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Research
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.
Donath Damian
University of Dar es Salaam
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0500-9886
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
Hard tick, genetic distance, phylogeny, 16S rRNA gene, Hyalomma, Rhipicephalus, Wildlife-livestock interface, Mikumi National Park
Ticks collection and identification
| Ward | Number of cattle infested with ticks | Number of ticks collected | Rhipicephalus | Hyalomma |
|---|---|---|---|---|
| Mikumi | 32 | 76 | 62 | 14 |
| Doma | 44 | 112 | 82 | 30 |
| Kidodi | 30 | 69 | 30 | 39 |
| Ulaya | 28 | 63 | 32 | 31 |
| Melela | 36 | 102 | 48 | 54 |
| Kisaki | 38 | 106 | 64 | 42 |
| Kilangili | 14 | 35 | 22 | 13 |
| Ruhembe | 20 | 37 | 18 | 19 |
| Tindiga | 10 | 30 | 13 | 17 |
| Total | 252 | 630 | 371 | 259 |
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 classified into two genera; Rhipicephalus (371) and Hyalomma (259) (Table 1). Due to morphological similarity among ticks, all specimens were identified to genus level. Specimens of Rhipicephalus and Hyalomma genera were grouped by similarity and 9 representative individuals from each group were randomly selected. To confirm results of morphological identification, partial mitochondrion 16S rRNA gene was used as a molecular marker in species identification. 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 identification are summarized in Table 2. BLASTn matched morphological results with identity varying between 92 and 100% (Table 2). Molecular results revealed that, among the specimens of the genus Rhipicephalus, five were R. microplus and one was R. evertsi, whereas, for Hyalomma genus, five were identified as H. marginatum, three were H. truncatum, two as H. rufipes and two as H. turanicam. R. microplus and H. marginatum out numbered all other tick species and together they represent 56% of the total ticks sequenced in the present study (Table 2).
| Sample accessions numbers | Sample collection ward | GenBank accession numbers | Percentage Identity | Tick species | Source country |
|---|---|---|---|---|---|
| MT079863 | Mikumi | EU918187 | 99.50 | R. microplus | Mozambique |
| MT079864 | Mikumi | MK737650 | 98.76 | H. rufipes | Egypt |
| MT079865 | Doma | KC170742 | 100 | R. micropmus | Thailand |
| MT079866 | Doma | KU130478 | 97.15 | H.truncatum | South Africa |
| MT079867 | Kidodi | KP776645 | 98.99 | H. marginatum | France |
| MT079868 | Kidodi | KT391063 | 94.24 | H.turanicum | Israel |
| MT079869 | Ulaya | KP776654 | 94.70 | H. marginatum | France |
| MT079870 | Ulaya | EU918187 | 99.75 | R. microplus | Mozambique |
| MT079871 | Melela | L34307 | 97.72 | H. marginatum | USA |
| MT079872 | Melela | KU130478 | 96.89 | H. truncatum | South Afica |
| MT079873 | Kisaki | KC170742 | 99.26 | R. microplus | Thailand |
| MT079874 | Kisaki | KU130465 | 99.49 | H. rufipes | South Africa |
| MT079875 | Kilangili | KJ613642 | 98.42 | R. evertsi | South Africa |
| MT079876 | Kilangili | EU918187 | 99.50 | R. microplus | Mozambique |
| MT079877 | Ruhembe | KT391063 | 91.54 | H. turanicum | Israel |
| MT079878 | Ruhembe | KP776645 | 98.50 | H. marginatum | France |
| MT079879 | Tindiga | KU130478 | 96.37 | H. truncatum | South Africa |
| MT079880 | Tindiga | KP776645 | 97.47 | H. marginatum | France |
Phylogenetic Analysis Of The Mitochondrion16s rRNA Gene
Eighteen sequences representing the two identified 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 five (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. rufipes, 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 rufipes and H. marginatum (Fig. 2).The high bootstrap value (100%) supported the close relationship between Rhipicephalus microplus and R. evertsi (Fig. 2).
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).
| Mean distance | Pairwise distance | ||
|---|---|---|---|
| Species | values | Minimum | Maximum |
| Rhipicephalus microplus | 0.01 ± 0.00 | 0.003 | 0.02 |
| Hyalomma marginatum | 0.04 ± 0.01 | 0.015 | 0.06 |
| Hyalomma truncatum | 0.01 ± 0.00 | 0.003 | 0.015 |
| Hyalomma rufipes | 0.02 ± 0.00 | 0.02 | 0.02 |
| Hyalomma turanicum | 0.03 ± 0.00 | 0.03 | 0.03 |
| Genus | |||
| Rhipicephalus | 0.04 ± 0.01 | 0.003 | 0.099 |
| Hyalomma | 0.06 ± 0.01 | 0.003 | 0.139 |
| Overall tick community | |||
| Tick community | 0.11 ± 0.01 | 0.003 | 0.234 |
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Research
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.
