Effective surveillance of invasive Aedes mosquitoes, particularly Ae. aegypti and Ae. albopictus, is essential for early detection, risk assessment, and optimization of control strategies such as the Sterile Insect Technique (SIT). Although BG-Sentinel 2 traps are widely used in Europe, their performance under eastern Mediterranean conditions remains insufficiently evaluated. We assessed the attractiveness of BG-Sentinel 2 traps baited with BG-Lure, dry ice (CO2), their combination, and unbaited controls using a Latin square field design in Larnaca and Nicosia, Cyprus. A total of 1,649 mosquitoes were collected. In Larnaca, Ae. aegypti occurred at low densities. Male captures were consistently low and unaffected by attractants, while female captures increased significantly only when BG-Lure and CO2 were combined (RR = 2.7, 95% CI: 1.6–4.5). In Nicosia, Ae. albopictus was abundant, but responses were sex-specific. Male captures were significantly reduced by CO2 alone (RR = 0.297, 95% CI: 0.175–0.467), with a weaker reduction when combined with BG-Lure, while female captures were not significantly affected by any treatment. For Culex pipiens, CO2 strongly increased female captures (RR ≈ 16), irrespective of BG-Lure, whereas males showed only a modest response to BG-Lure alone. Overall, attractant efficacy was highly species- and sex-specific. Standard baiting strategies did not consistently enhance detection of invasive Aedes mosquitoes and, in some cases, reduced male captures, with implications for SIT programs. These findings emphasize the need for local validation of surveillance protocols particularly at points of entry.

Ovitraps have been extensively utilized as low-cost and efficient surveillance method for container-breeding Aedes mosquitoes such as Aedes albopictus. However, variations in their design may affect their effectiveness in collecting mosquito eggs. Despite the widespread use of ovitraps in mosquito surveillance programs for Ae. albopictus, field based comparative assessment of key ovitrap components, particularly oviposition substrate and trap volume, remains limited. Herein, we report findings from two field trials conducted in Attica, Greece, aiming to compare ovitrap performance for Ae. albopictus egg collection. The first study evaluated the performance of two oviposition substrates (wooden strips vs. styrofoam) and three trap sizes (350, 600 and 1100 mL) following the Latin square rotation design. The second trial compared wooden strips with masonite in a randomized complete block design. In the first trial, ovitraps with wooden strips collected approximately twice as many eggs as those with styrofoam (50.5 ± 5.1 vs. 21.1 ± 3.1 eggs per trap per week) and markedly reduced zero-egg catches, indicating higher sensitivity for early detection at low population densities. In this trial, medium and large traps significantly outperformed small traps, collecting on average 44.4 ± 6, 38.5 ± 5.1, and 24.7 ± 5 eggs per week, respectively. In the second trial, masonite strips consistently yielded higher egg counts than wooden strips across both high and low oviposition periods. Our findings identify oviposition substrate and trap volume as key determinants of Ae. albopictus ovitrap performance that should be considered when refining ovitrap design for monitoring and control purposes.

Floods are among the most frequent and complex climate-related hazards, affecting health and health systems through infectious disease risks, water and chemical contamination, and disruption of essential services. Examining how international guidance reflects the operational realities offlood response, and how it supports countries in practice, is essential. While each event is unique, sharing experiences can strengthen preparedness and resilience.This paper draws lessons from an assessment of the response measures used during the 2023 floods in Greece.The coordination team rapidly adapted existing structuresfor infectious disease surveillance, vaccination and water quality monitoring to an emergency response of exceptional scale. To track flood-related diseases, the coordination team reconfigured syndromic surveillance tools initially designed for refugee camps, and compared proportional morbidity with baseline data to provide early warnings. The team also implemented multiplex polymerase chain reaction diagnostics and mobile vaccination campaigns targeting high-risk groups. Risk communication and community outreach combined repurposed materials with new flood-specific guidance. While there were no outbreaks of gastroenteritis, respiratory infections or mosquito-borne diseases, 45 leptospirosis cases and one fatal non-toxigenic Vibrio cholerae infection highlighted the need for sustained vigilance. The lessons learnt indicate that preparedness relies on flexible adaptation of existing systems, cross-governmental coordination and active community engagement. Gaps in global and regional guidance, especially regarding laboratory diagnosis, safe water restoration and chemical safety monitoring, complicate field decision-making. Strengthening international frameworks, embedding flood preparedness within climate adaptation strategies and applying One Health approaches are therefore critical to ensure health system resilience as flood events become increasingly frequent and severe.

