Phylogeny and genetic relationship between hard ticks (Ixodidae) infesting cattle collected from selected areas of a wildlife-livestock interface ecosystem of Mikumi National Park, Tanzania.


 Background:

There is substantial increase in the number of tick species and tick-borne infectious agents in Tanzania. Due to their impact on human, livestock and wild animal health, increased knowledge of ticks is highly needed. So far, no published data on the phylogeny and the genetic distance between hard ticks collected from cattle is available in Tanzania.
Methods

Ticks from cattle in 9 wards, which lie at the border of Mikumi National Park, were collected in dry season (November and December) 2019. Morphological identification of ticks was initially performed to genus level. To identify ticks to species level, molecular analysis based on mitochondrion 16S rRNA gene was performed. The evolutionary relationships and genetic distance between ticks were determined using Maximum Likelihood and Kimura 2-parameter methods respectively.
Results

On the basis of morphology, two genera (Rhipicephalus and Hyalomma) were identified of the 630 adult ticks collected from a total of 252 cattle. Six species; R. microplus, R. evertsi, H. marginatum, H. rufipes, H. truncatum and H. turanicum were confirmed by BLASTn and phylogenetic analysis. The considerably mean and pairwise genetic distances for Rhipicephalus and Hyalomma genera were observed, whereas, the high overall mean and pairwise genetic distances were also recorded.
Conclusion

The presence of different (clusters) phylogroups and considerably mean and pairwise genetic distances observed, reflecting possible biological diversity of hard ticks present in the study area. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Tanzania.


Conclusion
The presence of different (clusters) phylogroups and considerably mean and pairwise genetic distances observed, re ecting possible biological diversity of hard ticks present in the study area. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Tanzania.

Background
Ticks are obligate blood-sucking ectoparasites of mammals, birds and reptiles (1). They are responsible for severe economic losses in livestock production (2). Through blood sucking, ticks can cause reduction in livestock weight, limit livestock production and induce anemia (2). Tick bites may cause irritation which leads to secondary bacterial infections, together resulting in reduced quality of hides (2). Certain tick species inject toxin to animals, which cause paralysis when feeding blood (1). In addition, ticks are vectors of several pathogens; viruses, bacteria, protozoa and larial nematodes, which can cause diseases in human, livestock and wild animals (3). Till recently, about 800 tick species are known worldwide, the most of which belong to the two main families, Ixodidae (hard ticks) and Argasidae (soft ticks) (1). Ixodidae (hard ticks) is the largest family of ticks, which are of great economic importance due to their negative social-economic impact on agriculture (1,2,3). Acaricides have been the rst choice in tick control for cattle farmers, but hard ticks (Ixodidae) rapidly acquire resistance to these chemicals (4). Replication slippage and recombination drive genetic diversity in tick populations (4,5,6) generating point mutations and frame shifts within the genes targeted by acaricides, resulting in resistance (4,7). In addition, resistance can quickly accumulate in a population due to the mating structure of ticks and their ability to produce multiple generations within one season (5,8). It has, therefore, become increasingly important to clarify the level of genetic variation of hard tick population for the better development of tick control strategies.
Mikumi National Park is located in the Morogoro region of Tanzania and lies between latitudes 7°and 10°s outh of the equator and between longitudes 36° and 37°East of Greenwich (Fig. 1).This ecosystem is inhabited by a wide variety of wild animals and arthropods. Hence, the area is considered to be one of the epidemic foci of tick species and possibly tick-borne diseases (9,10). In areas which lie at the border of Mikumi National Park, people practice nomadic pastoralism, keeping large number of indigenous cattle.
Several cases of high tick infestation and tick-borne infections have been reported in livestock there in recent years (10,11). Moreover, cattle trade in the country is largely unregulated, creating risks of tick and tick-borne disease dissemination that require detailed investigation. Studies on ticks prior to the present study have been conducted and three tick genera; Hyalomma, Rhipicephalus and Ambryomma were reported to be endemic (12,13,14).
However, there is no phylogenic and genetic relationship (genetic distance) data for ticks of the family Ixodidae (hard ticks) infesting cattle in this area that have been published so far. Phylogeny utilizes the evidence of evolutionary relationship of species, whereas, genetic distance utilizes evidence from evolutionary divergence of species, both determine the genetic relationships of populations within species and was the main aspects of this study. The phylogenetic and genetic relationship data of Ixodidae ticks will provide valuable information to geneticists, farmers and acaricide suppliers about the differences and similarities of Ixodidae tick populations in the area. Therefore, accurate data from eld studies is needed to inform rational control strategies and establish models to predict the changing epidemiology of ticks, tick-borne diseases and economic impact on livestock production.
In the present study, we analyzed the phylogeny and genetic relationship (genetic distance) of the hard tick of the genera Hyalomma and Rhipicephalus infesting cattle from the wildlife-livestock interface ecosystem of Mikumi National Park, Tanzania. Phylogeny and genetic distance of ticks were performed using the mitochondrion 16S rRNA gene. Because of the genetically high conservation and strictly maternal inheritance, the 16S rRNA gene appear to provide a reliable and convenient method for distinguish the lineage among diverse populations of hard ticks.

