Aedes albopictus, an invasive mosquito species, is currently well established in many European countries, posing high risks to human health. A preliminary trial using repetitive releases of irradiated sterile male mosquitoes was designed, implemented and evaluated for the first time in Greece. The main scope of this trial was to investigate the efficacy of sterile insect technique (SIT) on wild population egg hatch rate in Greece using mass-produced sterile male mosquitoes from another country (Italy). The study was conducted in Vravrona area, close to Athens International Airport (Attica Region). The number of eggs laid in ovitraps was similar in all intervention and control plots. However, a significant reduction in egg hatch rate was recorded in the SIT plot in comparison with both control plots starting two weeks after the first release. This trial validates the logistics (transportation, releases handling and monitoring) as a major step towards implementing efficient, environmentally safe control approaches as an additional tool against the invasive Aedes species in Greece and more widely in Europe.

Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera: Culicidae), commonly known as the Asian tiger mosquito, is an invasive mosquito species of public health significance, well established in many countries worldwide. In Greece, it was first recorded in Corfu and Thesprotia between 2003 and 2004. In the following years, further distribution and establishment of Ae. albopictus in Greece have been confirmed in many Regional Units of the country. In the current study, we report the invasion history of Ae. albopictus in Greece, until 2016. The results from the entomological investigation following imported virus cases in 2014, 2015, and 2016 are also included. Moreover, its presence in Greece is demonstrated in a thematic map based on (a) information provided by pest control companies and/or citizens, (b) the official samples sent to Benaki Phytopathological Institute (BPI) and the National School of Public Health (NSPH), and

Negative impacts on the environment from the continuous use of synthetic insecticides against mosquitoes has driven research towards more ecofriendly products. Phytochemicals, classified as low-risk substances, have been recognized as potential larvicides of mosquitoes; however, problems related to water solubility and stability are limiting factors for their use in mosquito control programs in the field. In this context, many researchers have focused on formulating essential oils in nanoemulsions, exploiting innovative nanotechnology. In the current study, we prepared 4 (R)-(+)-limonene oil-in-water nanoemulsions using low and high energy methods, and we evaluated their physicochemical characteristics (e.g., viscosity, stability, mean droplet diameter, polydispersity index) and their bioactivity against larvae of two mosquito species of great medical importance, namely, Cx. pipiens molestus and Ae. albopictus. According to the dose–response bioassays with the limonene-based nanoemulsions and pure limonene (dissolved in organic solvent), the tested nanoformulations improved the activity of limonene against Ae. albopictus larvae, while the performance of limonene was either the same or better than limonene against Cx. pipiens molestus, depending on the applied system. Overall, we achieved the production of limonene-based delivery nanosystems, with sufficient lethal properties against mosquito larvae to consider them promising larvicidal formulations applicable to mosquito breeding sites.

Background of the Study. Following a large West Nile virus (WNV) epidemic in Northern Greece in 2010, an active mosquito surveillance system was implemented, for a 3-year period (2011, 2012, and 2013). Description of the Study Site and Methodology. Using mainly CO2 mosquito traps, mosquito collections were performed. Samples were pooled by date of collection, location, and species and examined for the presence of WNV. Results. Positive pools were detected in different areas of the country. In 2010, MIR and MLE values of 1.92 (95% CI: 0.00–4.57) and 2.30 (95% CI: 0.38–7.49) were calculated for the Serres Regional Unit in Central Macedonia Region. In 2011, the highest MIR value of 3.71(95% CI: 1.52–5.91) was recorded in the Regions of Central Greece and Thessaly. In 2012, MIR and MLE values for the whole country were 2.03 (95% CI: 1.73–2.33) and 2.15 (95% CI: 1.86–2.48), respectively, for Cx. pipiens. In 2013, in the Regional Unit of Attica, the one outbreak epicenter, MIR and MLE values for Cx. pipiens were 10.75 (95% CI: 7.52–13.99) and 15.76 (95% CI: 11.66–20.65), respectively. Significance of Results/Conclusions. The contribution of a mosquito-based surveillance system targeting WNV transmission is highlighted through the obtained data, as in most regions positive mosquito pools were detected prior to the date of symptom onset of human cases. Dissemination of the results on time to Public Health Authorities resulted in planning and application of public health interventions in local level.

