J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 51 http://jad.tums.ac.ir Published Online: March 31, 2023 Original Article Genetic Structure of Aedes (Stegomyia) albopictus Populations in Russia *Elena Shaikevich1, Ludmila Karan2, Marina Fedorova2 1Vavilov Institute of General Genetics, Gubkin str. 3, 119333 Moscow, Russia 2Central Research Institute of Epidemiology, Novogireevskaya str., 3a, 111123 Moscow, Russia *Corresponding author: Dr Elena Shaikevich, E-mail: elenashaikevich@mail.ru (Received 23 Nov 2021; accepted 31 Jan 2023) Abstract Background: Aedes (Stegomyia) albopictus was found for the first time in 2011 on the Black Sea coast in Russia, and dur- ing 2011–2019, the species expanded over two climate zones Cfa and Csa. Methods: Here, we studied the sequence diversity of the mitochondrial cytochrome c oxidase I (COI) gene, 1317–1433bp in length. In total, 131 specimens of Ae. albopictus sampled from 21 locations in Russia and Abkhazia were examined. Results: Two of the six identified mitochondrial haplotypes were detected for the first time. Four COI haplotypes were shared by at least two studied local populations. The most prevalent H1 and H2 haplotypes dominated in all the sampled localities in the Cfa zone. The H3 haplotype was prevalent in the Csa zone. Other haplotypes were rare. Phylogenetic analyses, spatial isolation and limited gene flow revealed that the samples from the Csa zone differed significantly from those from the Cfa zone. Conclusion: Two spatially isolated genetic lineages exist in Ae. albopictus population in southern region of Russia. One lineage obtained on the seacoast and inland (in valleys of the Caucasus Mountains and steppe zone) is widely distribut- ed worldwide including Mediterranean populations. This confirms the hypothesis that the emergence of Ae. albopictus population in southern region of Russia may be associated with the terrestrial spread of mosquitoes from the well- established European population due to human activity. The other lineage, discovered in Novorossiysk, a maritime port, is similar to Ae. albopictus from the USA and Japan, suggesting the independent introduction of these mosquitoes. Keywords: Aedes albopictus; Distribution; Population genetics; COI; mtDNA Introduction Aedes (Stegomyia) albopictus (Skuse, 1895) is recognized as a competent vector of arbo- viruses, including dengue, chikungunya, Zika and yellow fever viruses (1). Being initially na- tive to restricted tropical regions of Southeast Asia, Ae. albopictus spread widely during the last 30 years in tropical and subtropical climates owing to its ecological features and human ac- tivity (2). In Europe, established populations of Ae. albopictus were recorded for the first time in Albania (1979) and Italy (1991), but currently, the species is reported to occur in countries of the Mediterranean basin, Balkans and central Eu- rope, including the Czech Republic, and Germa- ny (3–5). Aedes albopictus introduction and ex- pansion led to epidemics of chikungunya fever in Italy and France and to autochthonous cases of dengue in France, Croatia and Spain (6–9). On the Black Sea coast Ae. albopictus was first observed in 2011 in Bulgaria, Turkey, Geor- gia, Abkhazia and Russia, and the following year was found in Romania (4, 10–12). In Rus- sia, it was originally recorded in only one lo- cation, Adler, near the Abkhazia border, but in subsequent years, Ae. albopictus spread along the coast, being discovered in 2015 in Gelen- dzhik and in 2016 in Novorossiysk (13–14). Monitoring of the species range showed that during the period 2011–2019, Ae. albopictus ex- panded along the coast for 400km and inland for 200km, establishing breeding populations in valleys of the Caucasus Mountains and in the steppe zone of the Krasnodar krai (15). The widespread distribution of Ae. albopic- Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 52 http://jad.tums.ac.ir Published Online: March 31, 2023 tus in southern Russia enhances the risk of au- tochthonous transmission of dengue, chikungu- nya and Zika viruses in a large area on the Black Sea coast, especially in summer, when many tourists from different countries visit the region (16). Such scenarios of disease outbreak occurred in Europe and in the USA (Florida) (6). The epi- demic potential of Ae. albopictus depends on the mosquito population’s vector competence and vectorial capacity, which are thought to vary in invading populations according to their genetic background and geographic origin (17, 18). Hence, information on the genetic diversity of invasive populations is important for survey of Ae. albopictus populations and disease risk as- sessment. Currently, both nuclear and mitochondrial