Established Population of the Invasive Mosquito Species Aedes albopictus in Romania, 2012–14 Author(s): Liviu F. Prioteasa, Sorin Dinu, Elena Fălcuţă, and Cornelia S. Ceianu Source: Journal of the American Mosquito Control Association, 31(2):177-181. Published By: The American Mosquito Control Association DOI: http://dx.doi.org/10.2987/14-6462R URL: http://www.bioone.org/doi/full/10.2987/14-6462R
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
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Journal of the American Mosquito Control Association, 31(2):177–181, 2015 Copyright E 2015 by The American Mosquito Control Association, Inc.
SCIENTIFIC NOTE ESTABLISHED POPULATION OF THE INVASIVE MOSQUITO SPECIES AEDES ALBOPICTUS IN ROMANIA, 2012–14 LIVIU F. PRIOTEASA,1 SORIN DINU,1,2 ELENA FA ˘ LCUT ¸A ˘1
AND
CORNELIA S. CEIANU1,3
ABSTRACT. During an entomological investigation carried out in Bucharest and surroundings in fall of 2012, 45 adult mosquitoes (38 females and 7 males) of Aedes albopictus were collected in a neighborhood from the southern area of the city. The morphological identification of the species was further confirmed by sequencing 2 mitochondrial DNA markers: the cytochrome c oxidase subunit I and NADH dehydrogenase subunit 5 genes. Aedes albopictus was collected again in 2013 in the same area from July until October. During late summer the species was found also in another location in the city, downtown Bucharest. Larvae were found in water barrels and other types of household containers, as well as in rain catch basins. In 2014, following a nuisance complaint of a Bucharest inhabitant, the entomological investigation found aggressive Ae. albopictus adults on his property that harbored many mosquito larvae in container-type breeding habitats. These findings are the 1st records of this invasive species and of its breeding population in Romania, and show maintenance of the species over 2 winter seasons. Surveillance of the species outside the area of the capital city was not performed, therefore it is not known whether Ae. albopictus has been introduced in other regions of the country. The presence of Ae. albopictus has been reported every year (2012–14) to competent public health authorities, stressing on the importance of surveillance and of implementation of control measures. KEY WORDS
Aedes albopictus, Asian tiger mosquito, invasive species, Romania, Bucharest
The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), (Stegomyia albopicta sensu Reinert et al. 2004), is a species native to temperate and tropical Asia. In the past 3 decades it has rapidly extended its distribution range, first in the New World and subsequently in Europe. Intercontinental expansion has been mainly attributed to a global trade of used tires (Reiter 1998) and intracontinental spread by passive transportation of Ae. albopictus in ground vehicles from infested areas into new areas (Scholte et al. 2008). The species was 1st detected in Europe in Albania in 1979, and the 1st subsequent record of its presence in Europe was in Italy in 1990. Since then it has spread with remarkable speed and is now present, often abundant, in Belgium (not established), Bosnia and Herzegovina, Croatia, France, Germany (not established), Greece, Malta, Monaco, Montenegro, the Netherlands (not established), San Marino, Serbia, Slovenia, Spain, Switzerland, Turkey, and Vatican City, and was recently recorded in Bulgaria, Czech Republic (not established), and southern Russia (Ganushkina et al. 2012, Medlock et al. 2012, Sˇebesta et al. 2012). In Europe, this vector species was respon1 Cantacuzino National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent¸ei, 050096, Bucharest, Romania. 2 Genetics Department, Faculty of Biology, University of Bucharest, 1–3 Aleea Portocalelor, 060101, Bucharest, Romania. 3 To whom correspondence should be addressed.
