Rhipicephalus is a genus of ticks that includes several species known to transmit a range of pathogens to both animals and humans (1). Some of the most common species of Rhipicephalus ticks include R. sanguineus sensu lato, R. microplus, and R. annulatus (2). These ticks are distributed in different regions worldwide, with R. sanguineus s.l. found in the temperate and tropical regions and R. microplus and R. annulatus being more commonly found in tropical and subtropical regions (3). Rhipicephalus ticks are known to transmit various pathogens, including bacteria, viruses, and protozoans, such as Crimean-Congo hemorrhagic fever virus, Babesia, Anaplasma, Ehrlichia, and Rickettsia species, which can cause diseases such as Crimean-Congo hemorrhagic fever, Lyme disease, Rocky Mountain spotted fever, and other tick-borne illnesses (3, 4).

The genus Hyalomma comprises several species of hard ticks that are widely distributed in Africa, Asia, and Europe (5). These ticks are known to transmit a number of pathogens, including viruses, bacteria, and protozoa, some of which can cause severe diseases in humans and animals (3). Hyalomma ticks are important vectors of the Crimean-Congo hemorrhagic fever virus, a highly pathogenic virus that can cause severe hemorrhagic fever in humans (6, 7). They are also known to transmit several other viral pathogens, such as the Nairobi sheep disease virus and the Alkhumra virus (8). In addition, Hyalomma ticks can transmit bacterial pathogens such as Rickettsia, Ehrlichia, and Anaplasma, as well as protozoan pathogens such as Theileria and Babesia (9–12). Some Hyalomma species are also associated with the transmission of tick-borne encephalitis virus, a flavivirus that causes a range of neurological symptoms in humans (13).

The identification of Rhipicephalus and Hyalomma species using morphological diagnosis keys might be constrained by several factors (14). For instance, species from the R. sanguineus group have very similar morphologies, making their distinction quite laborious (15) and requiring therefore advanced taxonomic knowledge (i.e., male sclerites) for reliable species identification. In addition, tick species morphological diagnosis could be rendered problematic due to the alteration of some key morphological traits during the course of engorgement, in particular, for female ticks. Moreover, some species may exhibit significant variations in morphology depending on their geographic location or the host they are feeding on (16). Finally, in comparison to adult ticks, species identification is more problematic with juvenile stages of Rhipicephalus and Hyalomma due to the absence of expression of some key morphological features present in adults. Accordingly, morphological diagnosis can be time-consuming, requiring specialized knowledge and training, and these taxonomic competences may not be readily available in some regions (17). These challenges underscore the importance of incorporating additional approaches to morphological diagnosis, including molecular and genetic techniques, to identify and classify tick species more accurately (17).

The use of DNA markers has significantly improved the reliability of the identification of tick species, including those of the genera Hyalomma and Rhipicephalus (18). Compared to morphological diagnosis, DNA-based identification was shown, for several tick species, to be highly accurate and able to differentiate between closely related species (19). Some of the DNA markers used for species identification in these genera include mitochondrial DNA (mtDNA) genes, such as cytochrome c oxidase subunit 1 (CO1) and 16S rRNA, as well as nuclear DNA markers like the internal transcribed spacer (ITS) region (20–22). These markers were successfully applied to species identification for instance with Hyalomma dromedarii and Rhipicephalus microplus (23–25).

However, and despite their usefulness, there are limitations to the use of existing DNA markers for tick species identification (26, 27). Indeed, there are still gaps in the available DNA sequence databases for some tick species, emphasizing accordingly, the need to design more reliable molecular markers for their identification (28). Moreover, the existence of hybridization and introgression events between different tick species can further complicate the identification process. Therefore, the development of more comprehensive DNA sequence databases and the use of multiple molecular markers can help to increase the accuracy and reliability of tick species identification (29).

In this context, the present study targeted an mRNA-based marker as a complementary tool to morphological diagnosis and to genomic and/or mitochondrial DNA markers. Unlike genomic DNA markers, which have several limitations, mRNA markers provide more specific and sensitive information for species identification. mRNA is a complementary copy of a gene that is used as a template for protein synthesis (30). mRNA-based markers are highly conserved among species and are often more species-specific than genomic markers (31, 32). These markers can be used in combination with traditional morphological methods and other molecular markers to improve the accuracy of tick species identification.

