The tick species that comprise the Amblyomma maculatum group (Acari: Ixodidae) are widely distributed in the Nearctic and Neotropical regions, ranging from the United States of America (USA) to Argentina. This group includes three species: Amblyomma maculatum, Amblyomma tigrinum, and Amblyomma triste, which parasitize a high number of vertebrates, including domestic mammals and humans, where they are the main vectors of Rickettsia parkeri s.s. The identification, distribution, and validity of the three species within the group have been controversial and continue to raise questions. Previous studies have mostly focused on the analysis of partial nuclear and mitochondrial genes, highlighting the need for new integrative analyses that contribute to clarifying their systematics and ecology. In this investigation, we obtained the first mitochondrial genome of A. triste (14,808 bp), which, upon comparison with the reported mitochondrial genomes of A. maculatum, exhibits a genetic distance of 4.2%, providing new evidence for the validity of the former species. The order, composition, and structure of the mitogenome of A. triste are consistent with the characteristics reported for most Metastriata including taxa within Amblyomma, that count with several mitochondrial genomes published in the last few years. The attributes of the mitogenomes, such as the absence of paralogs and a small genome, confirm the utility in studies involving complexes of cryptic species as it also serves as a robust tool for inferring phylogenies. Furthermore, we reported new molecular markers at the mitochondrial level which can be used in phylogenetic studies for other tick species, especially those with controversial or challenging taxonomy. We highlight the need of sequencing the mitochondrial genome of A. tigrinum, which is part of the A. maculatum group, as well as experimental crosses of populations from different places in the Americas to obtain additional evidence for species recognition.
Introduction
Ticks (Acari: Ixodida) are obligate hematophagous ectoparasites capable of transmitting multiple pathogens such as bacteria, helminths, protozoa, and viruses (de la Fuente et al. 2008; Baneth 2014; Bezerra-Santos et al. 2022). Ixodida comprises three families: Argasidae (soft ticks), Ixodidae (hard ticks), and Nuttalliellidae (Nava et al. 2017; Guglielmone et al. 2020; 2021; 2023). Currently, Ixodidae is the most diverse family with approximately 759 species and 15 genera, where Amblyomma is the third largest in terms of species number (136), surpassed by Ixodes and Haemaphysalis with 266 and 176 species, respectively (Guglielmone et al. 2023). Amblyomma species have received special attention due to their involvement in pathogen transmission (e.g., Anaplasma, Ehrlichia, Hepatozoon, Rickettsia) and by the presence of cryptic species (complexes or groups) in America, such as the Amblyomma cajennense, Amblyomma maculatum, and Amblyomma ovale (Dumler et al. 2001; Demoner et al. 2013; Rivera-Páez et al. 2017; Suwanbongkot et al. 2019), and the Amblyomma testudinarium and Amblyomma marmoreum complexes in Asia and Africa, respectively (Mohamed et al. 2022; Cotes-Perdomo et al. 2023b).
The A. maculatum group is widely distributed from the United States of America (USA), Argentina, Belize, Bolivia, Brazil, Chile, Colombia, Costa Rica, Ecuador, French Guiana, Guatemala, Honduras, Mexico, Nicaragua, Paraguay, Peru, Uruguay, and Venezuela (Mendoza-Uribe & Chávez-Chorocco 2004; Guzmán-Cornejo et al. 2006; Mertins et al. 2010; Abarca et al. 2012; Lado et al. 2018; Rivera-Páez et al. 2018; Ossa-López et al. 2022; Guglielmone et al. 2023). Currently, the phylogenetic position of the A. maculatum group within Amblyomma is unclear (Santodomingo et al. 2021; Cotes-Perdomo et al. 2023a). In addition, the three species of the A. maculatum group are morphologically and phylogenetically closely related, casting doubts on their identification and taxonomic recognition (Koch 1844; Mendoza-Uribe & Chávez-Chorocco 2004; Estrada-Peña et al. 2005; Guzmán-Cornejo et al. 2006; Mertins et al. 2010; Abarca et al. 2012; Paddock et al. 2015; Lado et al. 2018; Guglielmone et al. 2023). For example, some studies suggest the possibility that A. maculatum and A. triste are conspecific (Nava et al. 2017; Lado et al. 2018). Other species such as A. tigrinum always cluster into a well-defined monophyletic lineage (Lado et al. 2018), and differs from A. maculatum and A. triste in the adult morphology, and ecological preferences. However, the larvae and nymphs of these three species are extremely difficult to differentiate using morphological traits (Guglielmone et al. 2000; Mendoza-Uribe & Chávez-Chorocco 2004; Nava et al. 2007; 2017; Lado et al. 2018; Guglielmone et al. 2021; 2023).