sible for an epidemic of chikungunya fever with 217 confirmed cases in northern Italy in 2007 (Angelini et al. 2008), and 2 chikungunya fever cases locally transmitted from an imported case in France in 2010 (Grandadam et al. 2011). Autochthonous cases of dengue were reported in the south of metropolitan France in 2010 (La Ruche et al. 2010; Marchand et al. 2013), as well as in Croatia in 2010 (Gjenero-Margan et al. 2011). Here we report the 1st detection of Ae. albopictus in Bucharest, capital city of Romania, in autumn of 2012, and species activity recorded in 2013 and 2014. An entomological survey for West Nile virus vectors was carried out between August 26 and October 5, 2012, in the city of Bucharest and its suburban area, with 14 collection sites. Three types of mosquito traps were used: Centers for Disease Control and Prevention (CDC) gravid traps (John W. Hock Company, Gainesville, FL), CDC light traps (John W. Hock Company), and BG Sentinel traps with BG-lure (Biogents AG, Regensburg, Germany). Between September 4 and October 3, 2012, 38 Ae. albopictus females and 7 males were captured (Table 1). All Ae. albopictus individuals were captured at 7 collection points covering an area of about 3 km2 in the south of Bucharest city (Fig. 1). Most of them were caught with CDC gravid traps and only a few with BG Sentinel traps. Identification of the captured mosquitoes was performed using morphological identification keys (Becker et al. 2010). For the molecular identification, DNA was extracted each year
177
178
JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION Table 1.
Data on Aedes albopictus collected in different traps in Bucharest, Romania, 2012–14. No. of adult males
Year 2012 2013
2014
VOL. 31, NO. 2
Month September October July August September October August–September
No. of adult females
BG Sentinel traps
CDC gravid traps
BG Sentinel traps
CDC gravid traps
Preimaginal stages
2 1 1 — — — —
4 — 12 6 — — 3
4 3 — — — — —
30 1 24 42 — 2 35
— — — 102 2 — 70
(2012–14) from 3 female mosquitoes, and 2 mitochondrial DNA markers spanning cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes were amplified and sequenced with 2 sets of primers as previously described (Bonacum et al. 2001, Birungi and Munsterman 2002). The COI sequences were used for species-level identification with BOLD v3.0 Beta (www.boldsystems.org.). Both COI and ND5 sequences derived from all samples were identical. One sequence for each mitochondrial DNA marker (2012–14) was deposited in GenBank under the following accession numbers: HF536717, LN808745, LN808746 (COI) and HF536718, LN808747, LN808748 (ND5). Gene sequences of Ae. albopictus from
Romania are similar to sequences obtained from mosquitoes collected in temperate and subtropical regions. One hundred percent identity was observed between the sequences derived from Ae. albopictus specimens captured in Romania and those obtained from mosquitoes collected in Europe (Albania, France, Italy, Netherlands, and Greece), and also from some populations of Madagascar, La Reunion, North America, and Hawaii. In 2013, mosquito collection activity resumed on June 17 and continued until the end of October. Human-landing catches were performed on 4 different days in July 2013 and potential habitats for immature stages were investigated. Aedes albopictus was found at the same collection
Fig. 1. Distribution of areas found infested with Aedes albopictus in Bucharest in 2012–14 (hatched), 2013–14 (dotted), and 2014 (grid).
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SCIENTIFIC NOTE
points in downtown Bucharest as previously found in 2012 (Fig. 1). Immature stages were found as well, mostly in mixed populations with Culex pipiens (L.) either in domestic containers or rain catch basins (Table 1). Monitoring an area in the proximity of Bucharest, in Ilfov County, did not yield any Ae. albopictus. Thus, positive sites were all located in urban environment. In 2014, Ae. albopictus adults were sporadically captured from July through October in the same area. At the end of summer, a complaint of severe mosquito nuisance during daytime was submitted to our laboratory. According to the complainant, the nuisance started at the end of May and became severe in August, affecting mainly children showing unusual skin reaction to the mosquito bites. In an entomological investigation