sculptum [2]. While Cooley & Kohls [6] did not mention the specific origin of the described nymphs, it is possible that their nymphs were A. mixtum, the only species of A. cajennense (s.l.) known to occur in North America [2].
The present study aimed to determine the geographical distribution of A. cajennense (s.l.) in Brazil. For this purpose, all lots of A. cajennense (s.l.) in two large tick collections of Brazil were revised. New material was acquired during three field expeditions in the possible transition areas between the distribution ranges of A. cajennense (s.s.) and A. sculptum, where these two species might overlap. In addition, molecular analyses were performed on representative tick specimens from different regions, in order to confirm their taxonomic identification. Finally, a morphological description of the nymphal stage of A. cajennense (s.s.) is provided for the first time.

Examination of ticks deposited at tick collections
The following tick collections were assessed: the "Acari Colletion of the Instituto Butantan" (IBSP), São Paulo, Brazil; and the "Coleção Nacional de Carrapatos" (CNC) of the University of São Paulo, São Paulo, Brazil. In each tick collection, all lots containing adults of A. cajennense (s.l.) were examined morphologically under a stereomicroscope. Females were identified as A. cajennense (s.s.) or A. sculptum, based on the genital aperture morphology (Fig. 1), i.e. V-shaped in the former and Ushaped in the latter [2]. Due to the lack of discriminating characters to separate males of A. cajennense (s.s.) and A. sculptum (see Nava et al. [2]), males were morphologically identified as A. cajennense (s.l.) From each lot, the geographical coordinates of the municipality was retrieved from the "Instituto Brasileiro de Geografia e Estatística" (IBGE) website (www.ibge.gov.br).

Field expeditions for collection of ticks
Three field expeditions were performed in the possible transition areas of the distribution ranges of A. cajennense (s.s.) and A. sculptum in Brazil. One expedition, during January 2012, included areas of the states of Maranhão, Pará, Tocantins, and Goiás. These states were considered to represent the eastern transition area of the Fig. 1 Genital aperture of adult females. a "V" shaped in Amblyomma cajennense (sensu stricto), a specimen from Governador Jorge Teixeira, Rondônia, Brazil; b "U" shaped in Amblyomma sculptum, a specimen from Pirassununga, São Paulo, Brazil two species. A second expedition, also during January 2012, included areas of the states of Rondônia and Mato Grosso, which were considered to represent the western transition area of two species in Brazil. Finally, a third expedition, during February 2013, included the state of Mato Grosso, in areas that could represent the central transition area of the two species in Brazil. 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. cajennense (s.s.) [9] or A. sculptum [10]. 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. cajennense (s.l.) in Brazil [11,12]) for tick infestations, and/or for collecting questing ticks on the vegetation by dragging and visual search methods [13]. In addition, road-killed tapirs (Tapirus terrestris Linnaeus, 1758) and anteaters (Myrmecophaga tridactyla Linnaeus, 1758), two main hosts for A. cajennense (s.l.) [11,12] were occasionally examined for tick infestations. Every time A. cajennense (s.l.) ticks were found, they were collected in plastic vials containing absolute ethanol, and the geographic coordinates were recorded with the use of a Global Position System apparatus (GPS Garmin, model GPS V, Olathe, KA, USA). All ticks were taken to the laboratory for taxonomic identification as stated above.

Molecular analyses
For molecular analyses, we used one to three specimens from each given location. From the material collected in the field expeditions, we prioritised male specimens from locations where no female specimen was available, since species identification through morphological examination can be achieved only for females. In addition, we processed male and female specimens taken from the CNC tick collection, from different locations of Brazil, in order to have representative specimens from nearly all Brazilian states where A. cajennense (s.l.) has been reported.
Each tick specimen was individually submitted to DNA extraction by the guanidine thiocyanate method [14], and tested by a PCR assay targeting the entire tick ITS2 rRNA gene, with primers corresponding to the flanking regions (5.8S rRNA 3′-end, and 28S rRNA 5′-end), as previously described [15]. All PCR products of the expected size (≈1,100 bp) were purified with ExoSap (USB, Cleveland, OH, USA) and sequenced in an ABI automated sequencer (Applied Biosystems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyser, Foster City, CA, USA) with the same primers used for PCR.
The ITS2 alignment was used to construct a phylogenetic tree using maximum parsimony, as implemented in PAUP version 4.0b10 [18] with 500 bootstrap replicates, random stepwise addition starting trees (with random addition sequences) and TBR branch swapping. Bayesian analysis was performed using MrBayes v3.1.2 [19] with four independent Markov chain runs for 5,000,000 metropolis-coupled MCMC generations, sampling a tree every 100 th generation. The first 25 % of the trees represented "burn-in", and the remaining trees were used to calculate Bayesian posterior probabilities, which are presented in the consensus tree.

