Usutu virus (USUV) is a flavivirus of the Japanese encephalitis complex transmitted between Culex mosquitoes and birds, a transmission pattern similar to that of the West Nile virus (WNV). In Germany, the first case of USUV was detected in 2010 in mosquitoes collected in the town of Weinheim, and by 2018 the virus had spread to almost the entire country. Interestingly, the infection front exhibited a clockwise rotational spread pattern throughout the years, a pattern completely different from that of the WNV. This clockwise progression corresponded closely with the spatial temperature gradient, suggesting that warmer regions probably facilitated faster viral amplification and onward transmission. Understanding the drivers that influence the spreading patterns of arboviruses is important as it guides surveillance and implementation of control strategies. In this study, we develop a reaction-diffusion partial differential equation (PDE) model to investigate the spatial spread of USUV in Germany within an extended domain that includes some neighbouring countries (Belgium, the Netherlands, and Luxembourg), thereby capturing cross-border transmission processes. Mosquito parameters, i.e., extrinsic incubation rate, mortality and biting rates, are temperature-driven, as temperature plays an important role in the activity of mosquitoes. Our model qualitatively reproduced the main spatial trends of USUV in Germany and surrounding countries. The heterogeneous spread pattern arises from the interplay of diffusion and spatially varying temperature, which together may influence determine regions with higher transmission potential.

The globally distributed arbovirus West Nile virus (WNV) is expanding across Europe, with rising numbers of human cases and an increasingly broad geographic distribution. Transmitted primarily by Culex mosquitoes, WNV lineages 1 and 2 are pathogenic to humans, yet their differences in transmission dynamics remain unclear. As vertebrate hosts develop varying viremia, mosquitoes encounter a wide range of viral doses, which may influence transmission. Here, we investigated the dose-response differences between the lineages in Cx. pipiens biotype pipiens, Cx. torrentium, and Aedes albopictus. Mosquitoes were fed increasing titers of one strain of either WNV lineage, and infection and transmission were assessed after 14 days at 24 °C. Lineage 2 required lower infectious doses while causing higher transmission. Notably, Cx. torrentium was identified as a highly competent vector for lineage 2. These findings highlight important differences between WNV lineages, improve our understanding of dose-dependent transmission, and therefore support refined risk assessments in Europe.

Abstract Barmah Forest Virus (BFV), an arthropod-born virus transmitted by mosquitoes, is of significant public health concern in Australia and regions in the Pacific. Recent climate change and globalization raise the potential for BFV to extend its geographic distribution. Despite the rising importance of BFV, its vector dynamics remain poorly understood, particularly concerning the vector competence of different mosquito species. This study aims to investigate the vector competence of BFV across various mosquito species, beyond those endemic to the Australasian region, especially focusing on global relevant vector species. No transmission was observed for Culex quinquefasciatus and Cx. torrentium as well as Anopheles stephensi . In contrast, both investigated Aedes species, Ae. aegypti as well as Ae. albopictus , exhibited BFV-positive saliva across all four temperature profiles (18°C, 21°C, 24°C or 27°C) examined. These two invasive mosquito species must therefore be classified as potential vectors for BFV, indicating the potential risk of BFV transmission outside of Australia.