Seasonal patterns of mosquito population density and their vectorial capacity constitute major elements to understand the epidemiology of mosquito-borne diseases. Using adult mosquito traps, we compared the population dynamics of major mosquito species (Culex pipiens, Aedes albopictus, Anopheles spp.) in an urban and a wetland rural area of Attica Greece. Pools of the captured Cx. pipiens were analyzed to determine infection rates of the West Nile virus (WNV) and the Usutu virus (USUV). The data provided were collected under the frame of the surveillance program carried out in two regional units (RUs) of the Attica region (East Attica and South Sector of Attica), during the period 2017–2018. The entomological surveillance of adult mosquitoes was performed on a weekly basis using a network of BG-sentinel traps (BGs), baited with CO2 and BG-Lure, in selected, fixed sampling sites. A total of 46,726 adult mosquitoes were collected, with larger variety and number of species in East Attica (n = 37,810), followed by the South Sector of Attica (n = 8916). The collected mosquitoes were morphologically identified to species level and evaluated for their public health importance. Collected Cx. pipiens adults were pooled and tested for West Nile virus (WNV) and Usutu virus (USUV) presence by implementation of a targeted molecular methodology (real-time PCR). A total of 366 mosquito pools were analyzed for WNV and USUV, respectively, and 38 (10.4%) positive samples were recorded for WNV, while no positive pool was detected for USUV. The majority of positive samples for WNV were detected in the East Attica region, followed by the South Sector of Attica, respectively. The findings of the current study highlight the WNV circulation in the region of Attica and the concomitant risk for the country, rendering mosquito surveillance actions and integrated mosquito management programs as imperative public health interventions.

Background Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. Methods Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. Results The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. Conclusions The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities. Graphical Abstract

A 2-yr sand fly (Diptera: Psychodidae) seasonality study was performed in Attica Region, Greece, from June 2017 until November 2018, aiming also to detect the presence of Leishmania infection in the collected sand flies. In total, 701 sand flies were collected from urban areas within the Attica Region using BG-Sentinel traps, set weekly in eight fixed sites. Five species were identified morphologically and molecularly, namely Phlebotomus tobbi (Adler and Theodor), which was most the most commonly collected species, followed by P. Neglectus (Tonnoir), P. papatasi (Scopoli), P. simici (Theodor), and Sergentomyia minuta (Rondani). During both survey years sand fly populations peaked in late August to early September. Fifty-nine monospecific pools were examined for Leishmania detection by analyzing the ITS1 nuclear region using both RFLPs and sequencing, seven of which were found positive. Leishmania DNA was identified as L. infantum in six pools (five P. papatasi and one P. tobbi), whereas in one P. papatasi pool Leishmania DNA was identified as L. tropica. This is the first time that L. tropica has been detected in naturally infected sand flies from the Attica Region as well as in central Greece, while previously it has only been detected in sand flies collected from Central Macedonia (Northern Greece).

Aedes albopictus, also known as the Asian tiger mosquito, is an aggressive invasive mosquito species that transmits parasites that cause several major human diseases such as dengue, Chikungunya, and Zika. Its recent establishment in many European countries and the reported autochthonous cases of Aedes-transmitted arboviral diseases highlight the importance of effective surveillance programs to plan and implement efficient mosquito-control interventions. Aedes albopictus invaded the Greek island of Crete in 2014 and rapidly spread throughout; however, there are no comprehensive population-related data yet available. Our study focused on spatial and temporal dynamics of Ae. albopictus populations in rural and agricultural areas in Chania (western part of the Crete Island). An oviposition surveillance network, consisting of 51 ovitraps, was established in 2017 and systematically inspected for 2 years. Thirty ovitraps were established in rural areas and 21 in the surrounding agricultural areas. Eggs were collected weekly and transferred to the laboratory for counting and assessment of hatching rates. The spatial and temporal egg distribution was assessed by using geographical information systems and spatial statistical analysis tools. Kernel density and hot spot analysis were employed to identify clusters of high populations of eggs. Oviposition activity (eggs in traps) was recorded during April–May and December (of both years), while the maximum egg-laying rates were observed during September–October. The proportion of traps with eggs was similar between rural and agricultural areas, while the higher number of eggs was recorded in rural compared to agricultural areas during 2017. Egg hatch rates were high (&gt;90%) until the end of September. The proportion of diapausing non-hatched eggs rapidly increased at the end of November. All hatched larvae were identified as Ae. Albopictus, indicating a negative effect of the invasion on the population of the local species Ae. cretinus. This was a systematic attempt to monitor the seasonal and spatial patterns of the Asian tiger mosquito in the island of Crete, the southernmost area of Europe, and a study in Europe that assessed temporal and spatial dynamics of Ae. albopictus soon after its invasion and establishment in an area.