Application of DNA Barcoding and Use of Molecular Markers in the Identification of Fleas

Subject Areas :

saeid  Mohammadi ¹ (Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa)
Hica  loteman ² (Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa)
Nijel  bent ³ (Department of Zoology ,University of johansborg, South Africa)
betin  van woren ⁴ (Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa)

Received: 2021-08-06 Accepted : 2021-09-16 Published : 2021-09-23

Keywords:

Abstract :

Introduction & Objective: Fleas are small insects of the order Siphonaptera, wingless and oval, their bodies are compressed on both sides. Their role in transmitting some pathogens and causing zoonotic diseases has been confirmed. Using molecular markers and genetic barcoding using one or more small pieces of DNA as a standardized part of the genome is a good classification method to identify species. The aim of this study was to evaluate the phylogenetic relationships of an endemic flea in South Africa using sequencing of part of the cytochrome oxidase subunit 1 (COI) gene using morphometric and molecular methods.Materials and Methods: This study was conducted in 2017 in the Telperian/Ezemvelo Nature Reserve in South Africa. Sherman live traps were used to catch rodents. Fleas were removed from their hosts using forceps, and mice were released at their point ofcapture within the same day. Fleas were stored in 70% alcohol and transferred to the laboratory for morphological and molecular analysis.Results: The results of morphological analysis of the collected samples showed that there is no sexual dimorphism between males and females of this species except for the body length variable (P <0.05). The phylogenetic tree showed a slight difference in the study population. Despite minor differences, all specimens were placed in a clad.Conclusion: The results of this study confirm that the COI sequences can be used to study the genealogical relationships of fleas.

References:

1.Abramoff MD., Magelhaes PJ., Ram SJ. (2004). Image processing with ImageJ. Biophotonics International, 11; 36-42.

2.Billeter SA., Levy MG., Chomel BB., Breitschwerdt, EB. (2008). Vector transmission of Bartonella species with emphasis on the potential for tick transmission. Medical and Veterinary Entomology, 22; 1–15.

3.Bitam I, Dittmar K, Parola P, Whiting MF, Raoult D. (2010). Fleas and flea-borne diseases. International Journal of Infectious Diseases, 14(8); 667–76.

4. Chomel BB., Boulouis HJ., Maruyama S., Breitschwerdt, EB. (2006). Bartonella spp in pets and effect on human health. Emerging Infectious Diseases, 12; 389–94.

5.Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoeket R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5); 294–9.

6.Haeselbarth E., Segerman J., Zumpt F. (1966). The arthropod parasites of vertebrates in Africa south of the Sahara (Ethiopian Region). Vol. III. (Insecta excl. Phthiraptera). Johannesburg: Publications of the South African Institute for Medical Research, 13; 52.

7. Hatyoka LM., Brettschneider H., Bennett NC., Kleynhans DJ., Muteka SP., Bastos ADS. (2019). Bartonella diversity and zoonotic potential in indigenous Tete Veld rats (Aethomys ineptus) from South Africa. Infection, Genetics and Evolution, 73; 44-48.

8.Hebert PDN., Ratnasingham S., deWaard JR. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society B (Suppl. 1), S96–S99.

9.Hornok S, Beck R, Farkas R, Grima A, Otranto D, Kontschan J, et al. (2018). High mitochondrial sequence divergence in synanthropic flea species (Insecta: Siphonaptera) from Europe and the Mediterranean. Parasites & Vectors, 11(1).

10.Khoobdel M., Shayeghi M., Piazak N., Bazrafkan S. (2011). Diversity and relative abundance of medically important fleas in the rural areas of Kohgiloye-andBoyerahmad, Iran. Journal of School of Public Health and Institute of Public Health Research, 9(3); 63-72.

11. Krasnov BR., Burdelov SA., Khokhlova IS., Burdelova NV. (2006). Sexual size dimorphism, morphological traits and jump performance in seven species of desert fleas (Siphonaptera). Journal of Zoology, 261; 181-189.

12.Lawrence, AL., Brown GK., Peters B., Spielman DS., Morin-adeline V., Sˇlapeta J. (2014). High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers. Medical and Veterinary Entomology, 28; 330–336.

13.Lewis RE. (1999). Resume of the Siphonaptera (Insecta) of the World. Medical Entomology, 35; 377-389.

14.Linardi, PM., Santos JL. (2012). Ctenocephalides felis felis vs. Ctenocephalides canis (Siphonaptera: Pulicidae): some issues in correctly identify these species. Rev Bras Parasitol Vet, 21(4); 345-54.

15.Matthee, S., Horak IG., Beaucournu JC., Durden LA., Ueckermann EA., McGeoch MA. (2007). Epifaunistic arthropod parasites of the four-striped mouse, Rhabdomys pumilio, in the Western Cape Province, South Africa. Journal of Parasitology, 93; 47-59.

16.Matthee S., Krasnov BR. (2009). Searching for generality in the patterns of parasite abundance and distribution: Ectoparasites of a South African rodent, Rhabdomys pumilio. International Journal for Parasitology, 39; 781-788.

17.Poulin R. (2007). Evolutionary ecology of parasites. Princeton University Press, Princeton, New Jersey, 342 pp.

18.Pretorius AM., Beati L., Birtles RJ. (2004). Diversity of Bartonellae associated with small mammals inhabiting Free State province, South Africa. International Journal of Systematic and Evolutionary Microbiology, 54; 1959-1967.

19. Saccaggi DL., Kruger K., Pietersen G. (2008). A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae). Bulletin of Entomological Research, 98; 27– 33.

20.Segerman J. (1995). Siphonaptera of Southern Africa. Handbook for the identification of fleas, Publications of The South African Institute for Medical Research No. 57. Johannesburg, South Africa: South African Institute for Medical Research.

21.Service MW. (2008). Medical entomology for students. 4th ed.UK: University Press, 110-16.

22.Tamura K., Nei M. (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10; 512-526.

23.van der Mescht L. (2015). Exploring mechanisms that shape Siphonaptera composition and distribution patterns on small mammals across South Africa. PhD Thesis. Stellenbosch University, South Africa.

24.van der Mescht L., Matthee S. (2017). Host range and distribution of small mammal fleas in South Africa, with a focus on species of medical and veterinary importance: Flea vector distribution in South Africa. Medical and Veterinary Entomology, 31; 402-413.

25.Whiting MF, Whiting AS, Hastriter MW, Dittmar K. (2008). A molecular phylogeny of fleas (Insecta: Siphonaptera): origins and host associations. Cladistics, 24(5): 677-707.

26.Ying MA., Hai-Long L., Jian H., Yan-Mei Z., Han-Qing Y., Liang L., Qi-Yong L. (2018). Molecular identification and phylogenetic analysis of 44 species of fleas in Qinghai Province, western China based on mtDNA COI gene sequence. Acta Ecologica Sinica, 61(4); 488-497.

27.Yssouf A., Socolovschi C., Leulmi H., Kernif T., Bitam I., Audoly G., Almeras L., Raoult D., Parola P. (2014). Identification of flea species using MALDITOF/MS. Comparative Immunology, Microbiology and Infectious Diseases, 37; 153–157.

28.Zurita A, Callejon R, Garcia-sanchez AM, Urdapilleta M, Lareschi M, Cutillas C. (2019). Origin, evolution, phylogeny and taxonomy of Pulex irritans. Medical and Veterinary Entomology, 33(2); 296-311