(Diptera; Culicidae) and Culex Tritaeniorhynchus (Diptera; Culicidae) Expansion in Areas in Mainland Greece and Islands

VECTOR-BORNE AND ZOONOTIC DISEASES Volume 17, Number 3, 2017 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2016.1974

Records of Aedes albopictus (Skuse, 1894) (Diptera; Culicidae) and Culex tritaeniorhynchus (Diptera; Culicidae) Expansion in Areas in Mainland Greece and Islands

Eleni Patsoula,1 Stavroula Beleri,1 Annita Vakali,2 Danai Pervanidou,2 Nikolaos Tegos,1 Andreas Nearchou,3 Demetrios Daskalakis,4 Spiros Mourelatos,5 and Christos Hadjichristodoulou6

Abstract

Annual entomological surveillance programs aiming to monitor mosquito populations and record presence and absence of mosquito species have been performed in Greece. We report, in this study, new records and expansion of Aedes albopictus in the islands of Lesvos (region of North Aegean), Crete (region of Crete), and the regional units of Rodopi in East Macedonia-Thrace. Furthermore, Culex tritaeniorhynchus was recorded for the ﬁrst time in Arta (region of Epirus) in northwestern Greece.

Keywords: Aedes albopictus, Culex tritaeniorhynchus, entomological surveillance, pathogen vector, Greece

Introduction et al. 2005, Patsoula et al. 2006) and is now established in the capital city of Athens (Attica region), and in many regional osquito surveillance programs have been im- units in Central Macedonia, Thessaly, and Peloponese (ECDC Mplemented on an annual basis in Greece, since 2010 VBORNET 2016, Giatropoulos et al. 2012a,b, Giatropoulos when the West Nile virus (WNV) epidemic was recorded for 2013, LifeConops 2015). Worldwide, it is important for public the first time in Northern Greece. From 2010 to 2013, these health since it is known to transmit arboviruses, such as Dengue, programs were performed by the Public Health authorities, Chikungunya, and Yellow fever. It is also a vector of filarial the regions, and the MALWEST program (Marka et al. 2013, nematodes, Dirofilaria spp. (Cancrini et al. 2003a,b, Gratz Patsoula et al. 2016). During 2014 and 2015 transmission 2004, Becker et al. 2010, and ECDC 2012). In recent years, periods, entomological surveillance activities were carried cases of Dengue and Chikungunya fever have been recorded in out only in small scale, with the participation and cooperation Europe (Marchand et al. 2013, INVS 2014, Paty et al. 2014). of the subcontractors for the vector control programs. These Culex(Culex) tritaeniorhynchus Giles is a part of the Culex programs were funded by regions/regional units throughout vishnui subgroup (Toma et al. 2000), distributed throughout Greece and local public health authorities, in collaboration the Asian region, extending into the Middle-east, the Medi- with the National School of Public Health (NSPH) and the terranean and Afrotropical region, China, Russia, Japan, Hellenic Center for Disease Control & Prevention (HCDCP). Korea, Indonesia (Lee et al. 1989), Northeast Africa, India, One of the main objectives of entomological surveillance is Iran, Iraq, Israel, Jordan, Lebanon, Maldives Islands, Mo- to record presence/absence of mosquito species, monitor zambique, Saudi Arabia, Sri Lanka, Syria, Turkey, and mosquito populations and detect the geographical distribution, Turkmenistan (Walter Reed Biosystematics Unit). In Europe, and possible expansion of mosquito species in new areas. it has been reported in Albania (Danielovi and Adhami 1960, Aedes (Stegomyia) albopictus (Skuse, 1894), the Asian tiger Adhami 1987) and Greece since 2003 from samples origi- mosquito, is considered to be the most invasive mosquito nating from a coastal marsh in the area of Marathon in the species in the world (Gratz, 2004). Ae. albopictus first ap- Prefecture of Attica (Samanidou and Harbach 2003). It has peared in western Greece, in 2003 (Samanidou-Voyadjoglou also been recorded in rice fields in the regional unit of

1Department of Parasitology, Entomology and Tropical Diseases, National School of Public Health, Athens, Greece. 2Hellenic Center for Disease Control and Prevention, Athens, Greece. 3Andreas Nearchou Co-operative NOVA FARM SA—Geotechno Ygeionomiki O.E, Xanthi, Greece. 4Igionomiki Kritis-Daskalakis Demetrios, Chania, Crete. 5Ecodevelopment SA, Filyro Thessalonikis, Thessaloniki, Greece. 6Department of Hygiene and Epidemiology, University of Thessaly Medical School, Larissa, Greece.