Distribution maps
For construction of distribution maps of A. cajennense (s.s.) and A. sculptum in Brazil, we used the geographical coordinates of all lots of these two species in the IBSP and CNC tick collections, plus the coordinates of the ticks collected during the three field expeditions of the present study. Tick locations were plotted on four different maps: (i) geopolitical, (ii) biomes, (iii) climate, and (iv) remaining natural vegetation cover (forest remains). Map sources used in the present study were obtained from the "Instituto Brasileiro de Geografia e Estatística" (IBGE) website (www.ibge.gov.br) and the "Ministério do Meio Ambiente"(MMA) website (www.mma.gov.br). The distribution maps were constructed with the use of Arc-GIS version 10.0 (ESRI).
Description of the nymph of A. cajennense (s.s.) In July 2014, a tick colony of A. cajennense (s.s.) was established in the laboratory, started with adult ticks collected on the vegetation at Governador Jorge Teixeira municipality (10°31′S, 62°38′W), state of Rondônia, Brazil. Unfed F 1 nymphs, 15-30 day-old, were killed in hot water (70-80°C) and immediately preserved in 70 % alcohol until further processing for description. Nymphal description, based on optical microscopy, followed Martins et al. [7]. Measurements were taken from 10 specimens using the programme Image-Pro Plus 5.1 for analysis of images and morphometry, fitted to an Olympus SZX stereoscope microscope. All measurements are in micrometres and are given as the range followed by the mean in parentheses. These nymphs were deposited in the CNC (accession number CNC-2913). 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.

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).  Table S1).

Ticks collected during field expeditions
A total of 1,746 ticks from 77 localities were collected during the three field expeditions. These were morphologically identified as 249 females of A. cajennense (s.s.), 443 females of A. sculptum and 1,054 males of A. cajennense (s.l.) Detailed information on the ticks collected at each locality are listed in Table 1. Some of the above male specimens were identified to A. cajennense (s.s.) or A. sculptum by molecular methods, as stated in Table 2. At least 23 localities contained the presence of both A. cajennense (s.s.) and A. sculptum in sympatry, inferred by the presence of females of both species co-infesting the same individual hosts.

Molecular and phylogenetic analyses
DNA sequences of the ITS2 gene were generated for 24 A. cajennense (s.s.) from 14 different localities, and 26 A. sculptum from 16 localities ( Table 2). Phylogenetic analyses of these ITS2 sequences with corresponding sequences from GenBank formed six main clades, each corresponding to a species of A. cajennense (s.l.): A. cajennense (s.s.), A. sculptum, A. mixtum, A. tonelliae, A. internadinum and A. patinoi (Fig. 2). There was no polymorphism among the A. cajennense (s.s.) sequences, since all 24 tick specimens shared the same haplotype, which was 100 % identical to the GenBank ITS2 sequence of A. cajennense (s.s.) from French Guyana (JN866864). Conversely, the 26 A. sculptum presented polymorphism, as they yielded seven distinct ITS2 haplotypes. This polymorphism was extended to the A. sculptum sequences from GenBank, as they also represented distinct haplotypes in the phylogenetic tree (Fig. 2). The haplotypes of A. cajennense (s.s.) (haplotype A) and A. sculptum (haplotypes B-H) generated in the present study have been submitted to GenBank under the accession numbers KU169881-KU169888.