The globally distributed arbovirus West Nile virus (WNV) continues to expand across Europe, with rising numbers of human cases and an increasingly broad geographic distribution. WNV is primarily transmitted by mosquitoes of the genus Culex . Out of nine WNV lineages, human pathogenicity has been clearly established for lineages 1 and 2, but differences in their transmission dynamics, such as minimal infectious dose and transmission efficiency, remain poorly understood. In this study, we investigated how viral lineage, mosquito species, and blood meal titer influence the vector competence of two primary WNV vectors from Europe, Cx. pipiens biotype pipiens and Cx. torrentium , as well as the invasive mosquito species Aedes albopictus . Female mosquitoes were fed with increasing blood meal titers containing either of the WNV lineages. After an incubation period of 14 days at a mean temperature of 24°C, mosquito body titers were quantified, and the presence of infectious viral particles in the saliva was assessed. Our results revealed a clear difference between the two lineages. Lineage 2 resulted in higher transmission efficiencies across all three species and required lower infectious doses to cause transmission. Among the tested species, Cx. torrentium proved to be a highly competent vector (max. transmission efficiency = 30%, minimum infectious dose = 105 TCID50/mL), despite its underrepresentation in research. These findings provide detailed insights into how viral lineage, mosquito species, and blood meal titer might shape WNV transmission, informing future risk assessments and efforts to mitigate WNV transmission in Europe. ### Competing Interest Statement The authors have declared no competing interest. German Federal Ministry of Agriculture, Food and Regional Identity, FKZ2819107A22 German Federal Ministry of Research, Technology and Space, 01Kl2022 Klaus Tschira Boost Fund

Abstract The mosquito Aedes albopictus is expanding across Europe, raising concerns due to its ability to transmit arboviruses. The larvicide Bacillus thuringiensis subsp. israelensis (Bti) plays a central role in controlling the spread of Ae. albopictus. The objective of this study is to assess the temporal effectiveness of Bti as a control method using fine-scale spatiotemporal data and a distributed lag non-linear modelling framework. We analyzed 1,320 ovitrap observations from 195 traps alongside records of 4,387 Bti treatments and local environmental conditions for the entire 2023 mosquito season in Heidelberg, Germany. Bti treatments produced a clear reduction in egg counts, with the strongest marginal effects occurring 6–13 days after treatment with efficacy diminishing thereafter at lower treatment counts, while higher counts demonstrated sustained effects. Cumulatively, Bti was highly effective at reducing egg counts even at moderate treatment levels, and a counterfactual no Bti scenario suggested that Bti treatments reduced seasonal egg production by an estimated 41.9% (95% CI 24.4% – 58.5%) and prevented total establishment across the study site. These results demonstrate that Bti can substantially reduce Ae. albopictus populations in urban settings, though its limited temporal efficacy underscores the need for repeated interventions to prevent establishment and spread.

Native to East Asia, the Asian bush mosquito (Aedes japonicus) has recently expanded its global range, with established invasive populations in Europe and North America. Given its potential role as a vector of various arboviruses, understanding its invasion process and ecological dynamics is crucial for managing its spread and mitigating public health risks. In the Iberian Peninsula, the species was first detected in Asturias in 2018 and has since expanded to neighbouring regions. Here, we elucidate the invasion pathways and possible origins of Ae. japonicus populations in Spain using sequence data and microsatellite markers, and by screening for the presence of maternally transmitted bacteria of the genus Wolbachia. We analysed 635 Ae. japonicus from 14 countries, including Japan (native range), the United States, and 12 European countries. No clear association between haplotypes and geographical location was detected in any of the three genes analysed (nuclear ITS2, mitochondrial COI and ND4). Wolbachia was not detected in any of the screened samples. In contrast, microsatellite-based population structure analyses showed that most Spanish samples clustered closely with those from College Park, Maryland (USA), located near the Port of Baltimore, one of the largest ports in the United States and a recognised gateway for invasive species introductions. Northern Spain hosts major seaports such as Bilbao and Gijón, whereas the nearest established Ae. japonicus population in Europe lies over 1,000 km away in northeastern France. Taken together, these findings suggest that the most plausible invasion route of Ae. japonicus into Spain involves maritime transport from the eastern coast of the United States to northern Spanish ports, likely accompanied by additional minor introductions of European origin. The inclusion of additional microsatellite loci originally developed for Ae. albopictus yielded results consistent with those obtained using Ae. japonicus-specific loci, reinforcing the robustness of the observed patterns. This work provides new insights into the invasion process of Ae. japonicus in Europe and highlights the need for continuous monitoring and tailored interventions at key ports of entry.