The Mosquito Weather Index (MWI) is the first operational index to translate the combined effects of temperature, humidity and wind speed into a single measure of mosquito activity, communicated to the public and public-health stakeholders in an easy-to-act-on form. The index ranges from 0 to 1 and maps to five activity levels, from "no activity" to "very high activity." This study presents the first empirical evaluation of the MWI. We assess its predictions of vector mosquito counts from adult suction traps in Moschato-Tavros, Attica, Greece, during 2018 and 2019. We find the MWI is strongly associated with Aedes albopictus and Culex pipiens trap counts. The single MWI variable improves predictions of trap counts---both in cross-validation and in forward forecasts---from models that lack information on weather or seasonality, while adding little once such information is included. We also evaluate different methods, finding that temporally fine-grained (hourly) weather data is crucial for its performance. These findings suggest that the MWI functions as intended, offering a simple, interpretable predictor of vector mosquito activity that can be acted on by the general public and mosquito-control specialists.

Mosquitoes transmit numerous infectious diseases, with climate change expanding their global distribution through warmer environments. Next-generation sequencing offers significant advantages for mosquito genomic surveillance and potential early warning systems. In this study, a portable metagenomic sequencing approach using Oxford Nanopore Technologies (ONT) for field-based mosquito analysis (MosquitoID protocol) was developed, enabling species and host feeding patterns identification, and pathogen detection, all coming from a single amplification-free workflow. DNA was extracted from 62 mosquito samples (Aedes albopictus, Aedes cretinus, Culex pipiens, Culiseta longiareolata) from Greece and Spain, either single-species pools (1-10 specimens) or mixed-species pools, with reverse purification method or archived samples. Additionally, 30 pooled Aedes aegypti samples from Bangladesh underwent cDNA reverse purification. All samples were sequenced using ONT rapid barcoding kits. Offline bioinformatics analysis via Geneious screened custom BLAST databases for species, host, and virus identification. MosquitoID accurately identified mosquito species in 89% of samples overall, with main discrepancies in Aedes cretinus. Virus screening detected Phasi Charoen-like virus in cDNA samples. Host DNA sequences identified multiple species including horses, cattle, and ducks. This study demonstrates metagenomic ONT sequencing's effectiveness for rapid host, species, and virus identification. After further benchmarking, the approach shows potential for real-time disease monitoring and enhanced surveillance systems. Integrating portable next-generation sequencing with offline bioinformatics tools could significantly strengthen mosquito-borne disease prevention strategies, particularly for non-bioinformaticians and in resource-limited settings.

Mosquito-borne diseases are an emerging public health challenge in Europe, driven by the spread of invasive mosquito species capable of sustaining outbreaks of tropical arboviral diseases. Rising temperatures, shifting precipitation patterns, human-driven habitat changes, and prolonged transmission seasons have increased the risk of dengue, chikungunya, and West Nile virus outbreaks, among other vector-borne diseases. Effective control requires a multifaceted approach, combining traditional and novel methods with advanced surveillance technologies and community involvement. However, growing insecticide resistance and concerns about insecticide use highlight the need for more prudent management of current tools and the development of innovative alternatives. Genetic control strategies, including the Sterile Insect Technique (SIT), Wolbachia-based approaches, and genetically modified (GM) mosquitoes, offer promising solutions but still face scientific, regulatory, and societal challenges. This review explores the current landscape of mosquito-borne disease control in Mediterranean Europe, emphasizing key challenges and emerging solutions. An integrated approach that strengthens surveillance, promotes sustainable control methods, and incorporates novel biotechnological tools supported by smart technologies will be essential to reduce the future burden of mosquito-borne diseases in the region.