For the A. maculatum group, Lado et al. (2018) reported four morphotypes with distinct geographical distribution: i) A. triste s.s., defined as morphotype I, including specimens from Argentina, southern Brazil, Paraguay, and Uruguay; ii) A. maculatum s.s., defined as morphotype II and including specimens from Colombia (Department of Santander), eastern United States of America (USA), Guatemala, Honduras, Mexico, Nicaragua, and Venezuela; iii) morphotype III, which presents a combination of morphological characters limiting the strict assignment to A. maculatum s.s. or to A. triste s.s.; this morphotype includes ticks from southern and southwestern USA (Arizona and Texas), and northern Mexico; and iv) morphotype IV, which also exhibits a combination of characters of A. maculatum s.s. and A. triste s.s., and is found in Chile, Ecuador, and Peru. Ossa-López et al. (2022) confirmed the wide distribution of A. maculatum s.s. (morphotype II) in Colombia and indicated that the sequences of the individual from the Department of Santander analyzed by Lado et al. (2018), consistently clustered with other samples from Colombia (from the departments of Caldas, Cundinamarca, and Tolima) instead of clustering with sequences from the USA.
Similarly, Ossa-López et al. (2022) confirmed the presence of A. triste s.s. (morphotype I) in Colombia using morphological and molecular data, therefore, two out of the three species within the A. maculatum group are found in the country, and A. tigrinum is currently excluded from the Colombian territory due to lack of verified records (Guglielmone et al. 2011; Guglielmone & Robbins 2018; Guglielmone et al. 2021; Ortíz-Giraldo et al. 2021; Guglielmone et al. 2023).
The medical and veterinary importance of A. maculatum is well-documented due to its role as the principal reservoir of Hepatozoon americanum, and together with A. triste, both tick species are vectors of Rickettsia parkeri s.s. (Mathew et al. 1998; 1999; Paddock et al. 2004; Venzal et al. 2004; Paddock & Goddard 2015; Colombo et al. 2016; Romer et al. 2020). For that reason, more research on reproductive compatibility, distribution, taxonomy, and molecular affinities is needed to clarify the controversial specific taxonomy and to contribute to the understanding of their role in pathogen transmission (Mathew et al. 1998; 1999; Ewing et al. 2000; Paddock et al. 2004; Venzal et al. 2004; Colombo et al. 2016; Allerdice et al. 2020; Romer et al. 2020; Cuervo et al. 2021; Ossa-López et al. 2022; Guglielmone et al. 2023).
Mitochondrial genomes (mitogenomes) have proven to be important for the taxonomy, systematics, and population genetics of ticks, emerging as a valuable source of informative molecular markers (Wang et al. 2019; Uribe et al. 2020; Kneubehl et al. 2022; Cotes-Perdomo et al. 2023a). Mitochondrial genomes of 120 tick species have been used in phylogenetic analyses (Kelava et al. 2021), recovering controversial relationships at the family and genera levels. The absence of paralogous and small size make the mitogenomes a robust molecular marker for inferring phylogenetic hypotheses in studies of cryptic diversity (Zaharias et al. 2020; Cotes-Perdomo et al. 2023a; 2023b). In this study, sequencing techniques were combined to generate the first mitogenome of A. triste, aiming to evaluate its composition (content and organization) and establish its phylogenetic relationships and differentiation from other closely related species within the genus Amblyomma.