markers are used to analyse the genetic struc- ture and origin of invasive Ae. albopictus pop- ulations (19). The application of a nuclear mark- er, the ITS2 region of rRNA genes, displayed a low level of differentiation between geographical populations, indicating that highly related se- quences were distributed across native and in- vasive populations (19, 20). Analyses of mi- crosatellites and a panel of genome-wide distrib- uted Single Nucleotide Polymorphisms (SNPs) showed that low levels of intra-population dif- ferentiation were caused by multiple re-intro- ductions into invaded zones, which led to the formation of local populations consisting of in- dividuals with different origins (21–23). In Eu- rope, a high admixture and lack of geographical structure was found based on ITS2 and SNPs (20, 22). This result suggests a long-distance human-aided dispersal of Ae. albopictus in Eu- rope (22). Mitochondrial DNA genes are often used as genetic markers to test biodiversity, ancestry and demographic changes in populations due to their uniparental (maternal) inheritance and lack of recombination (24). The cytochrome c oxidase I (COI) gene in Ae. albopictus has been widely analysed not only for species identification as a BARCODE tool but also through phylogenetic and population studies (25–29). A short BAR- CODE fragment of 450–650bp was used to con- firm discovery of Ae. albopictus in Turkey (10), countries of the Balkan Peninsula (4, 30, 31). The limited sequence length and number of in- dividuals analysed did not reveal a high level of diversity. At the same time, it is shown that the long fragment (circa 1400bp) of COI gene can be used to analyse the population structure of Ae. albopictus. A high level of polymorphism was detected among long COI sequences of Ae. albopictus sampled in native areas in South Asian countries (25, 26, 32–34). More than a thousand published Ae. albopictus COI sequenc- es reflects the genetic diversity of populations both in native and non-native invasive areas, highlighting the reliability of the long COI frag- ment to identify genetic divergence among Ae. albopictus populations and facilitating the study of phylogeographic relationships and the prob- able origin of individuals in recently established populations (25, 27–29, 34). Our aim was to study the genetic diversity and population structure of Ae. albopictus in the Krasnodar krai, Russia, and in Abkhazia. Two hypotheses regarding the structure of Ae. al- bopictus populations can be assumed. The sim- ultaneous occurrence of the vector in the eastern Black Sea countries suggests a common single source of origin, from which the mosquitoes spread locally by different modes of transport. In this case, the population genetic diversity should be low, because the population has existed for less than 10 years, since 2011 (12). On the other hand, the possibility of independent importation by sea cannot be excluded, as both Russia and Abkhazia have ports on the Black Sea and then subpopulations and high genetic diversity can be expected due to multiple importations and mixing of individuals in local populations. To test these assumptions, we sampled mosquitoes in Krasnodar krai, Russia, and Abkhazia in 2018 and sequenced a long fragment of the COI gene. We compared Ae. albopictus COI haplotype com- position between local populations across the studied area and with the haplotypes known for other endemic and non-endemic territories. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 53 http://jad.tums.ac.ir Published Online: March 31, 2023 Materials and Methods Study area Our study sites covered an area from 45° 13’N to 43°10’ N and from 38°59’E to 40°20’ E (Fig. 1, Table 1). According to the Köppen- Geiger classification, most of this area lies in the Cfa climate zone, defined as temperate (C) climate without dry season (f) and with a warm summer (a) (35). The Caucasus Mountains cross the Cfa zone and form a watershed that divides the zone into three climate subzones. The cli- mate of seacoast of Abkhazia and the part of seacoast of Russia is close to subtropical cli- mate with an average January temperature of > 6 °C and annual rainfall above 1400mm and de- noted as the subzone Cfa-1 (Table 1). The val- leys located on the western and northern slopes of the mountains are characterized by a mild temperate climate (Cfa-2) with an average Jan- uary temperature of > 2.0 °C and annual rainfall of approximately 1000mm. The climate of the Zakubanskaya Plain located to the north and northwest of the Caucasus Mountains is mild continental (Cfa-3), with average January tem- peratures of > 0.0 °C and approximately 600– 700mm of precipitation per year. To the west of the