Our study focused on identifying and validating mRNA-based markers for the diagnosis of Hyalomma and Rhipicephalus species. The rational base of our study was to determine the minimum length of partial Bm86 cDNA fragments required for species identification in two tick genera, namely Rhipicephalus and Hyalomma. The decision to use Bm86 cDNA encoding the vaccine target gut protein is justified by the fact that the complete cDNA sequence perfectly aligns with the recent classification of hard ticks (31). This strategy offers several expected benefits, including the potential to detect several tick species using a single molecular marker (33). Although changes in climate and in the length of the different seasons will directly affect tick survival, activity, and development, our mRNA-based markers can be applied to both engorged and non-engorged ticks, making it useful for analyzing ticks in various developmental stages and environmental settings (34, 35).

Therefore, the aims of this study were to conduct an in silico analysis to identify the minimum partial Bm86 cDNA sequence required for the identification of species within the Hyalomma and Rhipicephalus genera, to develop a molecular method based on the amplification and sequencing of these two minimal sequences, and to apply this technique for the identification of tick field specimens belonging to the two genera collected from several hosts located in different Tunisian regions.

The identification of tick species has traditionally relied on morphological methods, but these methods can be difficult and subjective. Difficulties may arise during the morphological diagnosis of tick species due to the presence of significant morphological variations related to the stages of development, the state of engorgement of ticks, and the possible presence of hybrid specimens resulting from crossovers between two closely related species (52–54). In recent years, the use of DNA markers for determining tick species has gained widespread popularity due to its precision and efficiency compared to traditional morphological methods (17) and its ability to provide more detailed information about the evolutionary relationships between different species (55).

In recent years, several DNA markers such as the internal transcribed spacer (ITS) region, the cytochrome c oxidase subunit I (COI) gene, and the 16S rRNA gene have been used for tick species identification (15, 52, 56, 57). However, despite their wide use, these markers have been found to have limitations in terms of their discriminatory power and sensitivity (15, 27, 58, 59). For example, the ITS region has been shown to have limited resolution in differentiating between closely related species (27), while the COI gene has been found to have low sensitivity in detecting intraspecific genetic variations (27). Similarly, the 16S rRNA gene has limitations in its ability to differentiate between tick species that belong to different genera (60). In light of these limitations, there is a need for more effective DNA markers that can provide better tick species identification. These limitations are the reason where the proposed study comes in, as it aims to identify the minimum length of partial Bm86 cDNA fragments needed for species delineation in the tick genera Rhipicephalus and Hyalomma.

The Bm86 cDNA marker represents a new and innovative way of species delineation in ticks as it targets the tick's RNA banks that have been previously utilized in various research studies. In many research laboratories, these RNA libraries are for the most part widely studied for their potential in the development of anti-tick vaccines mainly based on proteins from the intestine and salivary glands of ticks (61–65) and in the search for RNA viruses that infect ticks such as tick-borne encephalitis virus (TBEV) and Kyasanur forest disease virus (KFDV) (66–71). In fact, the Bm86 cDNA marker could represent a significant advance in the field of delineating tick species that make up these banks, providing a sensitive and discriminatory tool that researchers can use in interspecific and intraspecific diversity analyses. Its ability to target the tick's RNA banks, which have already been utilized in previous research, offers new avenues for a more accurate and efficient method for tick species and isolate identification, which will have important implications for ticks and tick-borne disease research and control.

The selection of the Bm86 transcript as a species identification marker for ticks in the genera Hyalomma and Rhipicephalus was motivated by several factors. First, this transcript had been previously used in commercial and experimental anti-tick vaccines (48, 49, 72–75), making it a readily available target in tick RNA banks (64, 76). Second, research showed that the phylogeny based on the complete or almost complete sequence of this transcript was in perfect agreement with the recent taxonomy of hard ticks (31, 77). This made it an ideal candidate for species identification as it had already demonstrated its utility in representing the genetic diversity of these ticks (78–81). Furthermore, using this transcript as a marker for species identification would help to better understand the evolution and ecology of these ticks, which are important vectors of various diseases affecting livestock and humans.