Material and methods
Sample collection and DNA extraction
In the year 2018, two male ticks from the A. maculatum group were found parasitizing a capybara (Hydrochoerus hydrochaeris, Rodentia, Caviidae) at the vereda Las Plumas, municipality of Arauca, Department of Arauca (6◦36′ 18′'N; 70◦31′ 51′'W), Colombia (Ossa-López et al. 2022). The collections were executed within the framework permit granted by the National Environmental Licensing Authority (ANLA) to the Universidad de Caldas as stipulated in resolution 02497 of December 31, 2018. The specimens were stored in cryogenic tubes with absolute alcohol and were morphologically identified following the keys of Kohls (1956); Estrada-Peña et al. (2005); Barros-Battesti et al. (2006); Nava et al. (2017); Lado et al. (2018) and Guglielmone et al. (2021; 2023). The specimens were confirmed as A. triste, primarily based on the presence of a thick and highly sclerotized spine and a thin and weakly sclerotized seta on the tibia of legs II-IV. In addition, the spiracular plates (peritrema) are almost oval, with short and wide dorsal projection, and longer spines on coxa IV (Ossa-López et al. 2022). The total DNA from each individual was extracted using the DNeasy Blood and Tissue kit (Qiagen) following the manufacturer's recommended protocols, with an extended incubation period (16 h) in lysis buffer at 56 °C.
Amplifying and sequencing
The molecular confirmation was made with partial sequences of three mitochondrial genes, 12S, 16S and cox1 (Ossa-López et al. 2022). DNA from a single tick was used for subsequent molecular processes and the sequencing of the complete mitogenome. The fragments were obtained by conventional PCR and sanger sequencing (Macrogen Inc., South Korea) ( Supplementary Material 1 (Supplementary Material 1(saa.29.1.7p93–108).xlsx)). The sequences were analyzed using Geneious® Prime 2022.1 software (Kearse et al. 2012).
Two strategies were implemented for the amplification and sequencing of the mitogenome. Initially, two fragments that complete ∼10kb (70% of the mitogenome) were amplified by long-range PCR and pooled together at equimolar concentration to prepare a Nextera XT DNA library, which was sequenced using Nova Seq 6000 150 PE (150 × 2 bp; 10 Gb/sample) in Illumina platform. Subsequently, primers were designed along the missing region using as a reference the mitogenome of A. maculatum (MW719251; available in the National Center for Biotechnology Information – NCBI database) for primer-walking sequencing by Sanger ( Supplementary Material 2 (Supplementary Material 2(saa.29.1.7p93–108).xlsx)).
Assembly, annotation, and alignment
For each raw file from Illumina sequencing, the quality was checked using FastQC (Andrews 2010). The cleaning, filtering, and adapter trimming of each raw file was made across Trimmomatic (Bolger et al. 2014). The partial cox1 sequence was used as a scaffold for reference-guided assembly using the "Map to Reference(s)" option in Geneious® Prime 2022.1. Subsequently, the protein-coding (PCGs; coding sequences - CDSs), transference RNAs (tRNAs), and the two ribosomal RNAs (rRNAs) genes were identified using MITOS web server (Bernt et al. 2013). The annotation was manually verified with the ORFs searcher in Geneious Prime® 2023.2.1. The genes obtained from the Amblyomma aureolatum transcriptome from SRA repository in GenBank (SRR4301110; Martins et al. 2017) assembled de novo by Uribe et al. (2020) were included in our analysis, beside the following mitogenomes available in the NCBI: Amblyomma cajennense s.s. (OP901701; OP901707), A. maculatum (MW719251), Amblyomma mixtum (OP901702; OP901703), Amblyomma ovale (MT554102; MT554103), Amblyomma patinoi (OP901704), Amblyomma sculptum (NC_032369; OP901706), Amblyomma tonelliae (OP901705), and Dermacentor nitens (NC_023349) used as outgroup.