carried out between August 29 and September 2, 2014, we found multiple plastic containers with water (plastic toys) in the garden with larvae and pupae of both Cx. pipiens and Ae. albopictus, and we observed the presence of biting adults. Ten mosquito bites were acquired on legs by one of us during 30 min of exposure. Forty-four Ae. albopictus females and 26 males emerged in the laboratory from the collected larvae; the traps collected 50 Cx. pipiens and 38 Ae. albopictus adults (Table 1). The infested courtyard was situated in the same sector of the city as were the previous observations (Fig. 1). Every year during 2012–14, reports of the presence of Ae. albopictus and the associated vector risk, as well as methodological guidance for control (2013), were submitted to the local and national public health authorities. The rapid expansion of the distribution range of Ae. albopictus has been attributed mainly to the international trade of used tires, and to a lesser extent to the import of water plants such as lucky bamboo (Medlock et al. 2012). Importing used tires, mainly from Western Europe, has been a business in Romania in the late 20 years, and it might have been even stimulated by recently (2011) introduced legal obligation for use of winter tires by all car owners. Indeed, the proximity of stores selling used tires to the points where Ae. albopictus was collected in 2012 has been noticed in our investigation. Aedes albopictus might have been introduced as well by ground vehicles passing in close proximity to the city on the route linking Bulgaria, Greece, and Turkey with Central and Western Europe. The area is a neighborhood with small family dwellings with courtyards. The species was still present at same collection stations in July 2013 as detected by gravid trap, human-landing catch, and presence of immature stages. No nuisance complaint on behalf of the public was registered in 2012–13; however, in 2014 the 1st nuisance report from a Bucharest inhabitant led to a heavily infested property. All the sites found infested in 2012–14 were located in Sector 5 of the municipality of
179
Bucharest which includes both urban periphery and the downtown area. We conclude that the species survived the 2 winter seasons. Environmental parameters influence the establishment of Ae. albopictus in an area (Straetemans 2008). Winter temperatures influence the survival of diapausing eggs, and cold temperature in the winter could be limiting establishment of the species, e.g., 22uC for Japan (Kobayashi et al. 2002) and 25uC for North America (Nawrocki and Hawley 1987). Favorable areas for overwintering are those with the mean temperature in January $0uC. Optimal summer temperatures for the development of the species range between 25uC and 30uC, with an annual rainfall of at least 500 mm, including sufficient amount of rainfall in summer for maintenance of breeding sites. Bucharest is situated in southeastern Romania (44u249N, 26u59E) at an average altitude of 80 m above sea level, on the Romanian Plain, bordered to the south and east by the Danube River valley. The climate of Bucharest is temperate continental with some Mediterranean characteristics, and the urban area is a heat island (Ionac and Grigore 2013). As calculated from the statistical data (Romanian Statistical Yearbooks, 1963–2009), the average annual temperature in Bucharest was 11.4uC, ranging from 21.4uC in January (the coldest month) to 23uC in July (the hottest month). In this period of time the average annual rainfall was 617.4 mm; late summer droughts often occurred. During our investigation we set a LogTag device (LogTagH Recorder, Auckland, New Zealand, model TRIX-8) in a house yard for monitoring the air temperature from December 2012 until September 2013. The lowest registered temperature was 28.2uC on January 10, 2013, as compared to 218uC recorded at the meteorological station situated in the outskirts of the city. The lowest monthly average temperature in winter was recorded in January and was 20.65uC (range: 28.2uC to 8.9uC), and subzero minimum daily temperatures were recorded at this site until the end of March; the mean temperature in July was 24.98uC and the warmest month was August, with a mean temperature of 26.57uC (range: 17.8uC to 33.5uC). Therefore, the city infrastructure may indeed offer sanctuaries with a milder microclimate than reflected by meteorological records, both over winter and summer, that is suitable for the establishment of this invasive species. Similar entomological surveys were not performed in other regions, except for the natural areas in the Danube River delta, and it cannot be assessed whether the reported record was the only introduction event in this country. Mitochondrial markers have limited resolution to characterize Ae. albopictus populations, therefore tracing the origin of Bucharest population was not possible. However, the mitochondrial sequences clearly related