Caucasus Mountains, in Novorossiysk and its surroundings, the climate is close to the dry Mediterranean type (Csa), with precipitation of the driest month in summer < 40mm. Mosquito sampling Mosquitoes were sampled at 17 sites in Kras- nodar krai, Russia, and three sites in Abkhazia in 2018 (Fig. 1). The collection points were at a sufficient distance to avoid collecting sib- lings. The sampling sites were combined into seven groups on the basis of territory (Table 1). The collections were performed in August- September, when the population density of mos- quitoes was expected to be the highest (36). Adult females were collected with aspirators and the human landing catch approach. For the col- lection of mosquito larvae, we inspected contain- ers with water in cemeteries, used tires and plas- tic containers in yards or along roadsides, buck- ets and other types of containers. Mosquito lar- vae and pupae were harvested, brought to a lab, reared to adults and identified by morphologi- cal identification keys (37). DNA extraction, amplification, and sequenc- ing Each mosquito was homogenized in 300µl of saline buffer. The homogenate was centri- fuged at 800g for 2min, and the supernatant was retained. Total DNA was extracted from 100µl of supernatant using the RiboPrep kit (Am- plisense, Moscow, Russia) according to the man- ufacturer’s instructions. PCR was carried out in a 25μl volume with an Encyclo Plus PCR kit (Evrogene, Moscow, Russia). The COI gene fragments were amplified using primers 1454F and 2160R and 2027F and 2886R according to the original protocol (25). The amplification products were sequenced with forward and re- verse primers for each primers pair. Sequenc- ing of amplicons was performed using an ABI PRISM 310 sequencer and a BigDye Termi- nation kit as recommended by Applied Biosys- tems (United States). The sequences were edited manually with Chromas and then aligned using MAFFT v7 (https://mafft.cbrc.jp). The total length of the COI gene sequences was 1317– 1433bp. The nucleotide sequences were depos- ited in GenBank under accession numbers MZ 501500-MZ501561. Sequences MH817490– MH817558 from our preliminary study (38) were also included in the analysis. A total 131 sequences from individual mosquitoes were used for the genetic analysis. Analysis of mtDNA data The length of the aligned analysed sequenc- es was 1317bp. DNA polymorphism was eval- uated using DnaSP v5 (39). DnaSP software was used also to evaluate the values of number of migrants (Nm), level of population differentia- tion (Fst) and neutrality tests of Tajima D and Fu’s Fs to assess the genetic differentiation among the populations and deviations from se- lective neutrality. A chi-squared test was used to show the significance of differences in the http://jad.tums.ac.ir/ https://www.multitran.com/m.exe?s=Republic%20of%20Abkhazia&l1=1&l2=2 J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 54 http://jad.tums.ac.ir Published Online: March 31, 2023 haplotype distributions. A BLAST search was performed against GenBank sequences to iden- tify genetically related published haplotypes (Fig. 2). Genealogical relationships among the haplotypes were constructed using a median joining (MJ) network of COI haplotypes gen- erated in NETWORK ver. 4.6 (40) with a 95% probability. To reveal the evolutionary relation- ships between individuals from the Russian pop- ulations and from known established populations in Europe, the New World and Asia, our data were combined with previously published COI data: 6 haplotype sequences found in this study plus 26 genetically related sequences from Gen- Bank were used in this analysis (Fig. 2). Results The cytochrome c oxidase I gene diversity A 1317bp fragment of the COI gene was sequenced from the 131 mosquitos, 10–34 spec- imens for each of 7 localities (Table 1). The likelihood of amplification of the pseudogene was rejected as no insertions, deletions or stop codons were found in any of the samples. Eight polymorphic nucleotide sites and six haplotypes (H) were detected, and two haplotypes were unique (Table 2). Data on the nucleotide diver- sity (Pi), the average number of nucleotide dif- ferences (K) and the observed haplotype di- versity (Hd) are shown in Table 3. The highest values of Hd (0.757) and Pi (0.00182) were found in the Csa zone (Table 3). No obvious de- parture from neutrality was found in the inves- tigated populations (Table 3), although Tajima’s D statistic values were slightly positive (0.89987, P> 0.10) for the local population from the Csa zone and slightly negative for the local popu- lation from the Cfa-2 zone (-1.21122, P> 0.10) (Table 3). Tajima’s D and Fu’s Fs test values for all populations were not significant. Among the six Ae. albopictus COI haplo- types discovered in our study, four were pre- viously known in Ae. albopictus populations worldwide. The COI haplotypes designated in our work as H1, H2 and H5 correspond to H03, H17 and H54, respectively (25). Haplotype H3 is identical to the 26_J-Wa1 (41) and H79 (27). Two haplotypes, H4 and H6, are described for the first time. Thus, our results show that Ae. albopictus mosquitos collected in Russia in 2018 are related to populations involved in the world- wide expansion of this species through temper- ate regions. Population structure and differentiation Four out of six COI haplotypes were shared by at least two local populations (Table 1). In the Cfa zone, haplotype H1 dominated in all the sampled localities, with the highest frequency recorded in the Cfa-3 subzone (85%, 29/34), fol- lowed by Cfa-2 (73%, 32/44) and Cfa-1 (68%, 19/28). In the Csa zone, only 20% of individ- uals had haplotype H1, which was significant- ly lower than in the Cfa zone (χ2= 7.5902, p< 0.05). No significant differences were observed in the distribution of haplotype H2, which was detected in 19% of individuals. The H3 haplo- type was found in 10.7% (14/131) of individ- uals, of which 71.4% were collected in No- vorossiysk and its surroundings, i.e. in the Csa zone. The difference in the distribution of the H3 haplotype in the Csa zone compared with that in the Cfa zone was significant (χ2= 15.6415, p< 0.01). The remaining haplotypes were rare (Table 1). Five haplotypes, H1-H5, were detect- ed in Ae. albopictus sampled along the seacoast both in Cfa-1 and Csa zones, and only two hap- lotypes, H1 and H2, were found in individuals sampled in the steppe zone of Krasnodar krai (Cfa-3). No genetic differentiation (Fst) was observed between the samples collected in the three Cfa climatic subzones. The migration (Nm) values between the Cfa-1, Cfa-2 and Cfa-3 local pop- ulations indicate frequent gene exchange be- tween these populations (Table 4). The samples from the Csa zone differed significantly from those from the Cfa zone (Table 4). Phylogenetic analysis The network of mitochondrial haplotypes was generated from six COI haplotypes obtained http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 55 http://jad.tums.ac.ir Published Online: March 31, 2023 in our study and 26 identical, close related (dif- fered by one nucleotide substitution) and con- necting sequences from GenBank (Fig. 2). The H1 haplotype is the most widely distributed in Russia and identical sequence was found in Asia (Japan, Taiwan, and China), America (the USA and Canada) and Europe (Albania, Italy, Greece and Portugal). The sequence identical to H2 was also reported from China, Taiwan, the USA, Canada and Italy. The H3 was found in Japan and in the USA, whereas the H5 was revealed in the USA. The unique H4 and H6 haplotypes are connected by one mutation with H3 and H5, respectively. A total 32 COI haplotype sequenc- es form two clusters in the network diagram. The first cluster includes the haplotypes H1 and H2 distributed in Europe and in other continents. The second cluster contains H3-H6 and COI haplotypes found mainly in the northeastern United States and Canada. The populations of Italy are the most studied in terms of the genetic diversity of Ae. albopictus, but no individuals with H3-H6 haplotypes were found here. Blast search based on the short and widely studied 450bp 5' COI gene fragment revealed that hap- lotypes H1 and H2 are distributed globally, in- cluding local European populations in Italy, Al- bania, Romania, Portugal, Serbia, Greece, Tur- key and Abkhazia (GenBank acc. numb. JF 810659, HF912379, HQ906848, MG198595- 600, JQ 412504-6, LN808745-46, MK518354, MK 995313). This short fragment of COI se- quences, BARCODE fragment, is considered in- sufficient for phylogenetic analysis of Ae. al- bopictus, nevertheless haplotypes H3-H6 are quite different from H1 and H2 in this 5' gene fragment as well. These nucleotide and amino acid differences are shown in Table 2 including variable nucleotide positions in sites 72, 103, 110 and 276. Table 1. Collection information and distribution of Aedes albopictus mtDNA COI gene haplotypes in Russia and Abkhazia Localities Climate zone/ subzone Mean January Temp. (T°C) Annual rainfall (mm) No. of studied specimens No. of specimens with haplo- types: H1 H2 H3 H4 H5 H6 1 Abkhazia Cfa-1 ≥ 6,0 1200-1400 12 7 5 2 Sochi Cfa-1 ≥ 6,0 1200-1400 16 12 1 3 3 Mountains-1 Cfa-2 ≥ 2,0 1200-1000 10 8 2 4 Mountains-2 Cfa-2 ≥ 2,0 800-1000 34 24 6 1 1 2 5 Krasnodar Cfa-3 ≥ 0,0 600-800 21 19 2 6 Adygeya Cfa-3 ≥ 0,0 600-800 13 10 3 7 Novorossiysk Csa ≥ 2,0 400-600 25 5 6 10 1 3 Total 131 85 25 14 1 4 2 Table 