Accurate identification of mosquito species is essential for effective vector control and mitigation of mosquito-borne disease outbreaks. Traditional morphological identification requires highly specialized personnel and is time-consuming, while molecular techniques can be cost-effective and dependent on comprehensive genetic information. Wing geometric morphometry has emerged as a promising alternative, leveraging detailed geometric measurements of wing shapes and vein patterns to distinguish between species and detect intraspecies variations. This paper presents a curated dataset of 18,104 mosquito wing images, collected from 10,500 mosquito specimens, annotated with extensive meta-information, designed to support research in wing geometric morphometry and the development of machine learning models, ultimately supporting efforts in vector surveillance and research.

OBJECTIVES:The World Health Organization recommends three drug anti-malarial combinations: cloroquine+primaquine, artesiminin+primaquine, and cloroquine+tafenoquine. These combinations aim to eradicate Plasmodium by disrupting its life cycle within the human body. We evaluated the effect of these medications on the vectorial competence of two main vectors in the New World. METHODS:We recruited patients diagnosed with malaria vivax from a primary care in Manaus, Amazonas. To determine how different treatments affected vectors, we collected blood samples prior to treatment at the hospital and at six intervals over the next 68 hours at the patient's homes. These samples were used to infect Anopheles darlingi and Anopheles aquasalis. To assess the potential for Plasmodium transmission by bite to a new human host, we analyzed the infection intensity, infection rate, and presence of parasites in the salivary gland of the mosquitoes. RESULTS:The results show the infection of the mosquitoes fed with the patient's blood during the first days of treatment with all three drug combinations. However, the cloroquine+tafenoquine combination was the least effective while artesiminin+primaquine was the most effective. CONCLUSIONS:In the first few days of treatment, two main vectors continue to spread malaria vivax from patients, potentially contributing to the ongoing transmission in malaria-endemic regions.

Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. In this work, we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground mosquito. Population genomic analysis of ~350 contemporary and historical samples counters the popular hypothesis that molestus originated belowground in London <200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Mediterranean or Middle East over the course of more than 1000 years, possibly in association with ancient agricultural civilizations of the Middle East. Our results highlight the role of early human society in priming taxa for contemporary urban evolution. They also provide insight into whether and how molestus contributes to West Nile virus transmission in modern cities.

Tahyna virus (TAHV) was the first mosquito-borne virus isolated in Europe, and has since been found throughout Eurasia and Africa. Infections are mostly asymptomatic but can cause “Valtice fever”, characterized by influenza-like symptoms, mainly in children, with severe cases occasionally causing neurological symptoms. The virus is maintained in an enzootic cycle between small mammals and mosquitoes. Recent and comprehensive studies of vector competence for TAHV are scarce. To fill this gap, and to better understand the transmission cycle of TAHV, we studied ten taxa ( Ae. aegypti , Ae. albopictus , Ae. japonicus japonicus , Ae. koreicus , Ae. rusticus , Cx. pipiens biotype pipiens , Cx. torrentium , Cs. morsitans/fumipennis , An. daciae , and An. stephensi ) by orally infecting them with TAHV. All taxa were susceptible to TAHV infection. Additionally, the invasive species Ae. albopictus , and Ae. rusticus , a species native to Europe, were able to transmit the virus at 27 ± 5 °C, with transmission efficiencies of 3.3% and 14.3%, respectively. Therefore, it is plausible for TAHV to be transmitted by Ae. albopictus and Ae. rusticus in natural settings in Europe. At a lower temperature of 24 ± 5 °C, infection and transmission decreased in Ae. albopictus . This data will allow future risk models and early warning systems to better predict TAHV transmission.