Ticks collection
Ticks from cattle were collected in dry season (November and December) 2019 in 26 survey points of the 9 wards which lie at the border of Mikumi National park, Morogoro region, Tanzania (Geographical coordinates S 7°00 to 8°00 and E 037°00 to 037°40 ) (Fig. 1). Cattle were restricted and kept standing and all body parts were examined. Only adult ticks were collected by plucking using blunt forceps.
Collected ticks were preserved in 70% ethanol and stored at -20 °C in the laboratory, at the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, Tanzania.

Identi cation Of Ticks To Genus Level Based On Morphology
The morphological identi cation of tick genus was performed according to published taxonomic keys (1) using stereo microscopes with magni cation up to 100X. This identi cation was conducted at the Entomology unit, Department of Parasitology and Entomology, Sokoine University of Agriculture, Tanzania.

Identi cation of ticks to species level based on molecular techniques
To support morphological identi cation, the identi ed ticks were con rmed by molecular analysis of the partial sequences of the mitochondrion16S rRNA gene. For each genus of tick identi ed morphologically, nine representative individuals were randomly selected for molecular analysis.

DNA Extraction
A total of 18 adult ticks, representatives of the 2 genera identi ed morphologically, were washed twice in distilled water and air dried for 15 minutes. Each individual tick was transferred into a 2 ml micro-tube containing glass plating bead (ZymoResearch, California, USA). The tubes were frozen in liquid nitrogen for ten minutes and the ticks were ground into a powder using a Geno-grinder (SPEX Sample Prep; UK), followed by enzymatic digestion using Proteinase K (15). Genomic DNA extraction was then carried out using phenol-chloroform extraction (16). The DNA was then precipitated with absolute ethanol and resuspended in 200 µL of 1 × TE buffer (Tris 10 mM, EDTA 1 mM, pH8) (15).
Agarose Gel Analysis And Puri cation Of Amplicons PCR products were visualized in 1.4% agarose gel (CSL-AG500, Cleaver Scienti c Ltd) stained with EZ-vision® Blue light DNA Dye in Tris-Acetate-EDTA (TAE Buffer) under UV light after staining with Gel Red Nucleic Acid Stain(https://biotium.com/product/gelred-nucleic-acid-gel-stain/). A100bp DNA ladder was used as a standard marker. The amplicons were puri ed using QIAquick PCR Puri cation Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. The concentrations of puri ed PCR products were determined using a spectrophotometer.

Sequencing Of The 16s rRNA Amplicons
Fragments were sequenced at Inqaba South Africa using the same forward and reverse primers as used to generate the PCR products. The labeled products were then cleaned with the ZR-96 DNA Sequencing Clean-up Kit (http://www.zymoresearch). The cleaned products were injected on the Applied Biosystems ABI 3500XL Genetic Analyzer with a 50 cm array using POP7 (https://www.thermo sher.com). Sequence chromatogram analysis was performed using Finch TV analysis software (https://www.softpedia.com/get/Science-CAD/FinchTV.shtml).

Sequences Editing And Identity Con rmation
Sequences were edited using BioEdit in MEGA X software (17). To con rm the identity of each tick species, the sequences were compared with those available in the GenBank database using the BLASTn program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). For the BLASTn algorithm, a stringent E-value cut-off (10 − 6 ) was used as described previously (13). The identity of the query sequence was assigned to the best hit (highest bit score) returned from BLASTn. The query ID was regarded as con rmed when the best hit (highest bit score) had an E-value below 10 − 6 (18).

Sequences Alignment
Representative mitochondrion 16S rRNA gene sequences of Rhipicephalus microplus, R. evertsi, Hyalomma ru pes, H. marginatum, H. truncatum and H. turanicum were downloaded from GenBank. Downloaded sequences and sequences generated in the present study were aligned by multiple sequence alignment using MUSCLE in MEGAX software (17,19). The sequences acquired in this study have been deposited in the GenBank database with accession numbers MT0798663 to MT079880.