BackgroundAedes albopictus, the Asian tiger mosquito, which is listed among the world's 100 most dangerous invasive species, is the main vector of chikungunya, dengue and Zika viruses. This mosquito species has rapidly dispersed and invaded much of the globe assisted by its life history traits and high propagule pressure driven by human activities. Aedes albopictus is currently widespread across mainland Europe and the Mediterranean region, including the islands. Cyprus remained free of Ae. albopictus until October 2022, when specimens were recorded for the first time in Limassol district, including the port area. Understanding the processes associated with the introduction, expansion and establishment of this vector in Cyprus is of primary importance to mitigate its dispersal on the island, and to implement control methods to prevent disease outbreaks. A genetic analysis of these invasive specimens collected in Limassol district and in areas from the Central Mediterranean was performed to obtain a genetic portrait of the demographic history of the invasive mosquitoes on Cyprus.MethodsWe applied highly polymorphic simple sequence repeat (SSR) markers to the Ae. albopictus mosquitoes collected in Cyprus and to specimens from Italy, France, Switzerland, the Balkans, Greece and Turkey to construct an SSR individual genotype dataset that would enable the invasion pattern of Ae. albopictus in Cyprus to be traced. Bayesian clustering analyses using STRUCTURE and BayesAss version 3 were employed to derive information on the degree of ancestry among Cypriot and Mediterranean mosquitoes and on recent mosquito movements both within Cyprus and between Cyprus and the Central Mediterranean areas.ResultsThe Cypriot mosquitoes appear to be highly polymorphic with no signs of genetic drift due to recent founder effects. An ongoing mosquito dispersal within the Limassol district was detected, suggesting the presence of established, hidden adventive populations. These mosquitoes share a high degree of ancestry with those in the Balkans and parts of northern Italy that border the Adriatic Sea.ConclusionsConsidering the trade connections of Limassol port, Cyprus with the Balkans and the Adriatic Italian region, we hypothesise that these areas may be involved in the incursion of Ae. albopictus into Cyprus. As the Balkan and Italian mosquitoes display high competence for CHIKV, questions arise about possible arbovirus outbreaks in Cyprus and highlight the need to implement surveillance and control measures.

The mosquito species Aedes cretinus, which is native to Greece, has become increasingly scarce following the invasion of the highly competitive and widespread Aedes albopictus. The ability of mosquitoes to survive low winter temperatures plays a pivotal role in their population dynamics of the next season. In this study, we investigated the overwintering capacity of Ae. albopictus and Ae. cretinus adults under semi-fi eld, sheltered microclimatic conditions in the northern area of Attica, Greece, during the winter of 2023-2024. Our fi ndings revealed that 11% of Ae. albopictus females and 21.1% of Ae. cretinus females were capable of overwintering, highlighting the importance of sheltered microclimates in enabling overwintering survival under low outdoor temperatures. In contrast, males of both species failed to overwinter. The winter survival ability of adults was signifi cantly greater in Ae. cretinus than in Ae. albopictus, and this may account for the occurrence of Ae. cretinus in the cooler environments of vegetated and wooded locations in northern areas of the Attica region. Nevertheless, the ability of Ae. albopictus females to also overwinter under the same sheltered microclimatic environment may aff ect the potential of interspecifi c competition in these areas.

The Asian tiger mosquito Aedes albopictus is a highly invasive species capable of transmitting human pathogens. For population management, the sterile insect technique (SIT) is considered an effective and sustainable alternative to conventional methods, such as insecticides and reducing or eliminating breeding sites. The use of symbiotic bacteria to improve the application of SIT or design combined SIT/incompatible insect technique (IIT) approaches is currently considered. In this context, exploring the microbiota of local mosquito populations is crucial for identifying interesting components. This study employed 16S rRNA sequencing and microbiological methods to characterize the diversity of laboratory and wild Ae. albopictus in Greece. Differences were recorded between wild and lab-reared mosquitoes, with laboratory samples exhibiting higher diversity. Laboratory treatment, sex, and developmental stage also resulted in variations between communities. Populations reared in the same facility developed mostly similar bacterial profiles. Two geographically distant wild populations displayed similar bacterial profiles, characterized by seasonal changes in the relative abundance of Pantoea and Zymobacter. Wolbachia was dominant in most groups (63.7% relative abundance), especially in field-caught mosquitoes. It was identified with two strains, wAlbA (21.5%) and wAlbB (42.2%). Other frequent taxa included Elizabethkingia, Asaia, and Serratia. Blood feeding favored an increase in Serratia abundance. Various Enterobacter, Klebsiella, Aeromonas, and Acinetobacter strains were isolated from larval and adult mosquito extracts and could be further characterized as diet supplements. These findings suggest that the microbiota of local populations is highly variable due to multiple factors. However, they retain core elements shared across populations that may exhibit valuable nutritional or functional roles and could be exploited to improve SIT processes.

