Invasive mosquito species Aedes albopictus and Aedes aegypti on the Black Sea coast of the Caucasus: genetics (COI, ITS2), Wolbachia and Dirofilaria infections

The area of invasive species Aedes albopictus and Aedes aegypti is expanding. Precise identification and understanding of the genetic diversity of invasive mosquito populations allows us to develop appropriate control methods. Endosymbiotic bacterium Wolbachia pipientis has different effects on their arthropod hosts and can influence the transmission and spread of the pathogens. The objective of the presented study was molecular-genetic identification of the Aedes mosquitoes collected in sampling sites on the Black Sea coast from 2007 to 2017; determination of genetic variability of Ae. aegypti, Ae. albopictus and their symbiotic bacteria Wolbachia; assessment of mosquitoes ability to be infected and to spread parasitic Dirofilaria. Another objective was obtaining the genetic characteristic of laboratory strain Ae. aegypti IMPITM. We investigated two markers of nuclear and mitochondrial DNA from Ae. albopictus and Ae. aegypti and compared them to DNA from Ae. cretinus and Ae. koreiсus sympatrically inhabiting the territory, as well as to one of Ae. aegypti from a laboratory line. The study of nuclear and mitochondrial DNA revealed a low level of variability in the invasive mosquitoes Ae. albopictus and Ae. aegypti collected at different collection sites and in different years. More than a half of Ae. albopictus were infected with Wolbachia, two strains of bacteria, wAlbA and wAlbB, occur in the Ae. albopictus population on the Black Sea coast. Total infection of Ae. aegypti and Ae. albopictus with dirofilariae was 1.8 %. Dirofilaria immitis was found only in mosquito abdomen, larvae of infective stage L3 were not found. D. repens larvae developed to the infective stage in the mosquitoes of both species.                                         

1. Porter C.H., Collins F.H. Species-diagnostic differences inaribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 1991;45:271-279.

2. Adhami J., Reiter P. Introduction and establishment of Aedes (Stegomyia) albopictus Skuse (Diptera: Culicidae) in Albania. J. Am. Mosq. Control Assoc. 1998;14(3):340-343.

3. Riabova T.E., Yunicheva I.V., Markovich N.I., Ganushkina L.A., Orabey V.G., Sergiev V.P. Detection of Aedes (Stegomyia) aegypti L. mosquitoes in Sochi City. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2005;3:3-5 (in Russian)

4. Bargielowski I.E., Lounibos L.P., Shin D., Smartt C.T., Carrasquilla M.C., Henry A., Navarro J.C., Paupy C., Dennett J.A. Widespread evidence for interspecific mating between Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in nature. Infect. Gen¬et. Evol. 2015; 36:456-61. DOI 10.1016/j.meegid.2015.08.016.

5. Rishniw M., Barr S.C., Simpson K.W., Frongillo M.F., Franz M., Alpizar J.L. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet. Parasitol. 2006;135:303-314. DOI 10.1016/j.vetpar.2005.10.013.

6. Bennett K.L., Shija F., Linton Y.M., Misinzo G., Kaddumukasa M., Djouaka R., Anyaele O., Harris A., Irish S., Hlaing T., Prakash A., Lutwama J., Walton C. Historical environmental change in Africa drives divergence and admixture of Aedes aegypti mosqui¬toes: A precursor to successful worldwide colonization? Mol. Ecology. 2016;25:4337-4354. DOI 10.1111/mec.13762.

7. Sergiev V.P., Supriaga V.G., Bronshtein A.M., Ganushkina L.A., Rakova V.M., Morozov E.N., Fedianina L.V., Frolova A.A., Morozova L.F., Ivanova I.B., Darchenkova N.N., Zhukova L.A. Results of studies of human dirofilariasis in Russia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2014;3:3-9. (in Russian)

8. Bocková E., Rudolf I., Kočišová A., Betášová L., Venclíková K., Mendel J., Hubálek Z. Dirofilaria repens microfilariae in Aedes vexans mosquitoes in Slovakia. Parasitol. Res. 2013;112:34653470. DOI 10.1007/s00436-013-3526-9.

