Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece.

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas.

Title: Essai sur le terrain de la Technique de l'Insecte Stérile (TIS) ciblant la suppression d'Aedes albopictus en Grèce. Abstract: La technique de l'insecte stérile (TIS) consiste à libérer un grand nombre de mâles stériles pour supplanter les mâles sauvages lors de l'accouplement avec les femelles, entraînant ainsi un déclin des populations de nuisibles. Dans la présente étude, nous avons mené un essai de suppression en Grèce contre le moustique vecteur invasif de la dengue, Aedes albopictus (Skuse), par le biais de la libération hebdomadaire de mâles stériles pendant 22 semaines de juin à septembre 2019. Notre approche comprenait le transport sur de longues distances de moustiques stériles, et leur lâcher à une densité de 2 547 ± 159 mâles stériles par hectare et par semaine dans le cadre d'une stratégie de lutte intégrée contre les nuisibles à l'échelle de la zone (AW-IPM). Les lâchers répétés de mâles stériles ont entraîné une réduction progressive de la densité des œufs, atteignant 78 % de la mi-juin au début septembre. Cette réduction est restée entre 70 % et 78 % pendant quatre semaines après la fin des lâchers. De plus, dans la zone d'intervention de la TIS, l'indice d'oviposition, représentant le pourcentage de pièges contenant des œufs, est resté plus faible que dans la zone témoin tout au long de l'essai. Cet essai représente une avancée significative dans le domaine de la lutte contre les moustiques, car il explore la viabilité et l'efficacité de la production et du transport de mâles stériles depuis une installation éloignée vers la zone de lâcher. Nos résultats fournissent des informations précieuses pour les futurs programmes de TIS ciblant Ae. albopictus et la méthodologie que nous avons utilisée pourra servir de point de départ pour développer des protocoles de libération plus raffinés et plus efficaces, y compris le transport de mâles stériles sur de longues distances depuis les unités de production jusqu'aux zones d'intervention.

Field Evaluation of Commonly Used Adult Mosquito Traps in Greece.

Background: Entomological monitoring activities are a major part of mosquito and mosquito-borne diseases surveillance. Several trapping methods are implemented worldwide, aiming to gather data on species composition and their abundance in various study areas. Methods: Several methodological modifications, such as trapping systems baited with attractants or carbon dioxide, have been proposed to increase trap efficiency. The aim of this study was to test different trap types, commonly used in Greece to collect mosquitoes, with the addition of the Biogents Sentinel lure. Moreover, traps were placed in two distinct land types and two different heights above the ground to compare their efficacy. West Nile Virus is endemic in Greece, so we also aimed to detect viral presence and circulation in selected mosquito pools. Results: Adult mosquitoes of Aedes albopictus, Culex pipiens s.l., and Culiseta longiareolata were collected in both study areas. The trap type had a significant impact on the total collections, while the trap position and interaction between trap and position did not significantly affect mosquito catches. WNV was detected in Cx. pipiens s.l. pools examined from the two study areas. Conclusion: This study emphasizes the role of trapping methods as a key component for monitoring and surveillance of adult mosquito populations, reflecting that several trap types present with different mosquito species capture and catch rates.

Winter survival of adults of two geographically distant populations of Aedes albopictus in a microclimatic environment of Athens, Greece.

Overwintering capacity is a major step towards establishment of invasive mosquitoes from the tropics in temperate zone areas and one of the main elements determining next seasons' population size that regulates disease transmission of competent invasive vector species. The Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culiciidae) is an aggressive invasive species that has greatly expanded its geographical boundaries over the last few decades. The species' ability to induce photoperiodic-based egg diapause allows its overwintering in temperate regions, which favors its establishment in higher latitudes. In warmer temperate areas winter survival can be accomplished in the adult stage as well especially in human-made shelters. Aedes albopictus is already showing signs of adaptation to colder climates which may result in disease transmission in new areas. Although the Asian tiger mosquito has invaded Greece since 2003-4, little is known regarding its overwintering capacity in the country, especially as far as adults are regarded. We studied the survival of Ae. albopictus adults during winter in a protected shelter in Athens, the capital city of Greece. The study involved two geographically isolated populations originating from Chania (Crete, most southern part of Greece), and Palaio Faliro (Athens, Central Greece). We exposed different cohorts of F3 adults of the above two populations that emerged from field collected eggs to "winter condition" from November 2018 to beginning of January 2019. in a protected microclimatic environment. Adult mortality was recorded systematically until the death of the last individual in the cohort. Results demonstrated evidence of winter survival of adults for both populations. Longevity of both females and males of the Palaio Faliro population was longer than that of the Chania population for almost all installation dates. Survival curves, regardless of the date of installation, were steeper for females of the Chania population compared to those from Palaio Faliro. In conclusion, overwinter survival of both populations in the adult stage highlights the need for future studies, considering local and microclimatic condition that vary significantly between regions. Winter climatic conditions play vital role in adult survival of this highly important public health species concerning distribution limits and knowledge on winter survival of Ae. albopictus transmitted viruses.

Knowledge, Attitudes and Perception of Mosquito Control in Different Citizenship Regimes within and Surrounding the Malakasa Open Accommodation Refugee Camp in Athens, Greece.

The study aims to evaluate the Knowledge, Attitude and Perception (KAP) of different societal groups concerning the implementation of targeted community-based mosquito surveillance and control interventions in different citizenship regimes. Targeted surveys were carried out within Malakasa camp for migrants and refugees, neighboring residential areas and urban areas in the wider Athens metropolitan area to investigate different knowledge levels and the role that both local and migrant communities can play in the implementation of community-based interventions based on their attitudes and perceptions. A scoring system was used to rate the collected responses. Results indicate different levels of KAP among the various groups of respondents and different priorities that should be considered in the design and execution of community interventions. Findings indicate a lower level of Knowledge Attitudes and Perceptions for the migrants, while the rate of correct answers for Perception significantly improved for migrants following a small-scale information session. The study highlights disparities in the levels of knowledge for certain public health issues and the feasibility of certain approaches for alleviating health-related challenges such as mosquito-borne diseases. Findings suggest that essential preparedness is needed by public authorities to respond to public health challenges related to migration and the spread of vector-borne diseases.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

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.

Seasonal Phenological Patterns and Flavivirus Vectorial Capacity of Medically Important Mosquito Species in a Wetland and an Urban Area of Attica, Greece.

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.

Reduction in Egg Fertility of Aedes albopictus Mosquitoes in Greece Following Releases of Imported Sterile Males.

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.

Sand Fly (Diptera: Psychodidae: Phlebotominae) Population Dynamics and Natural Leishmania Infections in Attica Region, Greece.

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).

Physicochemical Characteristics of Four Limonene-Based Nanoemulsions and Their Larvicidal Properties against Two Mosquito Species, Aedes albopictus and Culex pipiens molestus.

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.

West Nile Virus Circulation in Mosquitoes in Greece (2010-2013).

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.

West Nile virus state of the art report of MALWEST Project.

During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen.