CC BY
0MOLECULAR-GENETIC ANALYSIS OF THE SPECIES OF THE GENUS RHIPICEPHALUS (IXODIDA) BASED ON NUCLEOTIDES OF THE COI AREA OF
MITOCHONDRIAL DNA
R.K. SHAPAOTOV
Doctoral student, Institute of Zoology Academy of Sciences of the Republic of Uzbekistan
J.Z. NORMATOV
teacher, Qarshi State University
Abstract. R. sanguineus, R. turanicus, R. rossicus, R. pumilio, R. bursa andR. annulatus ticks belonging to the genus Rhipicephalus were found in agricultural animals in Tashkent, Syrdarya and Namangan regions. According to the analysis of the nucleotide sequences belonging to the COI region of mDNA of these species and the nucleotide sequences obtainedfrom the GenBank database, it was found that the representatives of this genus are grouped into 5 clades (groups).
Keywords: Rhipicephalus, tick, clade, COI, DNA.
Introduction. Ixodidae mites are transient obligate hematophages and ectoparasites of vertebrates, which are widespread throughout the world [5,9]. About 900 species of mites belonging to the Ixodidae family have been identified in the world fauna [1,4]. According to the results of research carried out in recent years, there is a morphological variation between mite species, which prevents their morphological identification [3]. In this regard, in the research conducted by many scientists, the nucleotide sequence of the ITS region of the ribosomal rDNA of blood-sucking mites was studied, and it was noted that this region plays an important role in the identification of species [7,14]. In particular, the representatives of this family are attracting the attention of zoologists, parasitologists, entomologists, veterinary and medical specialists as carriers of many infectious and parasitic diseases. Mites of the genus Rhipicephalus belonging to the Ixodidae family are carriers of rickettsioses in our country [17].
In recent years, 82 species of mites belonging to the genus Rhipicephalus Koch., 1844 (Ixodidae) have been recorded in the world fauna [8]. Rhipicephalus sanguineus, R. turanicus, R. bursa, R. rossicum, R pumilio, R. leporis andR. schulzei mites belonging to the genus Rhipicephalus Koch., 1844 were found in the fauna of Uzbekistan [10]. The purpose of this research work is molecular-genetic identification of mites belonging to the genus Rhipicephalus in Uzbekistan based on nucleotides of the COI region of mitochondrial mDNA.
These research works were carried out in the spring, summer and autumn seasons of 2022-2023 in the territory of Tashkent, Syrdarya and Namangan regions. A total of 1400 head of animals were examined for mite samples from 6 farms and 27 private farms located in Yukarichirchik, Kuyichirchik, Parkent, Chinoz, Bekabad, Boka, Bostanlik districts of Tashkent region, Pop district in the Namangan region; Boyavut, Gulistan, Saykhunabad and Sirdarya districts in the Syrdarya region. In particular, based on route and stationary methods, 4961 specimens of mites belonging to the genus Rhipicephalus were collected from 247 head of Bos taurus (cattle), 38 head of Equus caballus (horse), 761 head of Ovis aries (sheep), 313 head of Capra hircus (goat) and 41 head of Canis lupus familiaris (dog). During the study, 801 specimens of mite samples were collected from 9 landscapes of plains, mountains and foothills Reshetnikov, 2020, [13]. The brought mite samples were placed in 70 and 96% ethyl alcohol solution and stored in marked glass and ordinary plastic containers. Species composition and morphological characteristics of mites were carried out based on the methods by Walker et al., 2003; Estrada-Peña et al. 2004 [16,6].
To carry out molecular genetic research methods, genome DNA was extracted from the legs of R. sanguineus, R. turanicus, R. rossicus, R. pumilio, R. bursa and R. annulatus species belonging to the genus Rhipicephalus. Genomic DNA extraction was performed using reagents of Thermo Scientific GeneJET PCR Purification Kit (Germany) [15,12,2]. COI fragments of mitochondrial
mRNA from Rhipicephalus species were isolated using primers COI-F 5' ATCATAAAKAYHTTGG 3', COI-R 5'GGGTGACCRAARAAHCA 3', which are widely used in molecular taxonomy for nucleotide sequences Lv J et al., 2014 [11]. When preparing Master-mix for PCR, water (distilled) -7.1 pl, 10x PCR buffer -1 pl, dNTP - 0.2 pl, primers - 0.5 pl, Taq-polymerase - 0.2 pl=10 pl were used. Polymerase chain reaction from isolated DNA samples was performed using an automatic programmable amplifier (PR-96E) in the following mode.