9. Shaikevich E.V. PCR-RFLP of the COI gene reliably differentiates Cx. pipiens, Cx. pipiens form molestus and Cx. torrentium of the Pipiens Complex. Eur. Mosq. Bull. 2007;23:25-30.

10. Bogacheva A.S., Shaikevich E.V., Rakova V.M., Ganushkina L.A. The fauna of bloodsucking mosquitoes in the Nizhny Novgorod Region, their infection with Dirofilaria and endosymbiotic bacteria. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2017;1:43-47. (in Russian)

11. Simon C., Frati F., Beckenbach A., Crespi B., Liu H., Flook P. Evo¬lution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 1994;87(6):651-701.

12. Bourtzis K., Dobson S.L., Xi Z., Rasgon J.L., Calvitti M., Moreira L.A., Baton L.A., Hughes G.L., Mavingui P., Gilles J.R. Harnessing mosquito-Wolbachia symbiosis for vector and disease control. Acta Trop. 2014;132:150-163. DOI 10.1016/j.actatropica.2013.11.004.

13. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013;30:2725-2729. DOI 10.1093/molbev/mst197.

14. Braig H.R., Zhou W., Dobson S.L., O’Neill S.L. Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J. Bacteriol. 1998;180:2373-2378.

15. Tokarev Y., Yudina M., Malysh J., Bykov R., Frolov A., Grushevaya I., Ilinsky Y. Prevalence rates of the Wolbachia endosymbiotic bacterium in natural populations of Ostrinia nubilalis and Ostrinia scapulalis (Lepidoptera: Pyraloidea: Crambidae) in South-Western Russia. Ekologicheskaya Genetika = Ecological Genetics (St. Petersburg). 2017;15(1):44-49. DOI 10.17816/eco¬gen15144-49. (in Russian)

16. Calba C., Guerbois-Galla M., Franke F., Jeannin C., Auzet-Caillaud M., Grard G., Pigaglio L., Decoppet A., Weicherding J., Savaill M.C., Munoz-Riviero M., Chaud P., Cadiou B., Ramalli L., Fournier P., Noël H., De Lamballerie X., Paty M.C., Leparc-Goffart I. Preliminary report of an autochthonous chikungunya outbreak in France, July to September 2017. Euro Surveill. 2017;22(39):17-00647. DOI 10.2807/1560-7917.ES.2017.22.39.17-00647.

17. Tsai P.-J., Lin T.-H., Teng H.-J., Yeh H.-C. Critical low temperature for the survival of Aedes aegypti in Taiwan. Parasit. Vectors. 2018; 11(1):22. DOI 10.1186/s13071-017-2606-6.

18. Calvitti M., Marini F., Desiderio A., Puggioli A., Moretti R. Wolbachia density and cytoplasmic incompatibility in Aedes albopictus: Concerns with using artificial Wolbachia infection as a vector suppression tool. PLoS One. 2015;10(3):e0121813. DOI 10.1371/journal.pone.0121813.

19. Yunicheva I.V., Ryabova T.E., Markovich N.I., Bezzhonova O.V., Ganushkina L.A., Semenov V.B., Tarkhov G.A., Vasilenko L.E., Guzeeva T.M., Sergiev V.P. First evidence for Aedes aegypti L. propagation in Greater Sochi and in some towns of Abkhasia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2008;3:40-43. (in Russian)

20. Cancrini G., Frangipane di Regalbono A., Ricci I., Tessarin C., Gabrielli S., Pietrobelli M. Aedes albopictus is a natural vector of Dirofilaria immitis in Italy. Vet. Parasitol. 2003;118(3-4):195202.