Phylogenetic Analysis
To determine the genetic relationship between different tick species and to infer their evolutionary history, a phylogenetic tree was constructed. To build the tree, reference sequences of the mitochondrion 16S rRNA gene downloaded from GenBank database were aligned along with the sequences generated in the present study. The evolutionary history was inferred by using the Maximum Likelihood (ML) method with 100 replications in the bootstrap test and Tamura 3-parameter mode (17). Initial tree for the heuristic search was obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value (17). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 24 nucleotide sequences, 18 generated from the present study and the rest from GenBank. Evolutionary analyses were conducted in MEGA X software (19).

Evolutionary divergence (genetic distance) of ticks
To further determine the evolutionary divergence of the ticks collected in this wildlife-livestock interface ecosystem, mean and pairwise genetic distance analysis were conducted using Kimura's 2-parameter (K2P) model (20). The analysis involved the sequences generated in the present study only. Standard error estimates were obtained by a bootstrap procedure (100 replicates). All ambiguous positions were removed for each sequence pair (pairwise sequence deletion option) (20). The mean and pairwise genetic distance analysis was conducted in MEGAX software (19). A total of 630 adult ticks were collected from 252 cattle in 9 wards, which lie at the border of Mikumi National Park, Tanzania (Table 1). On the basis of morphology, all ticks were classi ed into two genera; Rhipicephalus (371) and Hyalomma (259) ( Table 1). Due to morphological similarity among ticks, all specimens were identi ed to genus level. Specimens of Rhipicephalus and Hyalomma genera were grouped by similarity and 9 representative individuals from each group were randomly selected. To con rm results of morphological identi cation, partial mitochondrion 16S rRNA gene was used as a molecular marker in species identi cation. A total of 18 specimens were sequenced, edited, aligned and the BLASTn analysis was performed. The lengths of the aligned sequences varied from 399 to 453 base pairs and the nucleotide components indicate that mitochondrion16S rRNA gene of these ticks is highly A-T rich with average nucleotide frequencies of Thymine (36.77%), Cytosine (9.51%), Adenine (39.71%) and Guanine (13.99%). A bias toward Adenine and Thymine (76.48%) was consistent with the base composition of arthropod mitochondrial DNA. The results from molecular identi cation are summarized in Table 2. BLASTn matched morphological results with identity varying between 92 and 100% (  Eighteen sequences representing the two identi ed tick genera generated in the present study and six reference sequences downloaded from GenBank database were used to infer phylogenetic relationship of tick species. To determine the evolutionary relationship of species, sequences from each species generated in the present study were aligned along with the representative sequences downloaded from the GenBank. The Maximum Likelihood tree from mitochondrion 16S rRNA gene sequences derived from Rhipicephallinae and Hyalomminae sub-families generated ve (clusters) phylogroups (A, B, C, D and E) (Fig. 2). Group A, constitutes Rhipicephalus genus with two monophyletic groups; R. microplus and R. evertsi. Group B, was members of H. truncatum, group C comprises H. ru pes, group D comprises members of H. turanicum and group E comprises H. marginatum members (Fig. 2).There was strong bootstrap support among the species of Rhipicephalus microplus, R. evertsi, Hyalomma truncatum and H. turancum (Fig. 2). The results provided weak support in bootstrap analysis for the clusters containing Hyalomma ru pes and H. marginatum (Fig. 2).The high bootstrap value (100%) supported the close relationship between Rhipicephalus microplus and R. evertsi (Fig. 2).

Ticks collection and identi cation
Evolutionary divergence (genetic distance) of ticks Due to technical limitations, Table 3 is provided in the Supplementary Files section.
Using K2P model, sample divergences at various taxonomic levels are shown in Tables 3 and 4. To determine the evolutionary divergence (distance) within the species, sequences from each species generated in the present study were aligned and the K2P distance model was then used to determine the mean and pairwise distances. H. marginatum recorded the highest mean intraspecies distance value, whereas, R. microplus recorded the lowest (Tables 3 and 4).The highest pairwise intraspecies value was recorded in H. marginatum and the lowest pairwise intraspecies values were observed in R. microplus and H. truncatum (Tables 3 and 4). Likewise, 16S rRNA gene sequences from each genus generated in the present study were aligned to determine the evolutionary distance of tick within the genus. The K2P distance model was thereafter applied to infer the mean and pairwise distances. The highest pairwise intragenus value was recorded in Hyalomma genus, whereas, the lowest pairwise intragenus value was recorded in Rhipicephalus genus (Table 4). Notably, the mean evolutionary divergences were recorded to be considerably high in both genera (Table 4). Finally, to determine the overall genetic distance of the overall tick community, all 16S rRNA sequences generated in the present study were aligned and the K2P model used to estimate genetic distance. The overall high mean distance of 0.11 ± 0.01 (Table 4) and the high pairwise maximum distance value of 0.23were recorded in the present study (Table 3).