However, the purpose of our study was to identify the minimum length partial Bm86 cDNA fragments required to differentiate between species within the tick genera Rhipicephalus and Hyalomma. Using a shorter sequence has practical benefits as it only requires one primer pair for amplification and a single primer for sequencing. This reduces the cost of the test compared to analyzing the complete or nearly complete sequence, which requires multiple primer pairs.

Through a combination of genetic analysis and phylogenetic analysis, the study identified the conserved and variable regions within the Bm86 cDNA of these tick species. The crucial factor in choosing the partial sequences specific to Hyalomma and Rhipicephalus genera was the alignment of two genetically closest species Hy. excavatum and Hy. anatolicum (96.9% identity rate) and Rh. microplus and Rh. annulatus (98.6%), respectively. As a result of these alignments, one conserved and two variable regions were identified in each genus, as shown in Figure 2 for Hyalomma and Figure 1 for Rhipicephalus. Therefore, to ensure accurate results in future molecular diagnosis studies of Hyalomma and Rhipicephalus spp., we suggest avoiding the use of partial sequences within these conserved regions and instead focusing on amplifying and sequencing fragments found in the two variable regions of each genus (Figures 1, 2). By evaluating different fragment sizes within the variable regions, we determined that the minimum size for species delineation was 398 bp for Rhipicephalus spp. (from 1,487 to 1,884) and 559 bp for Hyalomma species (from 539 to 1,097).

The study also confirmed tick species identification through morphological characteristics and mitochondrial 16S rRNA partial sequences, with a 99.27–100% identity with the closest published Hyalomma and Rhipicephalus sequences in GenBank. Interestingly, we identified a Hyalomma anatolicum tick specimen based on its morphological characteristics, and its identity was validated by mitochondrial 16S rRNA partial sequences. The validation revealed a 100% match with the closest published Hyalomma anatolicum sequence in GenBank. Notably, this finding marks the second report of this tick species since the discovery of a single Hyalomma anatolicum specimen in 1970 by Van Den Ende in Southern Tunisia (82). This discovery highlights the risk that this tick becomes a vector of the apicomplexan hemoparasite Theileria annulata in Tunisia as already advanced by Gharbi et al. (83).

Furthermore, the field evaluation study successfully amplified and genetically analyzed the minimal length Bm86 cDNA partial sequences for the identified tick species, with 99.10–100% and 96.85–97.34% homology rates with previously published Hyalomma and Rhipicephalus sequences, respectively. These findings have important implications for the accurate identification and classification of tick species, which is crucial for understanding the epidemiology and transmission of tick-borne diseases.

In addition, our results also support the utility of Bm86 orthologs as a potential marker for tick isolate or strain identification. In particular, the results suggest that the studied minimal length Bm86 fragment is just as effective at discriminating between isolates and strains within each Hyalomma and Rhipicephalus species as the complete or nearly complete sequences. The phylogenetic inference based on the two partial sequences that were chosen showed the same pattern as previously observed when using the complete or nearly complete sequence (Figures 5, 6). No changes in the relationships between the isolates and strains of each species were detected, which suggests that there is no incongruity in the phylogeny. The trees generated using the neighbor-joining method had high support values at the terminal nodes and most of the deeper branches. There were also no instances of polytomies, which is likely because the selected fragments contained the most informative sites, resulting in a conservation of the phylogenetic signal. These findings were consistent with previous studies (84, 85). This is interesting because it suggests that this shorter fragment could be a more efficient and cost-effective reliable method for identifying and characterizing Hyalomma isolates and strains. In fact, our findings indicate that the two Hyalomma trees generated using the complete or nearly complete sequences and the minimal sequence showed similar results. Specifically, the genetic analysis revealed that the Hy. marginatum strain from Turkey (Kutahya) (86) is more closely related to the Hy. marginatum strain from France (80) than to all the Hy. marginatum Tunisian strains (31). Additionally, the isolate Ariana from Hy. scupense was relatively more distant from the isolates from Sousse, Manouba, and Beja (31).