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Etoloakarnania, Western Greece, from 2008 to 2013 (Lytra The entomological surveillance areas were chosen after and Emmanouel 2014). Cx. tritaeniorhynchus is a vector of evaluation of the existing data for vector-borne diseases in Japanese Encephalitis (JE) (Bram 1967, Self et al. 1973) and humans and animals from previous transmission periods. isolates of other viruses, including Sindbis and Dengue, have However, it was not possible to conduct the entomological been found in this species (Lee et al. 1989). surveillance in all areas recommended by the entomological Our findings reflect the expansion and further establish- protocol. The surveillance was carried out by the contractor ment of these two species in new areas of the country, derived companies, which were awarded tenders for environmental from the national entomological surveillance programs. applications and mosquito control in the regions. The traps used were either CO2 and/or Triple traps according to the contrac- tors’ availability of the traps. The trap locations are presented in Materials and Methods Figures 1 and 2, for the years 2014 and 2015, respectively. Entomological surveillance was carried out from May to Traps were placed at dusk from *6:00 pm and were col- November (period varies per regional unit) and included lected the following morning at 9:00 am. The contents of the placing traps for adult mosquito collections and identification whole trap were placed in a plastic container on dry ice and of mosquitoes up to species level (Patsoula et al. 2016). The sent to the Laboratory of Medical Entomology in NSPH for results from the entomological surveillance activities were identification. All mosquitoes were identified to the species directly communicated to all the involved local authorities level using morphological characters (Darsie and Samanidou- (regions and regional units, Departments of Public Health Voyadjoglou 1997, Harbach 1998, Samanidou and Harbach and Social Welfare, Directorates of Sanitation Control). 2001, 2003, Schaffner et al. 2001). Ae. albopictus and Cx. Traps for adult mosquito collections were placed in rural, tritaeniochynchus adults, which were collected for the first urban, and periurban sites every 15 days, in 28 regional units time in areas of the country, were also verified by polymerase in 2014 and 19 regional units in 2015. In 2014, a median chain reaction (PCR) methods. number of 54 (range: 7–68) traps (CO2 and Triple traps) were DNA was extracted from one leg of individual adult placed throughout the study period in the 28 regional units, mosquitoes following the protocol described in Patsoula et al. while in 2015, a median number of 8 (range: 0–31) traps were 2006. The nuclear ribosomal spacer gene ITS2 region of the placed in the 19 regional units. rDNA gene was amplified by PCR using 5.8S and 28S

FIG. 1. Sites of adult mosquito collections, 2014. RECORDS OF A. ALBOPICTUS AND Cx. TRITAENIORHYNCHUS EXPANSION IN GREECE 219

FIG. 2. Sites of adult mosquito collections, 2015.