Distribution maps
A total of 58 localities with the occurrence of only A. cajennense (s.s.), 184 for only A. sculptum, 31 for both A. cajennense (s.s.) and A. sculptum, and 36 for A. cajennense (s.l.) (only male specimens) were used to construct distribution maps according to geopolitical regions (Fig. 3), biomes (Fig. 4), climate (Fig. 5) and natural vegetation cover (Fig. 6). The three field expeditions encompassed transition areas of the two species in Brazil, since almost all localities with sympatric occurrence of A. cajennense (s.s.) and A. sculptum were derived from these expeditions (Fig. 3). Therefore, these transition areas correspond primarily to the states of Maranhão and Tocantins in eastern Brazil, Mato Grosso in central Brazil, and Mato Grosso and Rondônia in western Brazil. These transition areas correspond to the geographic boundaries of the Amazon and Cerrado biomes (Fig. 4). In addition to these transition areas, A. cajennense (s.s.) was restricted to areas within the Amazon biome, whereas A. sculptum was found in the Cerrado, Pantanal, and Atlantic forest biomes. Interestingly, the single record of A. sculptum in the Caatinga biome ( Fig. 4) was from an area of tropical climate, instead of the dominant semiarid climate of this biome (Fig. 5).
The distribution of A. cajennense (s.s.) was almost restricted to areas with equatorial climate (which generally coincides with Amazon biome), whereas A. sculptum was almost restricted to areas with tropical climate (which generally coincides with Cerrado and Atlantic forest biomes) (Fig. 5). There were very few records of A. sculptum or A. cajennense (s.l.) from areas with subtropical climate in southern Brazil. In addition, the Pampa biome (southernmost Brazil) was the only biome where A. cajennense (s.l.) was absent in Brazil (Fig. 4). In the Amazon biome, the occurrence of A. cajennense (s.s.) was generally restricted to areas where the natural vegetation cover was absent. The same applies to A. sculptum in the Atlantic forest biome. On the other hand, in the Cerrado and Pantanal biomes, A. sculptum was found in areas with or without natural vegetation cover (Fig. 6). [Based on 10 specimens; (Fig. 7).] Idiosoma. Length from apices of scapula to posterior body margin: Legs. Coxa I with 2 pointed spurs separated by space equal or greater than breadth of external spur base; external spur about 2× longer than internal; coxae II-IV with small triangular spur. Trochanters without spur; Remark: The nymph of A. cajennense (s.s.) is morphologically very similar to the nymph of A. sculptum, recently described [7]. Comparing the two descriptions, the only concrete difference is the scutal width/length ratio [< 1.3 in A. cajennense (s.s.) vs > 1.3 in A. sculptum]. Amblyomma cajennense (s.s.) can be separated from A. tonelliae by using the same criteria recently proposed to separate A. tonelliae from A. sculptum, based on scutum surface and the length of the anterolateral seta on coxa I [8].