21. Zabashta M.V. The expansion of Aedes (Stegomia) albopictus Skuse, 1885 on the Black Sea coast of Russia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2016;3:10-11. (in Russian)

22. Carrasquilla M.C., Lounibos L.P. Satyrization without evidence of successful insemination from interspecific mating between invasive mosquitoes. Biol. Lett. 2015;11(9):20150527. DOI 10.1098/rsbl.2015.0527.

23. Zhou W., Rousset F., O’Neil S. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. B. 1998;265(1395):509-515.

24. Chuchuy A., Rodriguero M.S., Ferrari W., Ciota A.T., Kramer L.D., Micieli M.V. Biological characterization of Aedes albopictus (Diptera: Culicidae) in Argentina: implications for arbovirus transmission. Sci. Rep. 2018;8(1):5041. DOI 10.1038/s41598018-23401-7.

25. Zitko T., Kovacic A., Desdevises Y., Puizina J. Genetic variation in East-Adriatic populations of the Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae), inferred from NADH5 and COI sequence variability. Eur. J. Entomol. 2011;108(4):501-508. DOI 10.14411/eje.2011.065.

26. Costa-da-Silva A.L., Ioshino R.S., Araújo H.R., Kojin B.B., Zanotto P.M., Oliveira D.B., Melo S.R., Durigon E.L., Capurro M.L. Laboratory strains of Aedes aegypti are competent to Brazilian Zika virus. PLoS One. 2017;12(3):e0174081. DOI 10.1371/journal.pone.0174081.

27. Dallimore T., Hunter T., Harbach R.E., Medlock J.M., Strode C., Vaux A.G. Discovery of a single male Aedes aegypti (L.) in Merseyside, England. Parasit. Vectors. 2017;10:309. DOI 10.1186/s13071-017-2251-0.

28. Delatte H., Paupy C., Dehecq J.S., Thiria J., Failloux A.B., Fontenille D. Aedes albopictus, vector of chikungunya and dengue viruses in Reunion Island: biology and control. Parasite. 2008;15(1):3-13. DOI 10.1051/parasite/2008151003.

29. Delisle E., Rousseau C., Broche B., Leparc-Goffart I., L’Ambert G., Cochet A., Prat C., Foulongne V., Ferre J.B., Catelinois O., Flusin O., Tchernonog E., Moussion I.E., Wiegandt A., Septfons A., Mendy A., Moyano M.B., Laporte L., Maurel J., Jourdain F., Reynes J., Paty M.C., Golliot F. Chikungunya outbreak in Montpellier, France, September to October 2014. Euro Surveill. 2015;20(17):pii=21108. DOI 10.2807/1560-7917.ES2015.20.17.21108.

30. Ermakova L.A., Tverdokhlebova T.I., Nagorny S.A., Pshenichnaya N.Yu., Boltachiev K.Kh. Analysis of incidence of larvae helminthiases (Echinococcosis, Toxocariasis, Dirofilariasis) in humans in the Russian Federation. Epidemiologiya i Vaktsynoprofilaktika = Epidemiology and Vaccinoprophylaxis. 2017;16(1):43-46. (in Russian)

31. Fedorova M.V., Ryabova T.E., Shaposhnikova L.I., Lopatina Yu.V., Sebentzova A.N., Yunicheva Yu.V. Invasive mosquito species in Sochi: larval development sites and counting methods. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2017a;4:9-15. (in Russian)

32. Fedorova M.V., Shvez O.G., Yunicheva Yu.V., Ryabova T.E., Medyanik I.М. Spreading of invasive mosquitoes Aedes (Stegomyia) aegypti (L., 1762) and Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera, Culicidae) in the southern Krasnodar region, Russia. Proc. of the II Symposium “Modern problems of gen-eral and special parasitology”. St. Petersburg, 2017b;268-271. (in Russian)

33. Ganushkina L.A., Bezzhonova O.V., Patraman I.V., Tanygina E., Sergiev V.P. Distribution of Aedes (Stegomyia) aegypti L. and Aedes (Stegomyia) albopictus Skuse mosquitoes on the Black Sea coast of the Caucasus. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2013;1:45-46. (in Russian)