Emerging vector-borne diseases (VBDs) are a major public health concern worldwide. Climate change, environmental degradation and globalisation have led to an expansion in the range of many vectors and an erosion of transmission barriers, increasing human exposure to new pathogens and the risk for emerging VBD outbreaks. Europe is potentially underprepared for the increasing threat of VBDs, due to attention and funding being diverted to other public health priorities. Proactive, rather than reactive, prevention and control approaches can greatly reduce the socio-economic toll of VBDs. Endemic countries globally have decades of experience in controlling VBDs, and Europe has much to learn from this knowledge. Here, we advocate for the expansion of transdisciplinary knowledge-sharing partnerships, to co-create proactive measures against VBDs. We present the experiences and expertise of our diverse international team and explore how an array of interventions can be applied and adapted to the European context.

Toward the discovery of novel efficient repellents, protein-directed dynamic combinatorial chemistry (pdDCC) coupled to saturation-transfer difference (STD) NMR spectroscopy was initially employed to identify modulators of the malaria vector Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1). A library of potential binders of AgamOBP1 (secondary amines) generated from two amines and seven aldehydes was designed aiming to enable interactions with critical amino acids at the DEET-site and to bridge the DEET- and Icaridin sIC-binding pockets, both implicated in repellents recognition. Solubility issues hindered the clear identification of binders among the DCL members, except for one sublibrary, leading us to shift our strategy towards the synthesis of the designed amines, followed by direct evaluation of their binding to AgamOBP1 using 1H STD NMR spectroscopy. The identified binders were further validated in vitro by fluorescence competition assays, and the most potent compounds which also possessed suitable vapor pressure were evaluated as repellents in arm-in-cage behavioral assays against Aedes albopictus. Amines 2A, 3A, 4A, and 6A showed significant repellent activity. The most potent was compound 4A (4-methyl-N-(pyridin-4-ylmethyl)aniline) which acted as a a DEET-like repellent at 0.4 μL cm- 2 dose. Thus, our strategy showcased a promising scaffold for further optimization toward efficient mosquito repellents.

Climate change significantly influences the spread of infectious diseases, including leishmaniasis, which is transmitted by phlebotomine sand flies. The geographical distribution of sand flies has expanded northward from the Mediterranean region, increasing the risk of leishmaniasis in areas that previously lacked systematic vector surveillance. This study presents FEPO (SandFlies Extreme POpulation prediction), a machine learning ensemble model that serves as a core component for developing early warning systems for vector-borne diseases. FEPO uses more than one thousand field trap records collected between 2011 and 2022, along with 1 km meteorological, hydrological, and morphological grids, to produce daily maps of sand fly density spanning 26 European countries. The model stacks gradient boosted decision trees using CatBoost and applies a tailored under and over sampling strategy to address the scarcity and skewness of observational data, where occasional population surges are buried among many zero and low abundance counts. Tenfold cross validation shows that FEPO achieves an 11% lower mean absolute error compared to baseline regression models. The model reveals persistent hotspots along the Mediterranean and Balkan coasts, as well as in parts of Central and Northern Europe, where environmental conditions favor vector proliferation. By delivering high resolution outputs, FEPO enables public health agencies to target trapping and mitigate outbreaks while also offering a transferable blueprint for early warning systems that address other climate sensitive disease vectors.

