J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … 270 http://jad.tums.ac.ir Published Online: September 30, 2020 Original Article Detection of the Invasive Mosquito Species Aedes (Stegomyia) aegypti and Aedes (Hulecoeteomyia) koreicus on the Southern Coast of the Crimean Peninsula Lyudmila Ganushkina1; Alexander Lukashev1; *Ivan Patraman1; Vladimir Razumeyko2; Еlena Shaikevich1,3 1Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov First Moscow State Меdical University, Moscow, Russia 2Department of Ecology and Zoology Taurida Academia, Vernadsky Cremian Federal University, Simferopol, Republic of Crimea 3Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russia *Corresponding author: Dr Ivan Patraman, E-mail: ivanpatraman@gmail.com (Received 24 Sep 2018; accepted 05 Sep 2020) Abstract Background: The incidence and area of arbovirus infections is increasing around the world. It is largely linked to the spread of the main arbovirus vectors, invasive mosquito of the genus Aedes. Previously, it has been reported that Aedes aegypti reemerged in Russia after a 50-year absence. Moreover, in 2011, Ae. albopictus was registered in the city of Sochi (South Russia, Black Sea coast) for the first time. In 2013, Asian Ae. koreicus was found in Sochi for the first time. Methods: Mosquitoes were collected using the following methods: larvae with a dip net, imago on volunteers and using bait traps. The mosquitoes were identified using both morphology and sequencing of the second internal transcribed spacer of the nuclear ribosomal RNA gene cluster. Results: In August 2016, Ae. koreicus larvae and imago and a single male of Ae. aegypti were found on the southern coast of the Crimean Peninsula, where they were not registered before. Newly obtained DNA sequences were registered in GenBank with the accession numbers MF072936 and MF072937. Conclusion: Detection of invasive mosquito species (Ae. aegypti and Ae. koreicus) implies the possibility of their area expansion. Intensive surveillance is required at the Crimean Peninsula to evaluate the potential for the introduction of vector-borne diseases. Keywords: Mosquito monitoring; Arbovirus vectors; Aedes aegypti; Aedes koreicus; DNA Introduction At present, the invasive species Aedes (Steg- omyia) aegypti, Aedes (Stegomyia) albopictus and Aedes (Hulecoeteomyia) koreicus are spread- ing across the world and have been registered in Caucasian coast of the Black Sea in south- ern Russia since 2011 (1-4). All of them are known as vectors of human pathogens (4). In- vasive Aedes species have adapted its habitat preferences to human made containers, such as water storage tanks, discarded tires and jars, and water-filled pots (4). Due to the changing climate and the introduction of artificial hatch- ing habitats, it was expected that these mosquito species would also spread to the southern coast of Crimea, which has a climate very similar to Caucasus. In 2010, an entomological survey in the southern coast of Crimea found 15 blood- sucking mosquito species, namely three spe- cies of the genus Anopheles (An. claviger, An. maculipennis, An. plumbeus), 3 species of the genus Culex (Cx. hortensis, Cх. pipiens, Cх. territans), five species of the genus Aedes (Ae. refiki, Ae. cantans, Ae. dorsalis, Ae. cataphylla, Ae. geniculatus), three species of the genus Culiseta (Cs. annulata, Cs. longiareolata, Cs. morsitans) and Uranotaenia unguiculata (5). The coastal area of Crimea, especially the south- ern coast, is densely populated and attracts ap- Copyright © 2020 Iranian Scientific Society of Biology & Control of Diseases Vectors, and Tehran University of Medical Sciences. 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/ J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … 271 http://jad.tums.ac.ir Published Online: September 30, 2020 proximately five million tourists each year. The goal of the present study was to survey the southern coast of Crimea on the presence of invasive mosquito species. Materials and Methods Mosquito collection was carried out in Au- gust, 2016. Mosquito sampling sites are shown in figure 1. The larvae were collected with a dip net, while the imago were collected either on volunteers using sucking tubes or “Electro- frog” bait traps (LMD-Komplekt Plus, Russia). Mosquito sampling sites had different con- ditions. Priboy vacation resort (Number 1 in Table 1, Fig. 1) is located 300–500 meters to the northwest of Sakskoe Lake, one kilometer to the east is the resort city Saki, a town with well-developed residential areas. Frunzenets vacation resort (Number 2 in Table 1, Fig. 1) is located one kilometer away from a village on the shores of Lake Bagaily (Number 7 in Table 1, Fig. 1), which were overgrown with reeds and the inshore depth reached 1 meter. Mosquito larvae were collected in the thickets of the reeds. Departure of imago from the lake was registered. Imagoes were collected in the peak mosquito activity period from 8:30PM to 9:30PM. The mosquitoes were identified by morpho- logical key (7) and DNA sequencing. The most reliable characteristics for Ae. koreicus larvae were the form and number of сomb scales on the