Donath Damian
University of Dar es Salaam
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0500-9886
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 28 May, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Parasitology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Ticks collection and identification
| Ward | Number of cattle infested with ticks | Number of ticks collected | Rhipicephalus | Hyalomma |
|---|---|---|---|---|
| Mikumi | 32 | 76 | 62 | 14 |
| Doma | 44 | 112 | 82 | 30 |
| Kidodi | 30 | 69 | 30 | 39 |
| Ulaya | 28 | 63 | 32 | 31 |
| Melela | 36 | 102 | 48 | 54 |
| Kisaki | 38 | 106 | 64 | 42 |
| Kilangili | 14 | 35 | 22 | 13 |
| Ruhembe | 20 | 37 | 18 | 19 |
| Tindiga | 10 | 30 | 13 | 17 |
| Total | 252 | 630 | 371 | 259 |
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 classified into two genera; Rhipicephalus (371) and Hyalomma (259) (Table 1). Due to morphological similarity among ticks, all specimens were identified to genus level. Specimens of Rhipicephalus and Hyalomma genera were grouped by similarity and 9 representative individuals from each group were randomly selected. To confirm results of morphological identification, partial mitochondrion 16S rRNA gene was used as a molecular marker in species identification. 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 identification are summarized in Table 2. BLASTn matched morphological results with identity varying between 92 and 100% (Table 2). Molecular results revealed that, among the specimens of the genus Rhipicephalus, five were R. microplus and one was R. evertsi, whereas, for Hyalomma genus, five were identified as H. marginatum, three were H. truncatum, two as H. rufipes and two as H. turanicam. R. microplus and H. marginatum out numbered all other tick species and together they represent 56% of the total ticks sequenced in the present study (Table 2).
| Sample accessions numbers | Sample collection ward | GenBank accession numbers | Percentage Identity | Tick species | Source country |
|---|---|---|---|---|---|
| MT079863 | Mikumi | EU918187 | 99.50 | R. microplus | Mozambique |
| MT079864 | Mikumi | MK737650 | 98.76 | H. rufipes | Egypt |
| MT079865 | Doma | KC170742 | 100 | R. micropmus | Thailand |
| MT079866 | Doma | KU130478 | 97.15 | H.truncatum | South Africa |
| MT079867 | Kidodi | KP776645 | 98.99 | H. marginatum | France |
| MT079868 | Kidodi | KT391063 | 94.24 | H.turanicum | Israel |
| MT079869 | Ulaya | KP776654 | 94.70 | H. marginatum | France |
| MT079870 | Ulaya | EU918187 | 99.75 | R. microplus | Mozambique |
| MT079871 | Melela | L34307 | 97.72 | H. marginatum | USA |
| MT079872 | Melela | KU130478 | 96.89 | H. truncatum | South Afica |
| MT079873 | Kisaki | KC170742 | 99.26 | R. microplus | Thailand |
| MT079874 | Kisaki | KU130465 | 99.49 | H. rufipes | South Africa |
| MT079875 | Kilangili | KJ613642 | 98.42 | R. evertsi | South Africa |
| MT079876 | Kilangili | EU918187 | 99.50 | R. microplus | Mozambique |
| MT079877 | Ruhembe | KT391063 | 91.54 | H. turanicum | Israel |
| MT079878 | Ruhembe | KP776645 | 98.50 | H. marginatum | France |
| MT079879 | Tindiga | KU130478 | 96.37 | H. truncatum | South Africa |
| MT079880 | Tindiga | KP776645 | 97.47 | H. marginatum | France |
Phylogenetic Analysis Of The Mitochondrion16s rRNA Gene
Eighteen sequences representing the two identified 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 five (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. rufipes, 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 rufipes and H. marginatum (Fig. 2).The high bootstrap value (100%) supported the close relationship between Rhipicephalus microplus and R. evertsi (Fig. 2).
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).
| Mean distance | Pairwise distance | ||
|---|---|---|---|
| Species | values | Minimum | Maximum |
| Rhipicephalus microplus | 0.01 ± 0.00 | 0.003 | 0.02 |
| Hyalomma marginatum | 0.04 ± 0.01 | 0.015 | 0.06 |
| Hyalomma truncatum | 0.01 ± 0.00 | 0.003 | 0.015 |
| Hyalomma rufipes | 0.02 ± 0.00 | 0.02 | 0.02 |
| Hyalomma turanicum | 0.03 ± 0.00 | 0.03 | 0.03 |
| Genus | |||
| Rhipicephalus | 0.04 ± 0.01 | 0.003 | 0.099 |
| Hyalomma | 0.06 ± 0.01 | 0.003 | 0.139 |
| Overall tick community | |||
| Tick community | 0.11 ± 0.01 | 0.003 | 0.234 |