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the Bucharest population to those distributed in other European countries, populations adapted to temperate climate, with eggs entering diapause and withstanding cold winter temperatures. In all the investigated sites in Bucharest municipality, Ae. albopictus larvae were found in mixed populations with Cx. pipiens. The fact that the urban vector of West Nile virus, Cx. pipiens, may find favorable habitats in peridomestic area and in the infrastructure of the city of Bucharest is well documented (Savage et al. 1999, Campbell et al. 2001). Rain drain catch basins and various domestic water collections devices are the main breeding sites in the city. Catch basins similar to those that are larval habitats for Culex mosquitoes in Bucharest proved to be the most productive habitats for Ae. albopictus in Italy (Carrieri et al. 2011). Bucharest, the capital city of Romania, with a population of 1.9 million, hosts the main international airport, and is the largest business, trade, tourist, and cultural center of the country. Travel to and from endemic areas of dengue and chikungunya viruses is steadily increasing. Dengue-imported cases, including viremic persons, have been diagnosed in our laboratory (Dinu et al. 2015). Stable populations of Ae. albopictus can initiate and maintain local transmission of imported pathogens such as dengue and chikungunya viruses. This vector is also significantly involved in the circulation of the dog heartworm, Dirofilaria sp., which has already been reported to be a threat to animal health in the region (Mircean et al. 2012). Surveillance, suitable control programs, and informed community participation are critical to preventing this invasive species from establishing as a stable population and colonizing other areas in Romania. Although the finding of Ae. albopictus are reported every year, the information is still not largely disseminated in the public domain to inform communities about measures for mosquito habitat suppression. Mosquito control in the city of Bucharest is mainly based on adulticidal treatments which have little efficacy for the control of the Ae. albopictus population. Further monitoring, risk assessment of its threat to public health, adequate operational control response, and increased awareness among affected communities, the local administration, and public health professionals are necessary. This study was funded by the European Union grant FP7-261504 EDENext, and it is catalogued by the EDENext Steering Committee as EDENext321. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. We are grateful to. Paul Reiter, Pasteur Institute, Paris, France, for useful discussions and advice and to Enkelejda Dikolli and Kujtim Mersinaj, Institute of Public Health,
VOL. 31, NO. 2
Tirana, Albania, for providing Ae. albopictus reference specimens. REFERENCES CITED Angelini P, Macini P, Finarelli AC, Pol C, Venturelli C, Bellini R, Dottori M. 2008. Chikungunya epidemic outbreak in Emilia-Romagna (Italy) during summer 2007. Parassitologia 50:97–98. Becker N, Petric’ D, Zgomba M, Boase C, Dahl C, Madon M, Kaiser A. 2010. Mosquitoes and Their Control. 2d ed. Heidelberg, Germany: Springer. Birungi J, Munstermann LE. 2002. Genetic structure of Aedes albopictus (Diptera: Culicidae) populations based on mitochondrial ND5 sequences: evidence for an independent invasion into Brazil and United States. Ann Entomol Soc Am 95:125–132. Bonacum J, DeSalle R, O’Grady P, Olivera D, Wintermute J, Zilversmit M. 2001. New nuclear and mitochondrial primers for systematics and comparative genomics in Drosophilidae. Dros Inf Serv 84:201–204. Campbell GL, Ceianu CS, Savage HM. 2001. Epidemic West Nile encephalitis in Romania: waiting for history to repeat itself. Ann N Y Acad Sci 951:94–101. Carrieri M, Albieri A, Angelini P, Baldacchini F, Venturelli C, Zeo SM, Bellini R. 2011. Surveillance of the Chikungunya vector Aedes albopictus (Skuse) in Emilia-Romagna (northern Italy): organizational and technical aspects of a large scale monitoring system. J Vector Ecol 36:108–116. Dinu S, Pa˘nculescu-Ga˘tej IR, Florescu SA, Popescu CP, Sıˆrbu A, Opris¸an G, Ba˘descu D, Franco L, Ceianu CS. 2015. Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013. Travel Med Infect Dis 13:69–73. Ganushkina LA, Tanygina EIu, Bezzhonova OV, Sergiev VP. 2012. Detection of Aedes (Stegomyia) albopictus Skuse mosquito in the Russian Federation. Med Parazitol (Mosk) 1:3–4. Russian. Gjenero-Margan I, Aleraj B, Krajcar D, Lesnikar V, Klobucˇar A, Pem-Novosel I, Kurecˇic´-Filipovic´ S, Komparak S, Martic´ R, Ðuricˇic´ S, Betica-Radic´ L, ˇ avlek T, Babic´-Erceg A, Okmadzˇic´ J, Vilibic´-C ˇ upanc T, Radic´ I, Ljubic´ M, Turkovic´ B, Avsˇic´-Z Sˇarac K, Benic´ N, Mlinaric´-Galinovic´ G. 2011. Autochthonous dengue fever in Croatia, August– September 2010. Eur Surveill 16(9):pii519805. Grandadam M, Caro V, Plumet S, Thiberge J-M, Souare`s Y, Failloux A-B, Tolou HJ, Budelot M, Cosserat D, Leparc-Goffart I, Despre`s P. 2011. Chikungunya virus, southeastern France. Emerg Infect Dis 17:910–913. Ionac N, Grigore E. 2013. Thermal differences within Bucharest town area case study: 01.07.2006– 31.03.2007. Present Environ Sustain Dev 7:21–36. Kobayashi M, Nihei N, Kurihara T. 2002. Analysis of northern distribution of Aedes albopictus (Diptera: Culicidae) in Japan by geographical information system. J Med Entomol 39:4–11. La Ruche G, Souare`s Y, Armengaud A, Peloux-Petiot F, Delaunay P, Despre`s P, Lenglet A, Jourdain F, Leparc-Goffart I, Charlet F, Ollier L, Mantey K, Mollet T, Fournier JP, Torrents R, Leitmeyer K, Hilairet P, Zeller H, Van Bortel W, Dejour-Salamanca D, Grandadam M, Gastellu-Etchegorry M. 2010. First two autochthonous dengue virus infec-
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tions in metropolitan France, September. Eur Surveill 15(39):pii519676. Marchand E, Prat C, Jeannin C, Lafont E, Bergmann T, Flusin O, Rizzi J, Roux N, Busso V, Deniau J, Noel H, Vaillant V, Leparc-Goffart I, Six C, Paty MC. 2013. Autochthonous case of dengue in France, October 2013. Euro Surveill 18(50):pii520661. Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, Van Bortel W. 2012. A review of the invasive mosquitoes in Europe: ecology, public health risks, and control options. Vector Borne Zoonotic Dis 12:435–447. Mircean V, Dumitrache MO, Gyo¨rke A, Pantchev N, Jodies R, Mihalca AD, Cozma V. 2012. Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania. Vector Borne Zoonotic Dis 12:595–604. Nawrocki SJ, Hawley WA. 1987. Estimation of the northern limits of distribution of Aedes albopictus in North America. J Am Mosq Control Assoc 3:314–317. Reinert J, Harbach RE, Kitching IJ. 2004. Phylogeny and classification of Aedini (Diptera: Culicidae), based on morphological characters of all life stages. Zool J Linn Soc 142:289–368.
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Reiter P. 1998. Aedes albopictus and the world trade in used tires, 1988–1995: the shape of things to come? J Am Mosq Control Assoc 14:83–94. Romanian Statistical Yearbooks, 1963–2009. National Institute of Statistics, ed. Bucharest, Romania. Savage HM, Ceianu C, Nicolescu G, Karabatsos N, Lanciotti R, Vladimirescu A, Laiv L, Ungureanu A, Romanca C, Tsai TF. 1999. Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes. Am J Trop Med Hyg 61:600–611. Scholte EJ, Dijkstra E, Blok H, De Vries A, Takken W, Hofhuis A, Koopmans M, De Boer A, Reusken CB. 2008. Accidental importation of the mosquito Aedes albopictus into the Netherlands: a survey of mosquito distribution and the presence of dengue virus. Med Vet Entomol 22:352–358. Sˇebesta O, Rudolf I, Beta´sˇova´ L, Pesˇko J, Huba´lek Z. 2012. An invasive mosquito species Aedes albopictus found in the Czech Republic, 2012. Eur Surveill 17(43):pii520301. Straetemans M. 2008. Vector-related risk mapping of the introduction and establishment of Aedes albopictus in Europe. Euro Surveill 13(7):pii58040.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of the American Mosquito Control Association, 31(2):177–181, 2015 Copyright E 2015 by The American Mosquito Control Association, Inc.