lateral view of larval abdominal segment (Fig. 2A), position of the siphonal tuft and the number of the pectin teech (Fig. 2B). Imago Ae. koreicus show a black and white pattern due to the presence of white scale patches on a black background and have clear longitudi- nal lines on the scutum (Fig. 2C). Two narrow white dorsocentral stripes are characteristic for Ae. aegypti (Fig. 2D). DNA extraction was performed using Dia- toms DNA prep kit (Isogen, Moscow). The Encyclo PCR kit (Evrogen, Moscow) and pri- mers complementary to 5.8S and 28S rRNA genes (8) were used for amplification of the se- cond internal transcribed spacer (ITS2) region of rRNA genes. Amplification products were visualized in 1% agarose gel and purified from the gel using a clean-up kit (Evrogen, Mos- cow). Sequencing was performed using ABI PRISM® 310 Genetic Analyzer with BigDye® Terminator kit (Applied Biosystems, USA). The obtained DNA sequences were registered in GenBank (accession numbers MF072936 and MF072937). The internet resource https://www.gismeteo.ru/diary/ was used for climate analysis. Results Imago and larvae of eight mosquito spe- cies, 661 individuals in total, were collected on the Crimean Black Sea coast and in Sim- feropol (Fig. 1, Table 1). In Simferopol, Cx. pipiens was collected in water storage tanks. In Lesnoy Village (№6 in Table 1, Fig. 1), located close to the town of Sevastopol, mosquitoes were collected in an artificial concrete basin with an area of 23m2 and a depth of 0.5m. In this artificial basin, the larval density per 1m2 was the highest: Ae. koreicus – 238 individuals; Cs. longiareolata – 256; and Cx. pipiens – 17. Among them, one male Ae. aegypti was dis- covered at Priboy vacation resort. Morpholog- ical identification was confirmed by DNA se- quencing. This Ae. aegypti ITS2 DNA sequence was deposited to GenBank (accession number MF072936). The ITS2 nucleotide sequence of the Crimean Ae. aegypti was identical to the one from Abkhazia, 2012 (MH142316) and Adler (Russia), 2013 (MH142319). There was one nucleotide substitution in ITS2 (99% identity) between Ae. aegypti found in Crimea (MF072936) and Ae. aegypti sequences regis- tered in GenBank: Rockefeller strain KF471581- KF471583; JX423805 and JX423807 from Saudi Arabia; KU497616 from China; and KF471579 from New Caledonia. Larvae of the species Ae. koreicus were found http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … 272 http://jad.tums.ac.ir Published Online: September 30, 2020 close to Sevastopol in Lesnoy Village (Table 1), in an artificial concrete basin. Aedes kore- icus imago were found next to this pond. The sequence of the ITS2 genome region of Ae. koreicus larvae found in Crimea was identical to the DNA sequence of Ae. koreicus from Belgium (KF471636 and JF430391) and Sochi (HG763830). The Crimean Ae. koreicus DNA sequence was deposited in GenBank with the accession number MF072937. In addition to these two invasive species, representatives of six autochthonous mosquito species were registered in the studied area. Ima- goes sampled at all locations were mainly rep- resented by the species Cx. pipiens, Cx. mod- estus, and a few individual Coquillettidia richi- ardii and Ae. caspius (Table 1). Culiseta longi- areolata and Cx. pipiens larvae were found in Lesnoy Village (Table 1). Fig. 1. Map of Crimean Peninsula with the mosquito sampling locations indicated as in Table 1. The map of Europe with the current known established populations of Aedes koreicus and Ae. aegypti in Europe (ECDC; 2020. Available from: https://ecdc.europa.eu/en/disease-vectors/surveillance-and-disease-data/mosquito-maps), Crimean Peninsula indicated with a circle. Locations of Ae. aegypti collection are shown with a square, Ae. koreicus - with a star. Fig. 2. Diagnostic characters of Aedes koreicus larva (A, B) and female (C), Ae. aegypti male (D) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … 273 http://jad.tums.ac.ir Published Online: September 30, 2020 Table 1. Composition of adults and larvae of 661 collected mosquitoes Number on the map Sample site Coordinates Mosquito species Ae. aegypti Ae. koreicus Ae. caspius Cx. pipiens Cx. modestus Cx. hortensis Cq. richiardii Cs. longiareolata 1 Priboy 45°13'05″N 33°52'22″ E 1♂ 3♀ 154♀ 5♂ 157♀4♂ 11♀ 2 Frunzenets 45°02′37″N 33°63′28″Е 1♀ 52♀3♂ 90♀3♂ 2♀ 3 Evpatoria 44°12'00″N 33°21'30″E 26♀ 21♀ 4 Koktebel 44°57'35″N 35°14'52″E 3♀ 3♀ 5 Simferopol 44°56'53″N 34°06'15″E 12♀10L 6 Sevastopol (Lesnoy Village) 44°43′55″N 33°34′55″E 3♀29L 2L 33L 7 Lake Bagaily 45°02′37″N 33°63′28″Е 8L 16L 9L Total number of individuals 1 32 4 275 294 9 13 33 Discussion Screening of mosquitos on the south coast of Crimean Peninsula found, among others, Ae. aegypti and Ae. koreicus, which have not been registered in Crimea before 2016 (5). In 2018, Kovalenko and Tikhonov (9) examined eight regions of the Crimea and collected sev- en Ae. koreicus females in Sevastopol and three in Simferopol. Although no Ae. koreicus lar- vae or breeding sites were found in this study, this data implied the spread of mosquitoes to the center of the peninsula. So, additional find- ing of Ae. koreicus imagoes in Crimea in 