Phylogenetic analyses
The genes were aligned and filtered separately using MAFFT (Katoh & Standley 2013) and BMGE (Criscuolo & Gribaldo 2010). The CDSs + rRNAs filtered genes were concatenated in a matrix at the nucleotide level (Matrix-NT), which was the subject of phylogenetic analyses by probabilistic methods such as Bayesian Inference (BI; Rannala & Yang 1996; Yang & Rannala 1997) and Maximum Likelihood (ML; Felsenstein 1981). BI analyses were performed using MrBayes v3.2.7a (Ronquist et al. 2012), two parallel runs, 2000000 generations, in which the initial 25% of sampled data were discarded as burn-in; and ML analyses was performed in IQ-TREE (Nguyen et al. 2015) and the robustness was assessed using 1000 bootstrap pseudoreplicates. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best evolutionary model based on the Bayesian Information Criterion (BIC; Schwarz, 1978): GTR+I+G+F for BI and GTR+F+I+G4 for ML. FigTree v. 1.4.3 (Rambaut 2007) was used to visualize all the phylogenetic trees. Genetic distances were estimated using the p-distance method with the MEGA 11 program (Tamura et al. 2021) and corroborated with the identity percentage matrix in Geneious Prime® 2023.2.1.
Results
Content and organization of the mitochondrial genome of Amblyomma triste
The complete mitogenome of the male tick sequenced is available in GenBank as record OR497835. This molecule is completely circular, has a length of 14,808 bp, and it contains (i) 13 CDSs: cox1-3, nad1-6, nad4L, atp6, atp8 and cytb; (ii) two rRNA genes: a small subunit ribosomal RNA gene (rrnS or 12S), and a large subunit ribosomal RNA gene (rrnL or 16S); (iii) 22 tRNAs; and (iv) two intergeneric spaces: between 12S and tRNA-Ile, and between tRNA-Leu and tRNA-Cys, corresponding to the non-coding regions 1 and 2 (NCR1 and NCR2) respectively (Figure 1; Table 1).
In the organization of the mitogenome there are two overlaps in the same strand between: atp6 and atp8, and between nad4 and nad4L; and six overlaps in different strands between: nad2 and tRNA-Trp; tRNA-Trp and tRNA-Tyr; tRNA-Tyr and cox1; tRNA-Arg and tRNA-Asn; tRNA-Asn and tRNA-Ser; and between tRNA-Glu and nad1. All the tRNAs vary in length from 54 to 68 nucleotides (Figure 1; Table 1). Prediction of the tRNA secondary structure showed that 20 of the tRNAs have the standard cloverleaf structure, while tRNA-S1 (trnS1) and tRNA-C (trnC) were missing the D-arm or DHU arm (Figure 2).
FIGURE 1.
Circular and linear representation of the mitochondrial gene order of Amblyomma triste. Gene scaling is approximate, and the end of the arrow indicates the direction of the genes (the genes encoded in the H-strand or “+” and L-strand or “-”). All genes have standard nomenclature including the 22 tRNA genes, which are designated by a three-letter code for the corresponding amino acid. The CDS genes are in yellow, the rRNAs in red, the tRNAs in pinkish-purplish color, and the two potential control regions (CR), are non-coding in gray.
FIGURE 2.
Predicted secondary structure of the mitochondrial tRNA genes of Amblyomma triste obtained using MITOS. A. trnS1(aga), lacks DHU arm; B. trnS2(tca), normal cloverleaf structure; C. trnC(tgc), lacks DHU arm.
TABLE 1.
Mitochondrial genome organization of Amblyomma triste.
Genetic distances and phylogenetic analyses
The concatenated matrix with CDSs indicates that A. triste has a 95.8% of identity with A. maculatum (MW719251), a record provided by the Oklahoma State University Tick Rearing Facility, USA (Brenner & Raghavan, 2021; Table 2). A. ovale has also a 80.6% of identity with A. aureolatum, and the species of the A. cajennense complex between 76.1 and 81.9% (Table 2).