The increasing incidence of arboviral diseases in Europe, driven by the expansion of mosquito vectors due to globalisation and global warming, poses a growing threat to public health. Notably, the invasive tiger mosquito Aedes albopictus , a primary vector of dengue, has been rapidly expanding its range, with outbreaks becoming more frequent in various parts of the world. Insecticides targeting adult mosquitoes are commonly employed as response and protective measures for vector control, but the effectiveness of such interventions may be undermined by rising insecticide resistance, a phenomenon increasingly reported worldwide. Another promising avenue for vector control is the use of Wolbachia , an endosymbiotic bacterium capable of reproductive manipulation in mosquitoes, offering potential for population suppression. We evaluated permethrin (a pyrethroid insecticide) resistance in key mosquito species, including Aedes and Culex , collected from Germany through generation of LC 50 curves utilising topical exposure assays. Additionally, the prevalence of Wolbachia in these populations was determined via PCR amplification of the 16S rRNA gene, followed by sequencing of selected samples. All Aedes populations tested exhibited susceptibility to permethrin, whilst a potential trend toward resistance was observed in the Culex pipiens complex, a vector of West Nile virus. However, low numbers of field-caught Ae. albopictus and Ae. japonicus preclude firm conclusions about resistance in these species. Furthermore, Wolbachia was detected across all tested mosquito populations, marking the first recorded presence of Wolbachia in Ae. japonicus . These findings highlight the continued efficacy of pyrethroids against Aedes populations in Germany and underscore the need for ongoing surveillance of insecticide resistance, particularly in Culex species. Additionally, the detection of Wolbachia in native and invasive mosquito populations opens new avenues for the exploration of biological vector control strategies in Europe. This study provides crucial preliminary data supporting the strategic use of pyrethroids and Wolbachia for arboviral outbreak prevention in Germany.

Oropouche virus (OROV) emerged as a significant health threat in Central and South America in 2024. Culicoides (Diptera: Ceratopogonidae) is considered the primary vector, while the role of mosquitoes in the transmission cycle is still unclear. This study evaluates the vector competence of 5 mosquito species common in Europe for OROV (old strain). While Culex torrentium, Culex pipiens biotype pipiens, Aedes aegypti, and Aedes japonicus showed no transmission, Aedes albopictus demonstrated low transmission at higher temperatures. Temperature-based risk analysis for areas with established A albopictus populations suggests that regions around the Mediterranean are at potential risk for OROV transmission.

O'nyong-nyong virus (ONNV) is a zoonotic vector-borne alphavirus with humans as primary hosts during urban outbreaks. While its enzootic transmission cycle is not well investigated, its pattern of disappearance and reemergence suggests overlooked but critical wildlife reservoirs. While ONNV travel-related cases were reported in Canada and Germany, autochthonous transmission is limited to sub-Saharan Africa. The lack of specialised vectors, such as anopheline mosquitoes, is considered to be one of the reasons. Here, we investigated the vector competence of native and invasive culicine mosquitoes from Germany. While the tested native culicine mosquitoes from Germany did not transmit ONNV, our results provide experimental evidence for potential ONNV transmission by invasive Ae. albopictus mosquitoes. We further investigated the role of temperature on ONNV transmission. Ae. albopictus mosquitoes are highly invasive and have become widespread in Europe in the last decade. Our results highlight that, combined with rising summer temperatures and the abundance of competent vectors, ONNV may emerge as a public health threat for Europe in the near future.

Abstract Background Batai virus (BATV) is a zoonotic arbovirus of veterinary importance. A high seroprevalence in cows, sheep and goats and infection in different mosquito species has been observed in Central Europe. Therefore, we studied indigenous as well as exotic species of the genera Culex and Aedes for BATV vector competence at different fluctuating temperature profiles. Methods Field caught Culex pipiens biotype pipiens, Culex torrentium, Aedes albopictus and Aedes japonicus japonicus from Germany and Aedes aegypti laboratory colony were infected with BATV strain 53.3 using artificial blood meals. Engorged mosquitoes were kept under four (Culex species) or three (Aedes species) fluctuating temperature profiles (18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C) at a humidity of 70% and a dark/light rhythm of 12:12 for 14 days. Transmission was measured by testing the saliva obtained by forced salivation assay for viable BATV particles. Infection rates were analysed by testing whole mosquitoes for BATV RNA by quantitative reverse transcription PCR. Results No transmission was detected for Ae. aegypti, Ae. albopictus or Ae. japonicus japonicus. Infection was observed for Cx. p. pipiens, but only in the three conditions with the highest temperatures (21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C). In Cx. torrentium infection was measured at all tested temperatures with higher infection rates compared with Cx. p. pipiens. Transmission was only detected for Cx. torrentium exclusively at the highest temperature of 27 ± 5 °C. Conclusions Within the tested mosquito species, only Cx. torrentium seems to be able to transmit BATV if the climatic conditions are feasible. Graphical Abstract