Vector-borne diseases significantly impact global public health, with mosquitoes playing a critical role in the transmission of various pathogens. This study focused on the mosquito fauna in the Attica region of Greece, conducting a two-year entomological survey from March 2021 to December 2022 as part of an ongoing mosquito-management program. The research employed stratified random sampling to establish 57 adult traps across the region, with additional traps on the islands of Argosaronikos and Kythira island. The BG-sentinel traps, enhanced with CO2 to attract multiple mosquito species, were utilized for mosquito collection. Morphological identification of the collected mosquitoes revealed the presence of various species, with Aedes albopictus, Culex pipiens s.l., and Culiseta longiareolata being the most prevalent. Notably, all of our traps tested 100% positive for these species. Molecular techniques, including PCR amplification of ITS2 and COI genes, confirmed species identification. The findings highlight significant variations in species composition across different locations and emphasize the presence of invasive species such as Aedes albopictus, posing public health concerns. This study underscores the importance of continuous mosquito surveillance and integrated management strategies to mitigate the risk of mosquito-borne diseases in the Attica region. The results contribute to the development of evidence-based mosquito-control programs, which are essential for safeguarding public health in urban and peri-urban environments.

The boosted Sterile Insect Technique (SIT) by releasing sterilized males coated with the larvicide pyriproxyfen has been suggested for the control of Aedes container-breeding mosquitoes. In 2023, a boosted-SIT field trial was applied in Greece against Aedes albopictus by weekly releases of pyriproxyfen-coated sterile males at the beginning (12 May-16 June) and peak (1 September-6 October) of mosquito activity, including two Mark-Release-Recapture sessions in July. No overall effect on egg densities was noted in the treated site, while a significant decrease compared to a control site was recorded in specific locations. The egg hatching rate in the treated site (56-57%) was lower than the control site (84-85%) during the release cycles and one month following the second release cycle. During the second release cycle and the following month, an overall suppression of the adult population was achieved (85-93% reduction compared to the control site), and high boosted sterile-to-wild male ratio (15:1) and competitiveness index of boosted-SIT males (0.46) were recorded, respectively. The suppression of both adult and egg populations was more pronounced a month after the conclusion of releases, indicating a residual activity of boosted-SIT. Our findings demonstrate the potential of boosted-SIT against Ae. albopictus particularly in suppressing adult population.

Mass production of Aedes albopictus for Sterile Insect Technique (SIT) requires cost-effective and nutritionally balanced larval diets to ensure high survival, optimal development and competitive adult fitness. This study evaluates the potential of insect-derived meals and dead autoclaved bacteria as the main protein sources in mosquito larval diets. Four isoproteinic diets were studied, each one incorporating different protein sources: Brewer's yeast (CAA), Tenebrio molitor meal (UTH-YM), Hermetia illucens meal (UTH-BSF) and Enterobacter spp. dry biomass (UTH-ENT). Results demonstrated immature survival higher than 78% for all diets. Developmental duration varied significantly across diets. The UTH-ENT diet extended larval development and conferred greater wing length. The highest protandry percentage was observed in the CAA diet, facilitating sex separation for SIT implementation. Adult survival over 20 days varied significantly among diets but not sexes. Males from the UTH-BSF diet exhibited the highest survival rate, while females from UTH-ENT diet showed the shortest lifespan. Given the rising costs and the variability in the quality of brewer's yeast, our findings support the integration of insect-based diets as sustainable protein source alternatives for Ae. albopictus mass-rearing. Further research should refine larval diet formulations considering that combining different protein sources may enhance rearing success, to facilitate SIT efficiency and sustainable mosquito suppression.

The Asian tiger mosquito, Aedes albopictus, is currently the most widespread invasive mosquito species in the world. It poses a significant threat to human health, as it is a vector for several arboviruses. We used a SNP chip to genotype 748 Ae. albopictus mosquitoes from 41 localities across Europe, 28 localities in the native range in Asia, and 4 in the Americas. Using multiple algorithms, we examined population genetic structure and differentiation within Europe and across our global dataset to gain insight into the origin of the invasive European populations. We also compared results from our SNP data to those obtained using genotypes from 11 microsatellite loci (N = 637 mosquitoes from 25 European localities) to explore how sampling effort and the type of genetic marker used may influence conclusions about Ae. albopictus population structure. While some analyses detected more than 20 clusters worldwide, we found mosquitoes could be grouped into 7 distinct genetic clusters, with most European populations originating in East Asia (Japan or China). Interestingly, some populations in Eastern Europe did not share genetic ancestry with any populations from the native range or Americas, indicating that these populations originated from areas not sampled in this study. The SNP and microsatellite datasets found similar patterns of genetic differentiation in Europe, but the microsatellite dataset could not detect the more subtle genetic structure revealed using SNPs. Overall, data from the SNP chip offered a higher resolution for detecting the genetic structure and the potential origins of invasions.

