Medical and Veterinary Entomology

In Germany, insecticide use is recommended during mosquito-borne disease outbreaks, yet nationwide data on insecticide resistance in key vector species are scarce. This is particularly relevant for Culex pipiens s.l., a vector of West Nile virus. Here, we provide the first broad-scale assessment of molecular insecticide resistance (kdr and ace-1 mutations) in Cx. pipiens f. pipiens across Germany. We associated insecticide resistances with environmental conditions, potential links with mitochondrial variation of the cytochrome c oxidase I (COI) gene as well as phenotypic resistance (CDC bottle assay with permethrin) of the Frankfurt population with elevated kdr frequencies. Kdr resistance alleles were detected in German Cx. pipiens f. pipiens reaching frequencies of up to 50% in the metropolitan areas of Berlin and Frankfurt am Main. Annual precipitation was negatively associated with kdr allele frequency. Interestingly, ace-1 resistance mutations were only present in the metropolitan area of Frankfurt am Main and in Muggensturm (Rhine valley, southwestern Germany). COI haplotype analyses revealed low genetic variability within German Cx. pipiens f. pipiens populations with no association to the insecticide resistance genotype. Phenotypic assays of the Frankfurt population showed early signs of permethrin resistance, but unmatched genotypic and phenotypic resistance state. This study provides the first broad-scale assessment of molecular insecticide resistance in Cx. pipiens f. pipiens across Germany, establishing a baseline for resistance surveillance and vector control. Further research is needed to identify factors shaping phenotypic resistance, as molecular markers alone may not reliably predict resistance. Since sampling was restricted to urban green spaces, broader sampling across environmental gradients is needed to identify additional environmental effectors.

In sub-Saharan Africa, human and animal health is threatened by African trypanosomoses. These diseases are caused by trypanosomes that are transmitted primarily by tsetse flies. Vector control remains one of the most effective strategies for reducing disease transmission, but requires a minimum level of knowledge of the population biology, ecology and dispersal patterns of the targeted vectors. In this respect, population genetic approaches using appropriate polymorphic genetic markers can provide valuable tools to inform and optimize control strategies. Senegal is currently engaged in a national tsetse control programme and has been identified as a study country within a European Union-funded research initiative, COMBAT (controlling and progressively minimizing the burden of animal trypanosomosis). Baseline entomological studies are currently underway in the Sine Saloum (Southern Senegal). This preliminary study aimed to characterize the population genetic structure and connectivity of tsetse fly species present in Southern Senegal, Glossina palpalis gambiensis and G. morsitans submorsitans, in order to assess their population sizes (geographically and demographically), population densities and dispersal capacities. The two species exhibited markedly contrasting population structures. The first, G. p. gambiensis, displayed an isolation by distance with rather dense subpopulations exchanging immigrants at short distances (1.6 km/generation) and continuously from Sine-Saloum to Casamance, in line with the apparent continuity of the mangrove network found along this coast. In contrast, G. m. submorsitans presented a relatively dense population over a 75 km wide area, fragmented into sparsely distributed, very dense and interconnected pockets. These findings indicate that G. p. gambiensis likely constitutes a large metapopulation comprising 3266 effective individuals on average (and up to 7765), whereas G. m. submorsitans appeared as a single and large population, scattered in small pockets that could be readily replenished by immigrants from distant areas in case of control. These results suggest that elimination is not a realistic option for any of these two populations. Alternatively, localized integrated pest management is likely to be the most effective approach for controlling the negative effects of these biting flies on domestic animals, by reducing trypanosome transmission. However, further entomological investigations and the use of more informative genetic markers are needed to obtain a more precise understanding of the epidemiological processes involved.

Aedes albopictus (Skuse), the Asian tiger mosquito, is a highly competent arboviral vector whose range has expanded substantially across the United States over the past four decades. Despite predictive models placing Nebraska within the species' climatically suitable range, its establishment status in the state has remained poorly characterized. Here, we report results from a nine-year mosquito surveillance program (2017-2025) conducted across 44 Nebraska counties in collaboration with the Nebraska Department of Health and Human Services. Ae. albopictus was detected in five counties, with sustained, annually increasing populations documented in Richardson, Douglas, and Lancaster counties. Richardson County recorded continuous detections during 2017-2025, with proportional representation rising to 60.50% of collected mosquitoes by 2025. In Douglas and Lancaster counties, temporal advancement of first seasonal detection in 2024 and 2025 provide evidence consistent with successful overwintering rather than annual reintroduction. A cumulative degree-day model predicted adult emergence in mid-May across all county-year combinations, consistently preceding trap deployment by two to seven weeks and revealing a systematic early-season surveillance gap. Generalized linear mixed-effects models indicated that trap-level detection persistence, rather than urban location, was the primary predictor of yearly Ae. albopictus positivity, suggesting that current invasion dynamics are driven by focal source populations. These findings provide strong evidence for the establishment of Ae. albopictus in eastern Nebraska and highlight the need for earlier seasonal surveillance and standardized criteria to define establishment in northward-expanding vector populations.