PCR was performed using an automatic programmable amplifier (PR-96E) in the following mode: Denaturation step 94°C, 5 min, followed by 5 cycles of 94°C, 30 s, 52°C, 30 s, and 68°C for 1 min; 5 cycles of 94°C, 30 s, 50°C, 30 s, and 68°C for 1 min; 94°C for 30 s, 5 cycles of 48°C for 30 s, and 68°C for 1 min, 25 cycles of 94°C, 30 s, 46°C, 30 s, and 68°C for 1 min; A final extension step at 68°C for 5 min. Nucleotide sequences of mites of the genus Rhipicephalus obtained from Sikvenes and DNA sequences obtained from the database of the International Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) were used.
According to the results of molecular genetic research, based on the results of molecular genetic research (sequence chromatography) on species of the genus Rhipicephalus, nucleotides with 664 base pairs belonging to the COI region of mRNA of R. sanguineus, R turanicus, R. rossicus, R pumilio, R. bursa and R annulatus were isolated (Table 1).
Table 1.
Comparison of species of the genus Rhipicephalus based on nucleotides of the COI region of
mDNA
№ Rhipicephalus avlodi turlari 1 2 3 4 5 6
1 R. sanguineus - 9,1 11,5 55,5 15,1 15,4
2 R. turanicus 51 - 8,6 56,6 14 14
3 R. rossicus 64 48 - 56,2 15,1 15,4
4 R. pumilio 305 311 309 - 58,1 56,4
5 R. bursa 84 78 84 319 - 12
6 R. annulatus 86 78 86 310 67 -
As can be seen from this table, there is a difference of 51 nucleotides between R. sanguineus species and R. turanicus species at 9.1%, 64 differences with the nucleotides of R. rossicus species at 11.5%, 305 differences with nucleotides of R. pumilio species at 55.5%, 84 differences with R bursa nucleotides at 15.1%, 86 differences with the nucleotides of the R. annulatus species at 15.4%.
There is a difference of 48 nucleotides between R. turanicus species and R. rossicus species at 8.6%, 311 differences with the nucleotides of R. pumilio species at 56.6%, 78 differences with the nucleotides of R. bursa and R. annulatus species, making up 14%.
The difference between the nucleotides of R rossicus and R pumilio species is 309 nucleotides at 56.2%, 84 differences with nucleotides of R. bursa species at 15.1%, 86 differences with the nucleotides of the R. annulatus species, which were recorded at 15.4%. There is a difference of 319 nucleotides between R. pumilio and R. bursa species at 58.1%, 310 differences with the nucleotides of the R. annulatus species, making up 56.4%. There were 67 differences between the nucleotides of R. bursa species and R. annulatus species, which was 12%. According to the results of molecular genetic research, based on the analysis of the nucleotide sequences of the mDNA COI region of the studied species belonging to the genus Rhipicephalus and the nucleotide sequences obtained from the GenBank database, it was found that the representatives of this genus are united into 5 clades (groups) (Fig. 2).
Figure 2. A phylogenetic tree of ticks of the genus Rhipicephalus developed based on the ML
(maximum likelihood-ML) method.
In the first group, the species R. rossicus showed 99% bootstrap support compared to the main joint, and 100% within the species. In the second group, R. turanicus species made 96% bootstrap support compared to the main joint, in the third group, R. linnaei and R. sanguineus species produced 97%, and R. sanguineus species produced 99% - 100% bootstrap support. In the fourth group, R. annulatus, R microplus and R. bursa species formed 98% bootstrap support and formed two subgroups. R annulatus and R. microplus in the first subgroup produced 98%, R annulatus species 99% - 100%, R. bursa species in the second subgroup also produced 99% - 100% bootstrap support. R. pumilio species in the fifth group combined to form a bootstrap support of 99% - 100%.