Additionally, our result suggests that the minimal length Bm86 transcript sequence selected in this study can be as effective as the complete or nearly complete sequence in discriminating between isolates and strains within Rh. microplus and Rh. appendiculatus species. First, the phylogenetic analysis based on the minimal length sequence was able to generate groups of isolates based on their geographic regions, which were similar to the groups generated by the analysis based on the complete or nearly complete sequence. Second, the results showed that the minimal length Bm86 transcript sequence specific to the Rhipicephalus genus was also effective in discriminating between the two genospecies groups of Rh. appendiculatus isolates (87) in a similar way than with the complete or nearly complete transcript Bm86 sequence (Figures 6, 7). This finding indicates that the shorter fragment could also be a useful tool for identifying and characterizing Rhipicephalus strains. Overall, these results highlight the potential benefits of using shorter sequence fragments to identify and distinguish between different strains and isolates of tick species belonging to these two genera, which could have important implications for tick control and disease prevention strategies. However, it is important to note that further research may be necessary to confirm these findings and to determine whether they are applicable to other tick species.

However, the primers we designed for the amplification of each Bm86 minimum partial sequence specific to Hyalomma and Rhipicephalus were found to be sensitive and specific for each targeted tick genus. We were able to confirm this by successfully obtaining an amplicon of the expected size for all samples when amplified with primers specific for their respective genus. This indicates that the two sets of primers we created are suitable for amplifying and sequencing our cDNA marker and are capable of differentiating between the two closely related tick genera.

The use of a minimal Bm86 cDNA sequence for tick species differentiation offers advantages such as the use of a single pair of genus-specific primers for amplification and only a single primer for sequencing, followed by a simple blast analysis to determine the species within each genus. However, it is important to acknowledge the limitations associated with cDNA, as researchers need to extract total RNA and obtain cDNA through reverse transcription. Nonetheless, the decision to exclusively use cDNA for both the Hyalomma and Rhipicephalus genera was made due to the presence of a large 2,888 bp intron at position 1,752 bp in Bm86 cDNA (88), which is located within the minimal sequence used to discriminate Rhipicephalus species. Consequently, amplification of gDNA using specific primers for the Rhipicephalus genus is not feasible. To maintain consistency, we chose to utilize cDNA exclusively for both Hyalomma and Rhipicephalus genera.

Furthermore, this cDNA marker is expected to be particularly useful in identifying tick specimens that are difficult to recognize morphologically, for instance, with fully engorged tick immature females, in case of occurrence of malformations, or in the occurrence of hybrids of two species of ticks that are taxonomically closely related (31, 89, 90). Our marker, which is able to accurately identify tick species that may be otherwise difficult to differentiate, could be used as an additional taxonomic tool for morphological diagnosis for improving the reliability of tick species identification, particularly when considering the increased risks for changes in tick fauna and the emergence of new tick species driven by the combination of climate change with anthropogenic factors.

Genomic and mitochondrial DNA markers have been shown to be effective in identifying tick species compared to traditional morphological methods. In addition to these existing markers, we have identified here a novel Bm86 cDNA marker that provides a new approach to tick species identification and can target tick RNA banks. In this study, we developed a molecular method based on the minimum length of partial Bm86 cDNA fragments needed for identifying accurately tick species within the Rhipicephalus and Hyalomma genera, increasing, therefore, the range of taxonomic tools applied to identify reliably hard tick species.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The collection of ticks from cattle was done in accordance with the Tunisian National School of Veterinary Medicine's guidelines for animal care and handling and required no ethical approval. Consent was sought from the farmers before sampling. The animals were gently restrained by their owners in the same way as during routine clinical examinations, and invasive sampling or tranquilizers were not used. Moreover, the sampling was overseen by veterinarians and veterinary technicians from the National School of Veterinary Medicine in Sidi Thabet.

MBS conceived the idea, conducted in silico analysis, and designed the experiments. SZ, MM, and MD performed the experiments. SZ and MBS wrote the manuscript. MAD collaborated with MBS in developing the study concept and in editing and finalizing the manuscript. All authors have reviewed and approved the current version of the manuscript.