Stink bugs (Hemiptera Pentatomidae) are major agricultural pests worldwide, including both native and invasive species. In France, for instance, Halyomorpha halys has rapidly expanded its range, affecting key crops such as hazelnuts, apples, and vegetables. Meanwhile, several native, polyphagous species - Nezara viridula, Palomena prasina and, to a lesser extent, Graphosoma italicum - are re-emerging and impacting diverse crops because of changes in agricultural practices and climatic conditions. Egg parasitoids represent promising biological control agents for these pests, be it through conservation, augmentation or introduction biological control. However, their actual potential is not precisely documented due to a lack of ambitious surveys and possible taxonomic issues. This study aims to address this gap by compiling results obtained from various of egg parasitism surveys of Pentatomidae in France and implementing a large-scale DNA Barcoding approach (2000 individuals) for the emerged parasitoids. Our results indicate a high diversity of egg parasitoids, and detected some taxonomic issues and molecular inconsistencies that warrant further study. Among the three families of egg parasitoids recovered (Encyrtidae, Eupelmide, and Scelionidae), Scelionidae was the most abundant and diverse with nine species of Trissolcus (including the two adventive species, Tr. japonicus and Tr. mitsukurii) and four Telenomus species (including two unidentified to species). Scelionidae dominated the parasitoid assemblages associated with most of the Pentatomidae host species, except for H. halys for which the eupelmid Anastatus bifasciatus was the most common species.

While mosquito invasions and associated pathogens are increasingly prominent in Europe, evidence gaps remain for many northerly regions where proactive management interventions may be most effective. At the regional periphery of Europe, there remains a significant knowledge gap concerning mosquito vector distributions in Northern Ireland. This study aimed to address this major gap by systematically surveying larval and adult mosquito populations in key habitats (ponds, reedbeds, saltmarsh, wet grasslands, wet woodlands, and urban gardens). We further identified environmental drivers of important vector species abundance and screened collected specimens with molecular techniques to identify presence of cryptic human-biting vector species. In Northern Ireland, mosquitoes (including potential bridge vectors of West Nile and Usutu viruses) were found to be widely distributed. Between May and August 2025, a total of 2430 mosquitoes (951 adults and 1479 larvae) were collected over 196 trap nights and 76 larval collections. Collections encompassed 17 species, with Culex pipiens/torrentium being the most widely distributed and abundant taxon recorded. Mosquito abundances were greatest in reedbed habitats, related negatively with rainfall and peaked in June and July. A subset of Culex pipiens/torrentium mosquitoes were identified to species/biotype level using PCR, with the majority (89.3%) identified as Culex pipiens pipiens and 9.3% as Culex torrentium. Importantly, the first detections of Culex pipiens/molestus hybrids (1.8%) on the island of Ireland were found. This work underscores the wide, yet previously undocumented abundance and distribution of potential zoonotic vectors in Northern Ireland and provides an important baseline for management planning and future research, while contributing to broader understandings of environmental population drivers and distributions.

The fieldwork was carried out between December 2023 and June 2025, with the aim, among others, of confirming the possible presence of the North African Gerbil (Gerbillus campestris) in Melilla. To this end, 56 camera trap stations were installed, each equipped with a camera trap. Of the total number of camera trap stations installed, the presence of Gerbillus campestris was detected in six of them, representing a capture success rate of 10.71%. Following the detection of the species, a specimen was captured using cage traps. For identification purposes, samples of epithelial tissue from the ear and faeces were collected. These samples were sent to the Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno, Czech Republic, for analysis. There, DNA was extracted from the samples using the Innu PREP DNA Mini Kit 2.0 (IST Innuscreen GmbH, Germany) according to the manufacturer's protocol. The results obtained confirm, for the first time through records of the species, the presence of Gerbillus campestris in Melilla and, therefore, in Spain and Europe.