primers (Porter and Collins 1991, Wesson et al. 1992, Collins culipennis s.l., Anopheles claviger, Uranotaenia unguiculata, and Paskewitz 1996) and a region of the mitochondrial cy- Coquillettidia spp.). tochrome oxidase I gene (COI) was also amplified using In the context of the entomological surveillance for the primers C1-J-1718 and C1-N-2191 (Simon et al. 1994). PCR years 2014 and 2015, Culex pipiens s.l. was the most abun- was carried out with the protocol and cycling conditions as dant species (89% and 66.9%, respectively) and was col- described in Patsoula et al. 2006. The PCR products were run lected in all study areas. on 2% agarose gels. Gels were stained with ethidium bromide During the entomological surveillance programs, in 2014 and bands were visualized under UV transillumination. and 2015, Ae. albopictus populations were recorded in areas Aedes albopictus products were subjected to a restriction with no previous official records of the species. In addition, fragment length polymorphism assay (RFLP) for further Ae. albopictus has been reported in areas where it has been verification of the PCR results (Patsoula et al. 2006). PCR sporadically found and has been established by now (ECDC products for both genes were cleaned using a commercially VBORNER). available kit (DNA isolation spin kit agarose, Applichem, Ae. albopictus has been detected for the first time in the Germany), according to the manufacturer’s instructions to be regional unit of Chania in Crete for two consecutive years, in analyzed by sequencing. the island of Lesvos in the North Aegean region, and in the regional unit of Rodopi in East Macedonia & Thrace region. The species has already been introduced and further records Results were performed in the regional units of Halkidiki, Pieria, and A total of 47,039 mosquitoes were collected in 2014, be- Kilkis in the region of Central Macedonia, and in the regional longing to 14 mosquito species (Culex pipiens s.l., Culex units of Drama, Xanthi, in the East Macedonia & Thrace theileri, Aedes caspius, Aedes dorsalis, Aedes albopictus, region. Aedes detritus, Aedes vexans, Culiseta annulata, Culiseta In addition, Cx. tritaeniorhynchus mosquitoes were re- longiareolata, Anopheles sacharovi, Anopheles hyrcanus, corded for the first time in the regional unit of Arta (Epirus Anopheles maculipennis s.l., Anopheles superpictus, Ano- region). pheles claviger). In Tables 1 and 2, dates of mosquito collections, region, In 2015, 2183 mosquitoes were collected. They belonged to regional unit and municipality, the trap type that was used, 13 mosquito species and 1 genus (Culex pipiens s.l.,Culex environmental site, longitude and latitude, and the number tritaeniorhynchus, Aedes caspius, Aedes dorsalis, Aedes al- of adult mosquitoes captured/trap/night are presented for bopictus, Aedes detritus, Aedes vexans, Culiseta annulata, Ae. albopictus and Cx. tritaeniorhynchus, respectively. The Culiseta longiareolata, Anopheles sacharovi, Anopheles ma- geographical distribution of mosquito traps that collected

Table 1. Data of Expansion of Ae. albopictus in Greece, 2014 and 2015 Environmental Number of adults Date Region/regional unit Municipality Trap type type Longitude Latitude captured/gender

July 28, 2014 Central Macedonia/Halkidiki Kassandra CO2 Natural, rural 23.316153886 40.1075490902 1\Ae. albopictus August 4, 2014 Central Macedonia/Kilkis Kilkis CO2 Periurban 22.9117564772 40.8384139886 3\Ae. albopictus, 2_ Ae. albopictus August 27, 2014 Central Macedonia/Halkidiki Poligyros CO2 Natural, rural 23.5534832056 40.2864091863 1\Ae. albopictus November 18, 2014 Crete/Chania Apokoronou Triple Periurban 24.298257 35.314194 1\Ae. albopictus September 17, 2014 Central Macedonia/Kilkis Kilkis CO2 Periurban 22.9117564772 40.8384139886 3_ Ae. albopictus October 13, 2014 Central Macedonia/Kilkis Peonia CO2 Periurban, rural 22.596035255 40.8577630747 1_ Ae. albopictus June 24, 2015 North Aegean/Lesvos Kalloni CO2 Marsh 26.252809622709492 39.21446337747715 1\Ae. albopictus 220 August 18, 2015 Crete/Chania Chania Triple Periurban 24.059203900000057 35.4937784 2\Ae. albopictus, 2_ Ae. albopictus September 8, 2015 Crete/Chania Chania Triple Urban 24.22473 35.311038 2\Ae. albopictus November 3, 2015 Crete/Chania Chania Triple Periurban 24.2638 35.3605 1\Ae. albopictus, 1 _ Ae. albopictus July 23, 2015 East Macedonia &Thrace/Drama Drama CO2/dry ice Urban 24.143649200000027 41.1407833 2\Ae. albopictus August 11, 2015 East Macedonia &Thrace/Drama Drama CO2/dry ice Urban 24.143649200000027 41.1407833 1\Ae. albopictus August 11, 2015 East Macedonia &Thrace/Xanthi Xanthi CO2/dry ice Urban 24.864466200000038 41.1198327 2\Ae. albopictus 11-9-2015 East Macedonia &Thrace/Drama Drama CO2/dry ice Urban 24.143649200000027 41.1407833 1\Ae. albopictus September 11, 2015 East Macedonia &Thrace/Drama Doxato CO2/dry ice Urban 24.16009650000001 41.02100069999999 4\Ae. albopictus September 11, 2015 East Macedonia &Thrace/Drama Prosotsani CO2/dry ice Urban 23.933067700000038 41.1912229 1\Ae. albopictus September 11, 2015 East Macedonia &Thrace/Rodopi Komotini CO2/dry ice Urban 25.405614000000014 41.1182717 1\Ae. albopictus September 11, 2015 East Macedonia &Thrace/Xanthi Kvdira CO2/dry ice Urban 24.903537099999994 41.0902074 1\Ae.albopictus October 6, 2015 Crete/Chania Chania Triple Periurban 24.059203900000057 35.4937784 3_ Ae. albopictus RECORDS OF A. ALBOPICTUS AND Cx. TRITAENIORHYNCHUS EXPANSION IN GREECE 221