34. Ganushkina L.A., Morozova L.F., Patraman I.V., Sergiev V.P. Assessment of the risk of expansion of the habitats of the mosquitoes Aedes aegypti L. and Aedes albopictus Skuse in Russia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2014b;4:8-10. (in Russian)

35. Ganushkina L.A., Patraman I.V., Rezza G., Migliorini L., Litvinov S.K., Sergiev V.P. Detection of Aedes aegypti, Aedes albopictus, and Aedes koreicus in the area of Sochi, Russia. Vector-Borne Zoonotic Dis. 2016;16(1):58-60. DOI 10.1089/vbz.2014.1761.

36. Ganushkina L.A., Rakova V.M., Ivanova I.B., Supryaga V.G., Sergiev V.P. Entomological monitoring of an area to assess Dirofilaria transmission risk. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2014a;3:9-12. (in Russian)

37. Ganushkina L.A., Tanygina E., Bezzhonova O.V., Sergiev V.P. Detection of Aedes (Stegomyia) albopictus Skuse mosquitoes in the Russian Federation. Meditsinskaya Parazitologiya i Paraz¬itarnye Bolezni = Medical Parasitology and Parasitical Disease. 2012;1:3-4. (in Russian)

38. Gutsevich A.V., Monchadskiy A.S., Shtakelberg A.A. Fauna of the USSR: Diptera. Mosquitoes. Vol. III, Iss. 4. Leningrad: Nauka Publ., 1970. (in Russian)

39. Jeffries C.L., Walker T. Wolbachia biocontrol strategies for arboviral diseases and the potential influence of resident Wolbachia strains in mosquitoes. Curr. Trop. Med. Rep. 2016;3:20-25. DOI 10.1007/s40475-016-0066-2.

40. Kamgang B., Brengues C., Fontenille D., Njiokou F., Simard F., Paupy C. Genetic structure of the tiger mosquito, Aedes albopictus, in Cameroon (Central Africa). PLoS One. 2011;6(5):e20257. DOI 10.1371/journal.pone.0020257.

41. Kampen H., Jansen S., Schmidt-Chanasit J., Walther D. Indoor development of Aedes aegypti in Germany, 2016. Euro Surveill. 2016; 21(47):30407. DOI 10.2807/1560-7917.ES.2016.21.47.30407.

42. Kartashev V., Sagach O., Nikolaenko S., Chizh N., Korzan A., Ambalov Y., Bastrikov N., Ilyasov B., González-Miguel J., Morchón G., Siles-Lucas M., Simon F. Emerging Human dirofilariasis as a medical problem. Proc. of 28th Annual Meeting of the German Society for Parasitology. Berlin, 21–24 March. 2018;161.

43. Kronefeld M., Kampen H., Sassnau R., Werner D. Molecular evidence for the occurrence of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany. Parasit. Vectors. 2014;7:30. DOI 10.1186/1756-3305-7-30.

44. Kuno G. Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains. J. Med. Entomol. 2010;47:957-971. DOI 10.1603/ME10152.

45. Marzinovsky E.I. Measures against dengue fever in Russia. Russian Journal of Tropical Medicine, Medical and Veterinary Parazitology. 1929;7(3):162-165. (in Russian)

46. Medlock J.M., Hansford K.M., Versteirt V., Cull B., Kampen H., Fontenille D., Hendrickx G., Zeller H., Van Bortel W., Schaffner F. An entomological review of invasive mosquitoes in Europe. Bull. Entomol. Res. 2015;105(6):637-663. DOI 10.1017/S0007485315000103.

47. Minard G., Van V.T., Tran F.H., Melaun C., Klimpel S., Koch L.K., Ly Huynh Kim K., Huynh Thi Thuy T., Tran Ngoc H., Potier P., Mavingui P., Valiente Moro C. Identification of sympatric cryptic species of Aedes albopictus subgroup in Vietnam: new perspectives in phylosymbiosis of insect vector. Parasit. Vectors, 2017;10(1):276. DOI 10.1186/s13071-017-2202-9.