TABLE 2.
Percent identity matrix of concatenated protein-coding genes (CDS).
FIGURE 3.
Phylogenetic tree based on the concatenation of 13 CDS and two rRNA genes. The tree was inferred with the best-fit substitution model and the topology shown corresponds to Bayesian Inference (BI: MrBayes v3.2.7a) and Maximum Likelihood (ML: IQ-TREE). Branch support indicated bootstrap percentages (ML/BI). Accession codes are in square brackets and mitogenome obtained in this study are shown in bold.
The interspecific genetic distances between A. maculatum and A. triste from each of the genes (CDSs and rRNA) are shown in Table 3. The nd3 and nd5 genes exhibited the greatest divergence, with 5.4% and 5% respectively. The phylogenetic relationships of 14 tick species based on the concatenated Matrix-NT, using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses, are shown in Figure 3. The topologies of the trees using ML and BI were identical, recovering a highly supported (100%) clade constituted by the two species of the A. maculatum group (A. maculatum and A. triste). Another well supported (73/100% respectively for ML/BI) was constituted by taxa of the A. ovale group. Similarly, the taxa of the A. cajennense complex were recovered in a separated clade with a 100% of support in both ML/BI reconstructions (Figure 3).
TABLE 3.
Pairwise distance (in percentage) of mitochondrial genes (CDS and rRNA) between A. maculatum and A. triste, according to the p-distance method.
Discussion
Content and organization of the mitochondrial genome of Amblyomma triste
The mitogenome of A. triste obtained in the present study is the first one recorded for this species, and it is like the mitogenomes reported for other arthropods including ticks (Montagna et al. 2012; Liu et al. 2013; Cameron et al. 2014; Brenner & Raghavan 2021).
In ticks, mitogenomes exhibit size ranges between 14 to 16 kb, circular organization, double-stranded DNA, with 37 genes: 13 CDSs, 22 tRNAs genes, and two rRNA genes, as well as two control or non-coding regions (NCRs); the arrangement of the genes of is similar to that found in the majority of species of Ixodidae ticks (Burger et al. 2013; Simon & Hadrys 2013; de Lima et al. 2017; Li & Liang 2018; Wang et al. 2019; Kelava et al. 2021; Cotes-Perdomo et al. 2023a).
In the A. triste mitogenome, the start codons (ATT and ATG) for the CDSs correspond to the typical start codons found in insects, which are the same as those commonly adopted by ticks (ATN) (Liu et al. 2013; Wang et al. 2019; Uribe et al. 2020; Brenner & Raghavan 2021). Regarding the termination codons (TAA and T), the CDSs correspond to the termination codons, which in ticks are mainly TAA and TAG, but sometimes “T” or “TA” may be converted into a complete termination codon by polyadenylation after translation (Montagna et al. 2012; Liu et al. 2013; de Lima et al. 2017; Uribe et al. 2020; Brenner & Raghavan 2021; Chavatte & Octavia 2021).
The mitochondrial rRNA genes (12S and 16S) of A. triste exhibit a complex functional structure with a relatively slow evolution rate; this is particularly important as these genes have long been used as markers in tick population genetics, and phylogenetic and systematic studies (Araya-Anchetta et al. 2015; Wang et al. 2019).
Tick mitogenomes contain unique copy of the 12S and 16S rRNA genes, and due to gene rearrangement, the position of the rRNA genes shifts, whereas the gene order and the location in the N strand remain unchanged; therefore, the combined use of CDSs and rRNA genes is recommended for taxonomic studies, particularly when involving cryptic species and complexes (Burger et al. 2014; Mans et al. 2019; Wang et al. 2019).