2. Variable sites of the Aedes albopictus COI gene sequences Haplotype No. of indi- viduals Variable nucleotide sites* АА** 72 103 110 276 732 861 888 1002 35 37 H1 85 T A T T A C T T I I H2 25 . . . . . T . . . . H3 14 . G . . G T . C V . H4 1 . G . . G T C C V . H5 4 . . C C . T . . . T H6 2 C . C C . T . . . T *Dots denote identity with the reference sequence (as in Zhong et al. 2013, GenBank accession no. JQ004525), and the base letters denote substitutions; **Variable amino acid sites http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 56 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 3. Estimates of genetic diversity for the mtDNA COI region for populations of Aedes albopictus Climate zone / subzone No. S Н Hd (±SD) К Pi (±SD) Tajima’s D Fu’s Fs Cfa-1 28 4 3 0. 5 (0.091) 1.048 0.0008 (0.00024) 0.05021 1.674 Cfa-2 44 7 5 0.445 (0.081) 0.891 0.00068 (0.00019) -1.21122 -0.548 Cfa-3 34 1 2 0.258 (0.086) 0.258 0.0002 (0.00019) 0.08512 0.555 Csa 25 7 5 0,757 (0.051) 2,393 0.00182 (0,00016) 0.89987 1.609 Total 131 8 6 0.534 (0.043) 1.294 0.00098 (0.00013) -0.27687 0.706 No.- number of tested specimens, S- number of polymorphic sites, Н- number of haplotypes, Hd- haplotype diversity, К- average number of nucleotide differences, Pi- nucleotide diversity (PiJC), Tajima's D and Fu's Fs statistics - neutrality tests, (Not significant, P> 0.10) Fig. 1. Map of the study area and sample sites. Isohytes show annual rainfall (mm), MJT - Mean January Temperature (T°C). The location of the region on the world map (below) is shown with a red circle http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 57 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 4. Pairwise differentiation (FST, below the diagonal), and gene flow (Nm, above the diagonal) among popula- tions of Aedes albopictus from different climatic zones/subzones Climate zone / subzone Fst (white) and Nm (gray) estimates Cfa-1 Cfa-2 Cfa-3 Csa Cfa-1 38.59 3.86 0.82 Cfa-2 0.00644 9.72 0.55 Cfa-3 0.06088 0.02507 0.34 Csa 0.23388 ** 0.31392 *** 0.42646 *** The significance of differences: ns - not significant; *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P< 0.001. Estimates of Fst: Csa-Cfa-1 Chi2= 15,817, P-value of Chi2: 0,0033 (P< 0.01) (df= 4); Csa-Cfa2 Chi2= 28,263, P-value of Chi2: 0,0000 (P< 0.001) (df= 5); Csa-Cfa3 Chi2= 66, P-value of Chi2: 0,0000 (P< 0.001) (df= 4) Fig. 2. The network diagram of the Aedes albopictus COI haplotypes obtained in this study and published data (25, 27, 28, 41). Colors correspond to different regions Discussion A high degree of genetic variability within and between populations of Ae. albopictus is usually observed in endemic areas. Thirty-five COI haplotypes of Ae. albopictus were detect- ed in the South Asian region, 33 in Malaysia, 42 in China, and 44 in Lao PDR (25, 26, 32, 34). In non-endemic areas where Ae. albopictus pop- ulations have recent origin and pass the bootle- neck, the genetic diversity is low. In Europe, 11 COI haplotypes were found in Italy (25). Five COI haplotypes were found in Portugal, being confirmed by subsequent analyses based on Ae. albopictus mitogenomes (28). In studied popu- lations, that spread along The Black Sea coast of the Caucasus, the genetic variability is also low that indicates its recent expansion. No significant differentiation was found among populations in Cfa zone despite the cli- matic differences between Cfa-1, Cfa-2 and Cfa- 3 subzones. The values of gene flow (Nm) and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 58 http://jad.tums.ac.ir Published Online: March 31, 2023 genetic differentiation (Fst) tests indicate the presence of a panmictic population in the Cfa zone (Table 4). Our conclusions are also support- ed by the results of the analysis of the genomes of individuals from Sochi and Krasnodar cities (Cfa zone), which showed that despite signifi- cant differences in the allele frequency spec- trum, Ae. albopictus from Sochi and Krasnodar were closely related, had a common origin and formed a clade related to mosquitoes from Italy and Greece (42). The lack of genetic structure or isolation by distance were observed in many studies on Ae. albopictus populations worldwide independently of whether the populations are na- tive or invasive. These data indicate ongoing and frequent gene flow among populations and seem to be due to a combination of low natural dis- persion capabilities and a high level of human- mediated spread (19). Haplotypes H1 and H2 dominated in all three Cfa subzones (Table 1) and these haplotypes are also wide distributed both in the native regions of Ae. Albopictus (Taiwan and China) and in the areas of intro- duction (the USA, Canada, and Europe) (25, 27, 