Discussion
In the present study, we analyzed the phylogenetic relationship and genetic distance of the hard ticks (Ixodidae) infesting cattle using the mitochondrion 16S rRNA gene. The phylogenetic and genetic relationship data will provide valuable information to geneticists, farmers and acaricide suppliers about the differences and similarities of Ixodidae tick populations in the area and could also be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Tanzania.
As shown in the results, the Maximum Likelihood tree generated in the present study recorded several clusters (groups) of the mitochondrion 16S rRNA gene sequences, indicating high divergence of the gene sequences of hard ticks (21,22,23) present in this wildlife-livestock interface ecosystem. In that way, clusters of similar sequences represent species clearly separated from other clusters (species) (24,25,26).Therefore, this is a re ection of high biological diversity of ticks within Ixodidae (hard ticks) (27,28,29) present in this wildlife-livestock interface ecosystem.
From the intraspecies genetic distance data generated in the present study; Hyalomma marginatum, H. ru pes and H. turanicum recorded considerably high mean and pairwise intraspecies genetic distances. On the other hand, Rhipicephalus microplus and Hyalomma truncatum recorded low mean and pairwise intraspecies genetic distances. The considerably high genetic distances recorded in H. marginatum, H.
ru pes and H. turanicum suggest that different groups of these species might represent distinct species or subspecies, or that the species are species complexes with high genetic diversity (30). The low value of divergence recorded in R. microplus and H. truncatum might indicate hybridization (30) or misidenti cation among the species (31,32).
From the intragenus genetic distance data generated here, Hyalomma spp., recorded considerably high mean and pairwise genetic distance. The considerably high mean and pairwise genetic distances recorded in Hyalomma genus suggest high biological diversity in this group (genus) of hard ticks (29,32). Therefore, either new species or subspecies closely related to those recorded in the present study might exist or that these ticks form species complexes with divergent lineages in this wildlife-livestock interface ecosystem (32). The interbreeding between Hyalomma ru pes, H. marginatum and H. turanicum populations has already been demonstrated (33,34) and these hybrid forms can be transported to different regions by migratory birds (34,35,36,37,38). Therefore, it is possible to nd hybrid forms as a result from the interbreeding of two or three species in regions where these species are found (37,38). This could explain for the wide sequence variability (divergences) for Hyalomma spp., particularly for Hyalomma marginatum. H. turanicum and H. ru pes (34,38) as observed in the current study.
The mean and pairwise genetic distances in the overall tick community recorded in the present study were highly similar to other studies (32,33). Such considerably genetic distance values re ect the high biological diversity within the hard tick community present in this wildlife-livestock interface ecosystem (32).
As expected, the mean genetic distances recorded in the present study increased with higher taxonomic ranking; 0.01 ± 0.00, 0.06 ± 0.01 and 0.11 ± 0.01 for intraspecies, intragenus and overall tick community respectively. This makes the mitochondrion 16S rRNA gene to be a good marker for supraspeci c differentiation and mainly, for taxa grouping purposes (28,29,39). According to Ros et al. (2007) (40), DNA barcoding assumes that the genetic distances between species are greater than within species.

Conclusion
The presence of different (clusters) phylogroups and considerable genetic distances observed, re ect the possible biological diversity of hard ticks present in the study area. Therefore, further work is required to delineate species boundaries and to develop a more complete understanding of hard tick diversity over larger scale.

Availability of data and materials
The data supporting the conclusions of this article are included within the article. The sequences generated in the present study were submitted to GenBank under the accession numbers MT079863 to MT079880 Author's contributions DD designed the investigation, performed the experiments, conducted the investigation and drafted the manuscript. MD designed the investigation, conceived the study and revised the manuscript. JJW and MB conceived the study, designed the investigation and revised the manuscript. All authors read and approved the nal manuscript.

Ethics approval and consent to participate
No speci c permits were required for this study. The study did not involve endangered or protected species. Therefore, the local ethics committee deemed that approval was unnecessary.

Consent to publication
Not applicable.

Competing Interests
The authors declare that they have no competing interests