48. Mousson L., Dauga C., Garrigues T., Schaffner F., Vazeille M., Failloux A.-B. Phylogeography of Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) based on mitochondrial DNA variations. Genet. Res., Camb. 2005;86(1):1-11. DOI 10.1017/S0016672305007627.

49. Patsoula E., Samanidou-Voyadjoglou A., Spanakos G., Kremastinou J., Nasioulas G., Vakalis N.C. Molecular and morphological characterization of Aedes albopictus in northwestern Greece and differentiation from Aedes cretinus and Aedes aegypti. J. Med. Entomol. 2006; 43(1):40-54.

50. Porter C.H., Collins F.H. Species-diagnostic differences inaribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 1991;45:271-279.

51. Riabova T.E., Yunicheva I.V., Markovich N.I., Ganushkina L.A., Orabey V.G., Sergiev V.P. Detection of Aedes (Stegomyia) aegypti L. mosquitoes in Sochi City. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2005;3:3-5 (in Russian)

52. Rishniw M., Barr S.C., Simpson K.W., Frongillo M.F., Franz M., Alpizar J.L. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet. Parasitol. 2006;135:303-314. DOI 10.1016/j.vetpar.2005.10.013.

53. Sergiev V.P., Supriaga V.G., Bronshtein A.M., Ganushkina L.A., Rakova V.M., Morozov E.N., Fedianina L.V., Frolova A.A., Morozova L.F., Ivanova I.B., Darchenkova N.N., Zhukova L.A. Results of studies of human dirofilariasis in Russia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2014;3:3-9. (in Russian)

54. Shaikevich E.V. PCR-RFLP of the COI gene reliably differentiates Cx. pipiens, Cx. pipiens form molestus and Cx. torrentium of the Pipiens Complex. Eur. Mosq. Bull. 2007;23:25-30.

55. Simon C., Frati F., Beckenbach A., Crespi B., Liu H., Flook P. Evo¬lution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 1994;87(6):651-701.

56. Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013;30:2725-2729. DOI 10.1093/molbev/mst197.

57. Tokarev Y., Yudina M., Malysh J., Bykov R., Frolov A., Grushevaya I., Ilinsky Y. Prevalence rates of the Wolbachia endosymbiotic bacterium in natural populations of Ostrinia nubilalis and Ostrinia scapulalis (Lepidoptera: Pyraloidea: Crambidae) in South-Western Russia. Ekologicheskaya Genetika = Ecological Genetics (St. Petersburg). 2017;15(1):44-49. DOI 10.17816/eco¬gen15144-49. (in Russian)

58. Tsai P.-J., Lin T.-H., Teng H.-J., Yeh H.-C. Critical low temperature for the survival of Aedes aegypti in Taiwan. Parasit. Vectors. 2018; 11(1):22. DOI 10.1186/s13071-017-2606-6.

59. Yunicheva I.V., Ryabova T.E., Markovich N.I., Bezzhonova O.V., Ganushkina L.A., Semenov V.B., Tarkhov G.A., Vasilenko L.E., Guzeeva T.M., Sergiev V.P. First evidence for Aedes aegypti L. propagation in Greater Sochi and in some towns of Abkhasia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2008;3:40-43. (in Russian)

60. Zabashta M.V. The expansion of Aedes (Stegomia) albopictus Skuse, 1885 on the Black Sea coast of Russia. Meditsinskaya Parazitologiya i Parazitarnye Bolezni = Medical Parasitology and Parasitical Disease. 2016;3:10-11. (in Russian)

61. Zhou W., Rousset F., O’Neil S. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. B. 1998;265(1395):509-515.

62. Zitko T., Kovacic A., Desdevises Y., Puizina J. Genetic variation in East-Adriatic populations of the Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae), inferred from NADH5 and COI sequence variability. Eur. J. Entomol. 2011;108(4):501-508. DOI 10.14411/eje.2011.065.