Table 2. Data on Cx. tritaeniorhynchus Mosquitoes in the Prefectural Unit of Arta, Prefecture of Epirus, Western Greece, 2015 Region/ regional Trap Environmental Number of Date unit Municipality type type Longitude Latitude adults captured August 24, 2015 Epirus/Arta Nikolaos Skoufas Triple Urban 210001.89 390331.62 40\ Cx. tritaeniorhynchus September 4, 2015 Epirus/Arta Nikolaos Skoufas Triple Urban 210001.89 390331.62 20\ Cx. tritaeniorhynchus September 5, 2015 Epirus/Arta Nikolaos Skoufas Triple Urban 210001.89 390622.95 29\ Cx. tritaeniorhynchus mosquitoes of the aforementioned species in different areas is 2014). Two COI and two ITS2 sequences have been sub- presented in Figure 3. mitted to the EMBL sequence database, with accession PCR amplification using both primer pairs (5.8S/28S and numbers LT221027-28 and LT221034-35, respectively. C1-J-1718/C1-N-219) produced fragments of the expected sizes, specific for Ae. albopictus. The RFLP assay verified Discussion and Conclusion that all samples examined were Ae. albopictus. Sequencing results of the four ITS2 and COI products revealed sequences Since the WNV outbreak in Greece in 2010 and the cases with 98–100% homology to the GenBank deposited ones for of autochthonous malaria due to Plasmodium vivax reported Ae. albopictus. One COI and four ITS2 sequences have been in Evrotas, Lakonia, there is increased concern among Public submitted to the EMBL sequence database, with the acces- Health authorities, regions regarding vector-borne diseases. sion numbers LT221029 and LT221030-33, respectively. Entomological surveillance programs are implemented to Sequencing of the two Cx. tritaeniorhynchus PCR prod- promptly monitor mosquito species’ presence/absence, de- ucts, examined for both genes, produced sequences sharing tect pathogen species, and support mosquito control actions. 95–99% homology with GenBank deposited ones. The two Our findings confirm the suspicion that Ae. albopictus has COI fragments share 99% similarities with GenBank se- been expanding in Greece, a fact that increases the possibility quences with accession numbers KC753196 and KC753197, for further local transmission of viruses transmitted through regarding Cx. tritaeniorhynchus samples collected in rice Ae. albopictus after their importation. Ae. albopictus is fields in Etoloakarnania, Greece (Lytra and Emmanouel known to be an effective vector of Dengue, Chikungunya,

FIG. 3. Geographical distribution of collection sites of Ae. albopictus and Cx. tritaeniorhynchus in new areas of the country. The yellow pins represent sites where Ae. albopictus adults were collected and red pins the sites of Cx. tritaeniorhynchus collections. 222 PATSOULA ET AL.

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