Discussion
In the present study, examination of all lots of A. cajennense (s.l.) from two large tick collections of Brazil, plus collection of new material during three field expeditions, confirmed that A. cajennense (s.l.) is currently represented in Brazil by only two species, A. cajennense (s.s.) and A. sculptum. This result was corroborated by molecular analyses inferred from the ITS2 ribosomal gene. We chose this molecular marker because a recent study revealed that the ITS2 gene fragment is suitable for evolutionary studies on A. cajennense [5]. If any other species of A. cajennense (s.l.) occurs in Brazil, it might have a much more restricted distribution, since our analyses encompassed areas throughout the Brazilian land.
As previously reported [2,5], our study confirms that A. cajennense (s.s.) occurs generally in the Amazon biome; however, almost all records were from the edge of this biome, among the so called "Seasonally Dry Tropical Forest" (SDTF) part of the Amazon biome; i.e., this tick is not found in thick of the rain forest [5], which generally means the inner part of the forest. While the absence of records from areas deeper inside the Amazon biome could be related to poor sampling in these less accessible areas, it is noteworthy that an extensive study in the state of Rondônia (Brazilian western Amazon) made multiple samplings throughout the state, and concluded that A. cajennense was generally absent from areas with dense Amazon forest [21]. The above statements are corroborated by our results shown in Fig. 6, where all records of A. cajennense (s.s.) are within areas with degraded forest cover, i.e. this species was not found at any area where natural vegetation cover remains in the Amazon biome. This geographic distribution could be related to particular abiotic conditions required by A. cajennense (s.s.), suggesting that this tick might not be adapted to extremely humid rainforests. On the other hand, the absence of A. cajennense (s.s.) identified in the present study. Dashed line ellipses include the localities that were sampled during three field expeditions for tick collections, the first expedition in green, the second in brown and the third in orange deeper in the Cerrado biome (Brazilian savannah) could be related to unsuitable abiotic conditions at the opposite extreme, namely, the much drier seasonally drought period, typical for the tropical climate that prevails in the Cerrado (Figs. 3, 4).
The distribution of A. sculptum in Brazil generally encompassed entire Cerrado and Pantanal biomes and great part of the Atlantic forest biome (Fig. 3). This distribution coincides with the tropical climate. Interestingly, the absence of A. sculptum in southern Brazil (Fig. 2) coincides with the subtropical climate (Fig. 4), suggesting that the cooler temperatures during autumn/ winter in this region might be the limiting factor for the establishment of A. sculptum. This assumption is corroborated by previous modelling studies [1,22]. While A. sculptum seems to be very adapted to the Cerrado biome, the same cannot be applied to the Caatinga biome, where the semiarid climate (Figs. 2, 3) is possibly the main limiting factor. Interestingly, our single record of A. sculptum in the Caatinga biome was from the state of Bahia (Figs. 2, 3), in an area that represents a narrow "invagination" of the tropical climate into the Caatinga (Fig. 4).
It is noteworthy that nearly all records of A. sculptum in the Atlantic forest biome were from areas where the natural vegetation cover has been degraded (Fig. 5), i.e. this tick was not found at any area where natural vegetation cover remains in the Atlantic forest biome. This result is corroborated by an extensive field study in a large Atlantic forest Reserve in Southern São Paulo, where A. sculptum (reported as A. cajennense) was never found inside the dense forest, where suitable hosts (e.g. tapirs) were abundant; on the other hand, A. sculptum was abundant in an open, degraded area within the Reserve [23]. The authors suggested that the distribution of A. sculptum has increased as a result of destruction of the Atlantic rainforest over the last few centuries. In fact, the Atlantic forest biome is indeed the most degraded Brazilian biome, which retains < 10 % of its original natural vegetation cover [24]. This condition can be clearly observed in Figs. 2 and 5. It seems likely that abiotic factors of these degraded areas of the Atlantic forest "simulate" the conditions of the Cerrado biome, where A. sculptum prevails on either natural or degraded areas, as shown in Fig. 5. This statement is corroborated by a previous study with the use of satellite imagery and the distribution of A. cajennense (s.l.) [1], which showed that both the Cerrado and the degraded Atlantic forest areas of Brazil presented similar normalised derived vegetation index (NVDI). This index is an indirect quantification of abiotic factors.
In the present study, we were able to find three transition areas of the distribution of A. cajennense (s.s.) and A. sculptum in Brazil, where these tick species were sympatric (Fig. 2). These results are in agreement with a recent environmental modelling study suggesting that A. cajennense (s.s.) and A. sculptum may overlap in parts of their range due to some similarities in abiotic variables [22]. Because these transition areas corresponded to the boundaries between the Amazon and Cerrado biome, or between the equatorial and tropical climate, we can infer that these transition areas represent extreme abiotic conditions for the distribution of the two species.
Continuing degradation of the Amazon forest, with replacement of the original forest cover by Cerrado-like vegetation cover may favour the expansion of A. sculptum into these areas. A potential example of this condition is the state of Rondônia, where Labruna et al. [21] did not find any A. sculptum during a large, extensive tick survey throughout the state during 2000-2005. Nearly one decade later, in 2012 (during our second field expedition), we were able to find an established population of A. sculptum infesting horses in Pimenta Bueno municipality (corresponds to the single record of A. sculptum in Rondônia in Fig. 2). Because Rondônia is one of the Brazilian states with the highest deforestation and forest degradation indices during the last two decades [25], this condition might have facilitated the expansion of A. sculptum into the state. This scenario has extreme medical relevance, since in Brazil A. sculptum is