SCIENTIFIC NOTE ESTABLISHED POPULATION OF THE INVASIVE MOSQUITO SPECIES AEDES ALBOPICTUS IN ROMANIA, 2012–14 LIVIU F. PRIOTEASA,1 SORIN DINU,1,2 ELENA FA ˘ LCUT ¸A ˘1
AND
CORNELIA S. CEIANU1,3
ABSTRACT. During an entomological investigation carried out in Bucharest and surroundings in fall of 2012, 45 adult mosquitoes (38 females and 7 males) of Aedes albopictus were collected in a neighborhood from the southern area of the city. The morphological identification of the species was further confirmed by sequencing 2 mitochondrial DNA markers: the cytochrome c oxidase subunit I and NADH dehydrogenase subunit 5 genes. Aedes albopictus was collected again in 2013 in the same area from July until October. During late summer the species was found also in another location in the city, downtown Bucharest. Larvae were found in water barrels and other types of household containers, as well as in rain catch basins. In 2014, following a nuisance complaint of a Bucharest inhabitant, the entomological investigation found aggressive Ae. albopictus adults on his property that harbored many mosquito larvae in container-type breeding habitats. These findings are the 1st records of this invasive species and of its breeding population in Romania, and show maintenance of the species over 2 winter seasons. Surveillance of the species outside the area of the capital city was not performed, therefore it is not known whether Ae. albopictus has been introduced in other regions of the country. The presence of Ae. albopictus has been reported every year (2012–14) to competent public health authorities, stressing on the importance of surveillance and of implementation of control measures. KEY WORDS
Aedes albopictus, Asian tiger mosquito, invasive species, Romania, Bucharest
The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), (Stegomyia albopicta sensu Reinert et al. 2004), is a species native to temperate and tropical Asia. In the past 3 decades it has rapidly extended its distribution range, first in the New World and subsequently in Europe. Intercontinental expansion has been mainly attributed to a global trade of used tires (Reiter 1998) and intracontinental spread by passive transportation of Ae. albopictus in ground vehicles from infested areas into new areas (Scholte et al. 2008). The species was 1st detected in Europe in Albania in 1979, and the 1st subsequent record of its presence in Europe was in Italy in 1990. Since then it has spread with remarkable speed and is now present, often abundant, in Belgium (not established), Bosnia and Herzegovina, Croatia, France, Germany (not established), Greece, Malta, Monaco, Montenegro, the Netherlands (not established), San Marino, Serbia, Slovenia, Spain, Switzerland, Turkey, and Vatican City, and was recently recorded in Bulgaria, Czech Republic (not established), and southern Russia (Ganushkina et al. 2012, Medlock et al. 2012, Sˇebesta et al. 2012). In Europe, this vector species was respon1 Cantacuzino National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent¸ei, 050096, Bucharest, Romania. 2 Genetics Department, Faculty of Biology, University of Bucharest, 1–3 Aleea Portocalelor, 060101, Bucharest, Romania. 3 To whom correspondence should be addressed.
sible for an epidemic of chikungunya fever with 217 confirmed cases in northern Italy in 2007 (Angelini et al. 2008), and 2 chikungunya fever cases locally transmitted from an imported case in France in 2010 (Grandadam et al. 2011). Autochthonous cases of dengue were reported in the south of metropolitan France in 2010 (La Ruche et al. 2010; Marchand et al. 2013), as well as in Croatia in 2010 (Gjenero-Margan et al. 2011). Here we report the 1st detection of Ae. albopictus in Bucharest, capital city of Romania, in autumn of 2012, and species activity recorded in 2013 and 2014. An entomological survey for West Nile virus vectors was carried out between August 26 and October 5, 2012, in the city of Bucharest and its suburban area, with 14 collection sites. Three types of mosquito traps were used: Centers for Disease Control and Prevention (CDC) gravid traps (John W. Hock Company, Gainesville, FL), CDC light traps (John W. Hock Company), and BG Sentinel traps with BG-lure (Biogents AG, Regensburg, Germany). Between September 4 and October 3, 2012, 38 Ae. albopictus females and 7 males were captured (Table 1). All Ae. albopictus individuals were captured at 7 collection points covering an area of about 3 km2 in the south of Bucharest city (Fig. 1). Most of them were caught with CDC gravid traps and only a few with BG Sentinel traps. Identification of the captured mosquitoes was performed using morphological identification keys (Becker et al. 2010). For the molecular identification, DNA was extracted each year
177
178
JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION Table 1.