2018 confirms the establishment of Ae. koreicus pop- ulation in Crimea. Native areas of Ae. koreicus are Korean pen- insula, China, Japan, and the Russian Far East. Recently, Ae. koreicus has been expanding to new areas due to exobiological plasticity. The species Ae. koreicus was first found in the Eu- ropean part of Russia in 2013 in the center of the town of Sochi (4, 9, 10). At present, Ae. koreicus has been registered in many Euro- pean countries including Belgium, northern Italy, Hungary, Slovenia and Germany (11- 18). It is known that Ae. koreicus can with- stand lower winter temperatures and is drawn to populated areas located near woods. Its lar- vae, as well as those of Ae. aegypti and Ae. аlbopictus, develop in artificial basins. Fe- males usually attack in the open air, occasion- ally indoors. Mosquitoes overwinter in the egg stage. Aedes koreicus has shown vector com- petence in the field and in laboratory expe- riments for a number of pathogens including dengue, West Nile fever, Japanese encephalitis viruses and Dirofilaria (12, 19). Therefore, discovery of Ae. koreicus in Crimea marks a significant threat of the introduction and spread of vector-borne diseases. Detection of a single Ae. aegypti male can be attributed to either breeding in the close-by residential area, which provides artificial res- ervoirs serving as breeding habitats, or an iso- lated introduction, possibly from the Black Sea coast of the Caucasian region, where these mosquitoes were recently registered (2, 4, 6). The latter possibility has been exemplified pre- viously by discovery of a single Ae. aegypti male in Northwest England (53°30′42″N, 2°59′01″W) in 2014, where climate, tempera- ture and humidity were generally unsuitable for this new invasive mosquito (20). In Cri- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … 274 http://jad.tums.ac.ir Published Online: September 30, 2020 mea, where the Ae. aegypti male was discov- ered in the present study, the highest tempera- ture during the summer season was registered in August 2016. At the time of mosquito col- lection, the temperature reached 36 °C during daylight hours and 28–31 °C at night. The rel- ative humidity in July and August (67–68%) was uncomfortable for these mosquitoes. Dur- ing May–August, it rained for only 2 days and the total amount of precipitation over the year was approximately 400mm whereas in Sochi, where Ae. aegypti mosquitoes were previous- ly registered, the annual rainfall exceeds 1500 mm. It is believed that Ae. aegypti distribution is limited to areas with a January isotherm of 10 °C and mean annual temperatures of 15 °C, whereas in Crimea in the winter 2015–2016 the lowest winter temperature reached -15 °C, and in subsequent years it was between -6 °C and -7 °C. Unfavorable conditions may indi- cate that Ae. aegypti was an occasional intro- duction to the peninsula. To our knowledge, there is no published data on findings of Ae. aegypti in Crimea (https://ecdc.europa.eu, 2018). At the same time, Ae. aegypti is well estab- lished on the Black Sea coast in Caucasus (4), and is now spreading along the Black Sea coast of Turkey and Georgia (6, 21). In 2014, we suggested that Ae. albopictus mosquitoes were likely to appear in Crimea (22), but neither larvae nor imago of this spe- cies were discovered during the present study. Nevertheless, climate change and the detec- tion of the more cold - sensitive Ae. aegypti highlights the risk of Ae. albopictus rooting in Crimea and calls for detailed entomological monitoring on the peninsula. The predomi- nance of Сх. pipiens and Cx. modestus ima- goes can be explained by the proximity of the sampling areas Frunzenets and Priboy to lakes (Fig. 1), which are a favorable environment for the development of the genus Сulex lar- vae, as evidenced by the presence of Culex hortensis larvae at Bagaily Lake (Table 1). The absence of Cx. hortensis imago among the collected samples (as compared with the larvae samples) is due to the fact that these spe- cies are reluctant to attack humans and spend most of the time in caves and similar shel- tered environments. Conclusion In the current study, larvae breeding habi- tats for Ae. koreicus and Ae. aegypti imago have been found on the southern coast of the Crimean Peninsula for the first time. The Ae. aegypti species was represented by a single male specimen; therefore, it is not clear if it has yet been established in Crimea. Our study indicates that Ae. koreicus is locally estab- lished in Crimea and its northward expansion is expected, highlighting the challenges for vector surveillance and control programs. Acknowledgements This work was supported by the RFBR grants No.16-04-00091 and No.19-04-00739. We are grateful to “LMD-Komplekt Plus” company, namely to A.S. Poznyshev, S.I. Ko- zhurin and R.A. Mingaliev, for providing Elec- trofrog traps for mosquito collection. We de- clare that we have no conflict of interest. References 1. Ganushkina LA, Tanygina EYu, Bezzhono- va OV, Sergiev VP (2012) Detection of Aedes Stegomyia) albopictus Skuse Mos- quitoes in the Russian Federation. 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