Conclusion. According to the results of the conducted molecular genetic research, the nucleotide sequences belonging to the mDNA COI region of species belonging to the genus Rhipicephalus were analyzed using bioinformatics methods. Nucleotide differences between species were found to range from 8.6% to 58.1%. In the phylogenetic tree, it was found that representatives of this family have 98%-100% bootstrap support.
REFERENCES
1. Barker S.C., Murrell A. (2004). Systematics and evolution of ticks with a list of valid genus and species names. Parasitology 129: s15—36.
2. Boom, R., Sol, C.J.A., et al., Rapid and simple method for purification of nucleic acids, J. Clin. Microbiol., Mar, 495-503, 1990.
3. Caporale DA, Rich SM, Spielman A, Telford IlIrd SR, Kocher TD. (1995). Discriminating between ixodes ticks by means of mitochondrial DNA sequences. Mol Phylogenet Evol 4:361-5.
4. Chen Z, Yang X, Bu F, Liu J. (2010). Ticks (acari: ixodoidea: argasidae, Ixodidae) of China. Exp Appl Acarol 51:393-404.
5. Chitimia L, Lin RQ, Cosoroaba I, Wu XY, Song HQ, Yuan ZG, Zhu XQ. (2010). Genetic characterization of ticks from southwestern romania by sequences of mitochondrial cox1 and nad5 genes. Exp Appl Acarol 52: 305-11.
6. Estrada-Peña A., Bouattour A., Camicas J.L., Walker A.R. Ticks of domestic animals in the Mediterranean region. A guide to identification of species // Univ. Zaragoza, Spain, 2004. - P. 131.
7. Feng Y, Li Q, Kong L, Zheng X. (2011a). DNA barcoding and phylogenetic analysis of pectinidae (mollusca: bivalvia) based on mitochondrial coi and 16s rrna genes. Mol Biol Rep 38:291-9.
8. Guglielmone A.A., Robbins R.G., Apanaskevich D.A., Petney T.N., Estrada-Pena A., Horak I.G., Shao R., Barker S.C. The Argasidae, Ixodidae and Nuttalliellidae (Acari:Ixodidae) of the world: a list of valid species names II Zootaxa, 2010.-V.2528.-P. 1-28.
9. Klompen J, Black IV WC, Keirans JE, Norris DE. (2000). Systematics and biogeography of hard ticks, a total evidence approach. Cladistics 16:79-102.
10. Kuklina T.E. Fauna of Ixodid ticks of Uzbekistan, Tashkent - 1976. 84-93. p.
11. Lv J, Wu S, Zhang Y, Chen Y, Feng C, Yuan X, et al. Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida). Parasit Vec. (2014) 7:1-11. 10.1186/1756-3305-7-93.
12. Marko, M.A., Chipperfield, R. and Birnboim, H.C., A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder, Anal. Biochem., 121, 382-387, 1982.
13. Reshetnikov A.D., Barashkova A.I. Method of collecting pasture ticks on a cylindrical drag // Russian Parasitological Journal. 2020. Volume 14. № 1. P. 41-45.
14. Schindel D, Miller S.E. (2005). DNA barcoding a useful tool for taxonomists. Nature 435:17.
15. Vogelstein, B. and Gillespie, D., Preparative and analytical purification of DNA from agarose, Proc. Natl. Acad. Sci. USA, 76, 615-619, 1979.
16. Walker A.R., Bouattour A., Camicas J.L., Estrada-Peña. A., Horak I.G., Latif A.A., Pegram R.G., Preston P.M:, Ticks of domestic animals in Africa: a guide to identification of species // Bioscience Reports, Edinburgh. 2003. P. 227.
17. Yarmukhamedova N.A., Mirzaeva A.U., Akramova F.J. Distribution of tick rickettsia in different regions of Samarkand region // Journal of biomedicine and practice, Tashkent -2022. P. 447-452.