The stable presence of the Aedes albopictus mosquito in Europe has set the stage for the emergence of tropical arboviral outbreaks (such as dengue and chikungunya), following the importation of infection by international travelers. Here, we leverage Ae.albopictus capture data collected weekly in Chania, Greece, in 2017 and 2018, to calibrate a model for assessing the potential epidemiological risks of mosquito-borne outbreaks such as dengue, chikungunya, and Zika. We estimated a peak density of female mosquitoes of 459 (95% Credible Interval, CrI: 424-508) per hectare in 2017 and 757 (95% CrI: 728-785) in 2018. The peak reproduction numbers occurred in early September and exceeded the epidemic threshold of 1 in 20-26% of the municipality area for dengue and in 40-70% for chikungunya (depending on the year). In contrast, we found a negligible risk of Zika transmission. We assessed the quantitative risks of outbreaks for both dengue and chikungunya, using two alternative measures, the Instantaneous Epidemic Risk (IER), and the Threshold Epidemic Risk (TER). We assessed quantitative differences in the two metrics and their determinants, showing that the IER tends to underestimate the risk of onward transmission early in the summer and to overestimate it in the second half of the season. This study identifies non-negligible risks of arboviral outbreaks in a country that, to date, has not recorded autochthonous transmission. It also underscores the importance of considering and adjusting for potential biases in traditional measures of epidemic risk.

The West Nile virus vector Culex pipiens and invasive species Aedes albopictus are abundant in Attica, Greece, which deploys an integrated vector management program for targeted vector control. The objective of our study is to evaluate the effects of this program on mosquito vector populations. Using mosquito surveillance and intervention occurrence data, we assessed the effects of mosquito control interventions on vector populations using a two-stage interrupted time series (ITS) approach. First, we fitted ITS models to 16 weeks of data centered on the week of each unique species-specific intervention. Second, we pooled the estimated coefficients in a meta-regression model. Following vector control interventions at the targeted intervention sites, we observed an overall 34% reduction (RR 0.66; 95% CI 0.50-0.89) in Culex pipiens counts and a nonsignificant 5% increase (RR 1.05; 95% CI 0.81-1.35) in Aedes albopictus counts, compared to the pre-intervention period. These results support the implementation in reducing Culex pipiens populations in Attica as part of an integrated public health plan to mitigate West Nile virus risk. However, the limited impact on Ae. albopictus suggests the need for complementary strategies beyond conventional biocides. Overall, this study could serve a model for evaluating IVM programs in diverse settings.

With the worldwide spread of the Asian tiger mosquito, Aedes albopictus, the number of autochthonous cases of exotic arboviral diseases, such as dengue or chikungunya, is increasing in temperate regions. In Europe, pyrethroids are the only insecticides allowed for the abatement of adult mosquitoes and are thus crucial for limiting ongoing arbovirus transmission. Despite this and the report of resistance rising in vector populations worldwide, information on the pyrethroid resistance status of vector populations and knowledge on resistance mechanisms is widely lacking. Genotyping of knockdown resistance (kdr) mutations situated within the target site of pyrethroids, i.e., the voltage-gated sodium channel (VGSC), and associated with pyrethroid resistance, is a cost-effective approach to investigate the spread of resistance in a population. Herein, we describe the European-wide distribution of two kdr mutations, i.e., I1532T and F1534C, in Ae. albopictus and evaluate their co-occurrence with another well-characterized kdr mutation, V1016G. Genotyping of the kdr mutation F1534C was performed by allele-specific PCR for 1732 Ae. albopictus specimens sampled in 19 European countries; for a subset of 419 specimens mutation I1532T was also genotyped by sequencing. For all samples, information on mutation V1016G was available, allowing evaluation of the co-occurrence of kdr alleles. Mutation 1534C was detected in nine sites from six countries at an overall frequency close to 5