Accurate species identification is crucial to assess the medical relevance of a mosquito specimen, but requires intensive experience of the observers and well-equipped laboratories. In this proof-of-concept study, we developed a convolutional neural network (CNN) to identify seven Aedes species by wing images, only. While previous studies used images of the whole mosquito body, the nearly two-dimensional wings may facilitate standardized image capture and reduce the complexity of the CNN implementation. Mosquitoes were sampled from different sites in Germany. Their wings were mounted and photographed with a professional stereomicroscope. The data set consisted of 1155 wing images from seven Aedes species as well as 554 wings from different non-Aedes mosquitoes. A CNN was trained to differentiate between Aedes and non-Aedes mosquitoes and to classify the seven Aedes species based on grayscale and RGB images. Image processing, data augmentation, training, validation and testing were conducted in python using deep-learning framework PyTorch. Our best-performing CNN configuration achieved a macro F1 score of 99% to discriminate Aedes from non-Aedes mosquito species. The mean macro F1 score to predict the Aedes species was 90% for grayscale images and 91% for RGB images. In conclusion, wing images are sufficient to identify mosquito species by CNNs.

Abstract Background The incidence of human infections caused by arthropod-borne viruses, such as the chikungunya virus (CHIKV), has increased globally due to a number of factors, such as climate change and globalization. The exotic mosquito species Aedes albopictus is a significant vector for CHIKV, raising concerns about its transmission potential in temperate regions, including Central Europe. We have therefore investigated the vector competence of Ae. albopictus for CHIKV at constant and fluctuating temperatures between 15 °C and 24 °C to assess the transmission risk in Europe. Methods Aedes albopictus mosquitoes were reared and artificially infected with CHIKV. Infection rates and transmission efficiencies (TEs) were determined after 14 days of incubation at constant and fluctuating (± 5 °C) mean temperatures of 15 °C, 18 °C, 21 °C and 24 °C. In addition, mosquito locomotor activity was measured under the same fluctuating temperature conditions. A risk map for CHIKV transmission in Europe was generated combining temperature data and the current distribution of Ae. albopictus. Results CHIKV transmission was observed at all tested temperatures. The highest TEs were recorded at fluctuating temperatures of 18 °C (54.3%) and 21 °C (58.6%), while the lowest TE was observed at a constant temperature of 15 °C (5.6%). TEs at fluctuating temperatures of 15 °C and 24 °C were the same (32.5%). Mosquito activity showed a nocturnal unimodal activity pattern with a peak during the start of the scotophase (hour 20). The proportion of active mosquitoes per hour increased with temperature and was nearly zero at 15 °C. The risk map indicated that regions in Southern and Central Europe, including recently invaded areas north of the Alps, have temperatures theoretically allowing CHIKV transmission for at least some days per year. Conclusions While CHIKV can be transmitted by Ae. albopictus at 15 °C, the activity of this mosquito is strongly decreased at this temperature, likely reducing the transmission risk. These findings emphasize the importance of considering both vector competence and mosquito activity when assessing the risk of arbovirus transmission in temperate regions. Further studies are needed to validate these laboratory findings under field conditions. Graphical abstract