Data on Aedes albopictus collected in different traps in Bucharest, Romania, 2012–14. No. of adult males
Year 2012 2013
2014
VOL. 31, NO. 2
Month September October July August September October August–September
No. of adult females
BG Sentinel traps
CDC gravid traps
BG Sentinel traps
CDC gravid traps
Preimaginal stages
2 1 1 — — — —
4 — 12 6 — — 3
4 3 — — — — —
30 1 24 42 — 2 35
— — — 102 2 — 70
(2012–14) from 3 female mosquitoes, and 2 mitochondrial DNA markers spanning cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes were amplified and sequenced with 2 sets of primers as previously described (Bonacum et al. 2001, Birungi and Munsterman 2002). The COI sequences were used for species-level identification with BOLD v3.0 Beta (www.boldsystems.org.). Both COI and ND5 sequences derived from all samples were identical. One sequence for each mitochondrial DNA marker (2012–14) was deposited in GenBank under the following accession numbers: HF536717, LN808745, LN808746 (COI) and HF536718, LN808747, LN808748 (ND5). Gene sequences of Ae. albopictus from
Romania are similar to sequences obtained from mosquitoes collected in temperate and subtropical regions. One hundred percent identity was observed between the sequences derived from Ae. albopictus specimens captured in Romania and those obtained from mosquitoes collected in Europe (Albania, France, Italy, Netherlands, and Greece), and also from some populations of Madagascar, La Reunion, North America, and Hawaii. In 2013, mosquito collection activity resumed on June 17 and continued until the end of October. Human-landing catches were performed on 4 different days in July 2013 and potential habitats for immature stages were investigated. Aedes albopictus was found at the same collection
Fig. 1. Distribution of areas found infested with Aedes albopictus in Bucharest in 2012–14 (hatched), 2013–14 (dotted), and 2014 (grid).
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SCIENTIFIC NOTE
points in downtown Bucharest as previously found in 2012 (Fig. 1). Immature stages were found as well, mostly in mixed populations with Culex pipiens (L.) either in domestic containers or rain catch basins (Table 1). Monitoring an area in the proximity of Bucharest, in Ilfov County, did not yield any Ae. albopictus. Thus, positive sites were all located in urban environment. In 2014, Ae. albopictus adults were sporadically captured from July through October in the same area. At the end of summer, a complaint of severe mosquito nuisance during daytime was submitted to our laboratory. According to the complainant, the nuisance started at the end of May and became severe in August, affecting mainly children showing unusual skin reaction to the mosquito bites. In an entomological investigation carried out between August 29 and September 2, 2014, we found multiple plastic containers with water (plastic toys) in the garden with larvae and pupae of both Cx. pipiens and Ae. albopictus, and we observed the presence of biting adults. Ten mosquito bites were acquired on legs by one of us during 30 min of exposure. Forty-four Ae. albopictus females and 26 males emerged in the laboratory from the collected larvae; the traps collected 50 Cx. pipiens and 38 Ae. albopictus adults (Table 1). The infested courtyard was situated in the same sector of the city as were the previous observations (Fig. 1). Every year during 2012–14, reports of the presence of Ae. albopictus and the associated vector risk, as well as methodological guidance for control (2013), were submitted to the local and national public health authorities. The rapid expansion of the distribution range of Ae. albopictus has been attributed mainly to the international trade of used tires, and to a lesser extent to the import of water plants such as lucky bamboo (Medlock et al. 2012). Importing used tires, mainly from Western Europe, has been a business in Romania in the late 20 years, and it might have been even stimulated by recently (2011) introduced legal obligation for use of winter tires by all car owners. Indeed, the proximity of stores selling used tires to the points where Ae. albopictus was collected in 2012 has been noticed in our investigation. Aedes albopictus might have been introduced as well by ground vehicles passing in close proximity to the city on the route linking Bulgaria, Greece, and Turkey with Central and Western Europe. The area is a neighborhood with small family dwellings with courtyards. The species was still present at same collection stations in July 2013 as detected by gravid trap, human-landing catch, and presence of immature stages. No nuisance complaint on behalf of the public was registered in 2012–13; however, in 2014 the 1st nuisance report from a Bucharest inhabitant led to a heavily infested property. All the sites found infested in 2012–14 were located in Sector 5 of the municipality of