The size and structure of the tRNA genes in this study (54 to 68 nucleotides) are consistent with the ranges reported for other tick species (50 to 90 bp), and most tRNA genes have a complete cloverleaf structure (Liu et al. 2013; Wang et al. 2019; Chavatte & Octavia 2021), except for trnS1 and trn-C, which lacks DHU arm or D-arm. This is a common feature in most animal species, including ticks (Liu et al. 2013; Cameron et al. 2014; Williams-Newkirk et al. 2015; Wang et al. 2019; Chavatte & Octavia 2021). Mitochondrial tRNA-C secondary structures are variable among tick species with some missing D-arm and/or T-arm and some having standard cloverleaf structure (Burger et al. 2012; Montagna et al. 2012; Williams-Newkirk et al. 2015).
Similarly, tick mitogenomes may contain two non-coding regions (NCRs), as in Metastriata, or one as in Ixodes (non-Australasian Prostriata), and Argasidae. In both cases, these regions have a conserved location within the mitogenome (Montagna et al. 2012; Kelava et al. 2021). Alternatively, they may be specific to the group, for example, Metastriata and Australasian Ixodes (Prostriata) have two non-coding regions, but with different locations within the mitogenome (Montagna et al. 2012; Liu et al. 2022). Changes in the non-coding regions of the mitochondrial DNA suggest potential differences in replication/transcription mechanisms, as NCRs contain regulatory elements (Burger et al. 2012; Liu et al. 2013; Wang et al. 2019; Chavatte & Octavia 2021).
Genetic distances and phylogenetic analyses
Mitogenomes can provide a deeper understanding of tick systematics, allowing the resolution of phylogenetic relationships that are problematic when using partial genes (Wang et al. 2019; Kelava et al. 2021; Mohamed et al. 2022). In this study, the interspecific distances of CDSs+rRNA genes between A. triste and A. maculatum were 4.2%, similar to the reported by Cotes-Perdomo et al. (2023b) when comparing the complete genomes of two species of the A. marmoreum complex (Amblyomma nuttalli and Amblyomma sparsum) with an interspecific distance of 4.4%. In comparison with other Amblyomma species, the intra-specific distances found were: A. ovale (2.2%); A. cajennense s.s. (0.2%); A. mixtum (4.4%); A. sculptum (0.6%), divergence percentage is consistent with the findings reported by Cotes-Perdomo et al. (2023a) for the A. cajennense complex. For other tick genera, Reynolds et al. (2022) reported intra-specific divergences ranging from 5.8% to 10.6% for Dermacentor albipictus, suggesting the existence of a species complex, and recommend further evaluation of other genes (nad1, nad2, nad5, cox1, and atp8) that may contribute to highlighting the genetic differences.
Traditionally, the A. maculatum group has been studied using divergences of gene fragments such as 12S, 16S, and cox1, which have been the most commonly used and reported genes for hard ticks (Beati & Keirans 2001; Marrelli et al. 2007; Beati et al. 2013; Paternina et al. 2016; Rivera-Páez et al. 2017; 2018; Lado et al. 2018). Our results demonstrate that these genes do not exhibit the highest interspecific divergences. For that reason, further research involving mitogenomes or other genes that display greater genetic distances should be conducted. This could be useful for differentiation of cryptic species such as A. cajennense complex, A. marmoreum complex and A. ovale complexes.
As reported by Ossa-López et al. (2022), the genetic distances of each of the evaluated genes (1.5–1.8% for 12S; 2.3–7.1% for 16S, and 4.9–7.0% for cox1) obtained between A. triste and A. maculatum from Colombia are larger than those reported by Lado et al. (2018), although they are lower than those found between A. tigrinum compared to all morphotypes that comprise the A. maculatum group (3.9–4.3%). The phylogenetic results also confirmed differences between A. maculatum from USA and Colombia based on genetic distances of each of the evaluated genes (1.2–1.8 for 12S; 1.5–5.8% for 16S, and 3–3.7% for cox1).