41). These results suggest two possibilities for the origin of the Russian Ae. albopictus popu- lations in the Cfa zone. First, mosquitoes might have been introduced through seaports located on the Black Sea coast, such as Sochi, Russia and Batumi, Georgia from areas where Ae. al- bopictus with COI haplotypes H1 and H2 are widely distributed, for example, from Southeast Asia, China or the USA. Maritime transportation of tires and ornamental plants is known as the main dispersal mechanism of Ae. albopictus (43). On the other hand, we cannot completely rule out the possibility of European origin of Ae. albopictus Сfa population. In countries of the Black Sea basin the mosquito was record- ed first in 2005 in Greece, then in 2011 in Tur- key, Georgia and Abkhazia suggesting its rap- id and successive dispersion along the eastern Black Sea coast. The previous genetic studies confirmed the identity of short fragments of Ae. albopictus COI sequences, 450bp barcording fragment, from Greece, Turkey, Serbia, Roma- nia, and Abkhazia (4, 10, 30, 31, 44). The 1317 bp fragments, identical to our H1 and H2 se- quences, were also detected in Ae. albopictus from Albania, Greece and Italy (25, 41). Thus, the emergence of the Russian population can be associated with the terrestrial spread of mos- quitoes due to human activity. This mechanism of Ae. albopictus dispersion has been observed in France and Spain and was suggested for Ro- mania (4). If the mosquitoes of the Russian pop- ulation are of European origin, their vector com- petence to dengue, Chickungunya and Zika vi- ruses may be high enough for local transmis- sion in the case of the occurrence of infected people as it took place in Italy, France and oth- er Mediterranean countries (6). Our results show the presence of spatial iso- lation and limited gene flow between popula- tions from Cfa and Csa climatic zones. In Csa zone the haplotype diversity is twice as high as in the Cfa subzones with H3 being the most common haplotype, which is currently only found in the USA, Ohaio (27) and Japan, Waka- yama (41). Haplotype H5 is also found in No- vorossiysk and solely in USA, New York (25). These data indicate the presence of genetic struc- ture in Russian population. The COI-based pop- ulation structure has been found in Portugal, Chi- na, and Laos and is usually explained by climat- ic factors. Though the presence of a genetic structure in our population can also be associ- ated with adaptation to the dry Mediterranean climate of Novorossiysk, this explanation seems unlikely. The history of introduction of Ae. al- bopictus to Russia has only about 10 years, and in Novorossiysk mosquitoes were recorded on- ly in 2016. This time is not enough to accumu- late adaptive mutations which is known to take a long time to occur. The introduced popula- tions are more likely to establish if they already possess alleles advantageous in the invaded en- vironment (45). In Europe, the spread of the mosquito began after the importation in the 1990s in Italy, which became the source for the rest of the European populations (46). In subse- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 51–62 E Shaikevich et al.: Genetic Structure of … 59 http://jad.tums.ac.ir Published Online: March 31, 2023 quent period, the mosquito has spread widely in the areas with a dry Mediterranean climate, but haplotypes H3 and H5 distributed in No- vorossiysk population were found neither in neighbouring countries (Turkey and Greece) no in Europe in general. Presumably, these hap- lotypes are rare and either have not been in- troduced in Europe or have not a selective ad- vantage over other haplotypes in the specified climate. This suggests the independent introduc- tion of Ae. albopictus to Novorossiysk, the larg- est seaport in southern Russia. Conclusion The key finding of this work is the exist- ence of two spatially isolated genetic lineages in Ae. albopictus population in southern re- gion of Russia. Phylogenic analysis indicated that one of the lineages is similar to Ae. al- bopictus from USA and Japan whereas the sec- ond lineage is related to Ae. albopictus from Mediterranean countries. Our data for the first time confirm the hypothesis that the emergence of Ae. albopictus population in southern region of Russia may be associated with the terrestri- al spread of mosquitoes from the well-estab- lished European population due to human ac- tivity. This finding is important for predicting the spread of invasive Ae. albopictus mosqui- toes and the subsequent distribution of the med- ically important arboviruses that they transmit. Further research is needed to confirm this hy- pothesis. 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