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Bucharest which includes both urban periphery and the downtown area. We conclude that the species survived the 2 winter seasons. Environmental parameters influence the establishment of Ae. albopictus in an area (Straetemans 2008). Winter temperatures influence the survival of diapausing eggs, and cold temperature in the winter could be limiting establishment of the species, e.g., 22uC for Japan (Kobayashi et al. 2002) and 25uC for North America (Nawrocki and Hawley 1987). Favorable areas for overwintering are those with the mean temperature in January $0uC. Optimal summer temperatures for the development of the species range between 25uC and 30uC, with an annual rainfall of at least 500 mm, including sufficient amount of rainfall in summer for maintenance of breeding sites. Bucharest is situated in southeastern Romania (44u249N, 26u59E) at an average altitude of 80 m above sea level, on the Romanian Plain, bordered to the south and east by the Danube River valley. The climate of Bucharest is temperate continental with some Mediterranean characteristics, and the urban area is a heat island (Ionac and Grigore 2013). As calculated from the statistical data (Romanian Statistical Yearbooks, 1963–2009), the average annual temperature in Bucharest was 11.4uC, ranging from 21.4uC in January (the coldest month) to 23uC in July (the hottest month). In this period of time the average annual rainfall was 617.4 mm; late summer droughts often occurred. During our investigation we set a LogTag device (LogTagH Recorder, Auckland, New Zealand, model TRIX-8) in a house yard for monitoring the air temperature from December 2012 until September 2013. The lowest registered temperature was 28.2uC on January 10, 2013, as compared to 218uC recorded at the meteorological station situated in the outskirts of the city. The lowest monthly average temperature in winter was recorded in January and was 20.65uC (range: 28.2uC to 8.9uC), and subzero minimum daily temperatures were recorded at this site until the end of March; the mean temperature in July was 24.98uC and the warmest month was August, with a mean temperature of 26.57uC (range: 17.8uC to 33.5uC). Therefore, the city infrastructure may indeed offer sanctuaries with a milder microclimate than reflected by meteorological records, both over winter and summer, that is suitable for the establishment of this invasive species. Similar entomological surveys were not performed in other regions, except for the natural areas in the Danube River delta, and it cannot be assessed whether the reported record was the only introduction event in this country. Mitochondrial markers have limited resolution to characterize Ae. albopictus populations, therefore tracing the origin of Bucharest population was not possible. However, the mitochondrial sequences clearly related
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the Bucharest population to those distributed in other European countries, populations adapted to temperate climate, with eggs entering diapause and withstanding cold winter temperatures. In all the investigated sites in Bucharest municipality, Ae. albopictus larvae were found in mixed populations with Cx. pipiens. The fact that the urban vector of West Nile virus, Cx. pipiens, may find favorable habitats in peridomestic area and in the infrastructure of the city of Bucharest is well documented (Savage et al. 1999, Campbell et al. 2001). Rain drain catch basins and various domestic water collections devices are the main breeding sites in the city. Catch basins similar to those that are larval habitats for Culex mosquitoes in Bucharest proved to be the most productive habitats for Ae. albopictus in Italy (Carrieri et al. 2011). Bucharest, the capital city of Romania, with a population of 1.9 million, hosts the main international airport, and is the largest business, trade, tourist, and cultural center of the country. Travel to and from endemic areas of dengue and chikungunya viruses is steadily increasing. Dengue-imported cases, including viremic persons, have been diagnosed in our laboratory (Dinu et al. 2015). Stable populations of Ae. albopictus can initiate and maintain local transmission of imported pathogens such as dengue and chikungunya viruses. This vector is also significantly involved in the circulation of the dog heartworm, Dirofilaria sp., which has already been reported to be a threat to animal health in the region (Mircean et al. 2012). Surveillance, suitable control programs, and informed community participation are critical to preventing this invasive species from establishing as a stable population and colonizing other areas in Romania. Although the finding of Ae. albopictus are reported every year, the information is still not largely disseminated in the public domain to inform communities about measures for mosquito habitat suppression. Mosquito control in the city of Bucharest is mainly based on adulticidal treatments which have little efficacy for the control of the Ae. albopictus population. Further monitoring, risk assessment of its threat to public health, adequate operational control response, and increased awareness among affected communities, the local administration, and public health professionals are necessary. This study was funded by the European Union grant FP7-261504 EDENext, and it is catalogued by the EDENext Steering Committee as EDENext321. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. We are grateful to. Paul Reiter, Pasteur Institute, Paris, France, for useful discussions and advice and to Enkelejda Dikolli and Kujtim Mersinaj, Institute of Public Health,
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