Although this study emphasizes the divergence percentages for the CDSs + rRNA genes, with 12S and 16S showing low percentages of 2.1% and 2.9% respectively, in comparison to the CDSs: nd3, nd5, atp8, and cox1 stand out with interspecific divergences of 5.4%, 5.0%, 4.3%, and 4.2% respectively. Three of these genes (nd5, atp8, and cox1) align with the recommendations of Reynolds et al. (2022), and should be explored in phylogenetic studies, using the primers designed in our study for A. maculatum (MW719251), which can be implemented for other populations or species ( Supplementary Material 2 (Supplementary Material 2(saa.29.1.7p93–108).xlsx)).
Finally, there are still several questions and gaps in the systematics and diversity of hard ticks. This study shows that the A. maculatum group comprises at least two species in Colombia. We highlight the need to establish both intra- and inter-specific divergences among the species within the A. maculatum group, new information on the mitogenomes of the different morphotypes reported by Lado et al. (2018), which originate from diverse geographic areas, is needed. Also, obtaining and evaluating the mitogenome of A. tigrinum, is crucial for elucidating both intra- and interspecific divergences within the species that comprise the A. maculatum group, and to carry out experimental crosses to obtain additional evidence for species recognition.
Acknowledgments
Ministerio de Ciencia, Tecnología e innovación of Colombia - Minciencias Program “Relación, distribución, taxonomía de especies de garrapatas asociadas a mamíferos silvestres en zonas endémicas de rickettsiosis en Colombia. Un acercamiento a la comprensión de la relación vectores patógenos-reservorios” (Code: 120385270267 and CTO 80740- 200-2021) – Project “Garrapatas asociadas a mamíferos silvestres en el departamento de Caldas: Diversidad, detección de patógenos y distribución (Code:71717)”. Minciencias for funding the PhD in Science-Biology of Paula Andrea Ossa López "Convocatoria del Fondo de Ciencia, Tecnología e Innovación del Sistema General de Regalías para la conformación de una lista de proyectos elegibles para ser viabilizados, priorizados y aprobados por el OCAD dentro del Programa de Becas de Excelencia cohorte 1–2019". We thank the Universidad de Caldas, Unidad Administrativa Especial de Salud de Arauca. Award “For Women in Science 2022” conducted in collaboration with L'Oréal, Minciencias, ICETEX and La Comisión Nacional de Cooperación con la UNESCO. Part of the laboratory work was conducted in and with the support of the Molecular Systematics Lab ( https://www.mncn.csic.es/en/investigación/servicios-cientifico-tecnicos/molecular-systematics-laboratory) of MNCN-CSIC, José Gutiérrez Abascal, Madrid, Spain.
© Systematic & Applied Acarology Society
Funding
This project was funded by the Vicerrectoría de Investigaciones y Posgrados - Universidad de Caldas proyecto “Morfología interna y marcadores moleculares en garrapatas (Acari: Ixodidae): una aproximación a las interacciones con pequeños mamíferos y sus patógenos” [code 0318322]. Ministerio de Ciencia, Tecnología e Innovación of Colombia - Minciencias Program “Relación, distribución, taxonomía de especies de garrapatas asociadas a mamíferos silvestres en zonas endémicas de rickettsiosis en Colombia. Un acercamiento a la comprensión de la relación vectores patógenos-reservorios”, granted by the (Code: 120385270267 and CTO 80740- 200-2021). Minciencias for funding the PhD in Science-Biology of Paula Andrea Ossa López "Convocatoria del Fondo de Ciencia, Tecnología e Innovación del Sistema General de Regalías para la conformación de una lista de proyectos elegibles para ser viabilizados, priorizados y aprobados por el OCAD dentro del Programa de Becas de Excelencia cohorte 1–2019". Part of this work was funded by Award “For Women in Science 2022” conducted in collaboration with L'Oréal, Minciencias, ICETEX and La Comisión Nacional de Cooperación con la UNESCO. Comunidad de Madrid for Atracción de Talento contract (REFF 2019-T2/AMB-13166) of JEU.