The Usutu Virus (USUV) is a mosquito-borne flavivirus originated in Africa. The virus circulates in Germany since 2010. It is primarily transmitted and maintained in the natural cycle by Culex mosquitoes and primarily affects birds, particularly Eurasian blackbird (Turdus merula), leading to significant mortality. Several studies have reported a high co-infection rate of European birds with both USUV and haemosporidians. Haemosporidians are blood parasites which maintain an enzootic life cycle with birds via different arthropod vectors. This study conducted screenings of birds from Germany received through a citizen's science project for both, USUV and haemosporidians between 2016 and 2021. The prevalence of USUV reached its peak in 2018, when it was first detected throughout most parts of Germany rather than being limited to localised hotspots. Subsequently, USUV prevalence consistently declined. On the other hand, the prevalence of haemosporidians initially declined between 2016 and 2019, but experienced a subsequent increase in the following years, exhibiting a more or less inverse pattern compared to the prevalence of USUV. In 2020, a statistically significant positive association between both pathogens was found, which was also detected across all years combined, indicating if at all a weak relationship between these pathogens.

Snowshoe hare virus (SSHV) is a zoonotic arthropod-borne virus (arbovirus) circulating in colder areas of the Northern Hemisphere. SSHV is maintained in an enzootic cycle between small mammals and mosquitoes, assumably of the genera Aedes and Culiseta. Symptoms of SSHV human infection can range from asymptomatic to severe neuroinvasive disease. Studies on SSHV transmission are limited, and there is no information available on whether mosquitoes of the genus Culex are able to transmit SSHV. Therefore, we investigated six mosquito species via salivation assay for their vector competence. We demonstrated that SSHV can be transmitted by the abundant European Culex species Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, and Cx. torrentium with low transmission efficiency between 3.33% and 6.67%. Additionally, the invasive species Ae. albopictus can also transmit SSHV with a low transmission efficiency of 3.33%. Our results suggest that local transmission of SSHV after introduction to Europe seems to be possible from a vector perspective.

Background Mosquito research in Europe has a long history, primarily focused on malaria vectors. In recent years, invasive mosquito species like the Asian tiger mosquito ( Aedes albopictus ) and the spread of arboviruses like dengue virus, chikungunya virus or bluetongue virus have led to an intensification of research and monitoring in Europe. The risk of further dissemination of exotic species and mosquito-borne pathogens is expected to increase with ongoing globalization, human mobility, transport geography, and climate warming. Researchers have conducted various studies to understand the ecology, biology, and effective control strategies of mosquitoes and associated pathogens. Main body Three invasive mosquito species are established in Europe: Asian tiger mosquito ( Aedes albopictus ), Japanese bush mosquito ( Ae. japonicus ), and Korean bush mosquito ( Aedes koreicus ). Ae. albopictus is the most invasive species and has been established in Europe since 1990. Over the past two decades, there has been an increasing number of outbreaks of infections by mosquito-borne viruses in particular chikungunya virus, dengue virus or Zika virus in Europe primary driven by Ae. albopictus . At the same time, climate change with rising temperatures results in increasing threat of invasive mosquito-borne viruses, in particular Usutu virus and West Nile virus transmitted by native Culex mosquito species. Effective mosquito control programs require a high level of community participation, going along with comprehensive information campaigns, to ensure source reduction and successful control. Control strategies for container breeding mosquitoes like Ae. albopictus or Culex species involve community participation, door-to-door control activities in private areas. Further measures can involve integration of sterile insect techniques, applying indigenous copepods, Wolbachia sp. bacteria, or genetically modified mosquitoes, which is very unlike to be practiced as standard method in the near future. Conclusions Climate change and globalization resulting in the increased establishment of invasive mosquitoes in particular of the Asian tiger mosquito Ae. albopictus in Europe within the last 30 years and increasing outbreaks of infections by mosquito-borne viruses warrants intensification of research and monitoring. Further, effective future mosquito control programs require increase in intense community and private participation, applying physical, chemical, biological, and genetical control activities.