Neoehrlichia mikurensis is widely detected in tick populations across Europe, yet its host range beyond small rodents remains poorly understood. Large wild mammals are frequently exposed to tick vectors, but their role in the transmission cycle of this bacterium is unclear. Wild boars (Sus scrofa) are among the most abundant large mammals in southern Sweden and commonly inhabit tick-infested environments, making them a potentially relevant host species. In this study, spleen samples from 87 wild boars collected between 2023 and 2025 across southern and central Sweden were analysed for the presence of N. mikurensis DNA using quantitative polymerase chain reaction. N. mikurensis DNA was not detected in any of the analysed samples, despite sampling in regions where the bacterium is known to be prevalent in ticks. These results indicate that wild boars may have a limited role in the maintenance of N. mikurensis in Sweden, although the possibility of rare or transient infection cannot be excluded. Despite being based on negative findings, this study contributes to a more comprehensive understanding of the host range of N. mikurensis and highlights the importance of continued evaluation of wildlife species in the transmission dynamics of tick-borne pathogens.

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

Feathers incorporated into bird nests are thought to provide multiple benefits, including thermal insulation and protection against nest-dwelling ectoparasites. For instance, in Swallows, it has been suggested that feathers influence infestations by Protocalliphora blow flies either by acting as a physical barrier between larvae and nestlings or through chemical compounds associated with preen oil. However, empirical evidence for an antiparasitic effect of feathers remains equivocal. Using 1,100 naturally infested Tree Swallow (Tachycineta bicolor) nests collected from a nest-box study system monitored in southern Quebec, Canada, between 2004 and 2018, we tested whether nest mass and feather abundance influenced hematophagous ectoparasites (Protocalliphora spp.) loads. We further assessed whether feather abundance directly affected body size using measurements of 1,513 P. sialia puparia, and emergence success using data from 961 Protocalliphora spp. puparia. Nest mass was positively associated with feather abundance, and both variables showed very weak positive relationships with parasite abundance. Feather abundance was unrelated to puparium size nor parasite emergence success. Our results provide no support for an anti-parasitic effect of feather lining within Tree Swallow nests. Using one of the largest datasets assembled to date on feather-lined nests and avian blow flies, we found no evidence that feather abundance impairs Protocalliphora density, development or emergence success. However, our findings do not preclude potential effects of feathers on other ectoparasites or microorganisms with different life histories. Future studies simultaneously monitoring multiple parasite taxa (such as fleas, mites, or bacteria) may therefore be needed to fully evaluate the ecological consequences of feather lining within avian nests.

Morphological variation in insects can indicate responses to habitat change and land use intensification, with potential consequences for trophic interactions. In insect host-parasitoid systems, host body is often a key determinant of offspring development and emergence success, as well as parasitoid host-selection behavior. Yet, most studies in agricultural landscapes have focused on size variation in crop-associated insects (i.e.: pests, pollinators and enemies), leaving those relying on non-crop resources largely unexplored. Here, we investigated how agroenvironmental contexts and trophic resource availability influence puparial size in Protocalliphora sialia (Diptera: Calliphoridae), an obligate hematophagous ectoparasite of Tree Swallow (Tachycineta bicolor) nestlings. We further tested whether puparial size affects emergence probability and risk of parasitoidism by Nasonia spp. (Hymenoptera: Pteromalidae) jewel wasps. Using 1,513 P. sialia puparia from 88 nests within a nest-box monitoring system spanning a 10,200-km^2 east-west intensification gradient in southern Quebec, Canada, we evaluated how agrointensive contexts, avian host availability, and ectoparasite densities influence puparial size, parasitoidism risk, and emergence success. The agricultural context appears to shape puparial size through survival biases and/or altered growth rates. In agroextensive contexts (i.e.: pastures, forages with high tree cover), where Protocalliphora are more numerous, P. sialia puparia were smaller and exhibited greater size variation. Conversely, in agrointensive environments (maize and soybeans monocultures with higher doses of pesticides), where Protocalliphora are fewer, small individuals were underrepresented, suggesting a selective mortality in P. sialia population. However, puparial size explained less than 10% of the variation in emergence or parasitism, likely reflecting consistently high parasitic pressure from Nasonia wasps, which infested 50-90% of puparia, depending on site and year. Our findings highlight the importance of selective mortality in structuring trait distributions and modulating multi-trophic interactions in human-modified landscapes.

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.

Ground-nesting wild bees require suitable soil conditions for their below-ground development and overwintering life stages. Understanding how soil translocation and disturbance affect individuals and the following generations of bees is important for identifying conservation measures for ground-nesting wild bees. We investigated the effects of soil translocation and disturbance on the ground-nesting solitary spring mining bee Colletes cunicularius. In a replicated controlled design, we compared the effects of soil translocation with different degrees of disturbance on the number of surviving bees and the emergence time. We found that soil translocation and disturbance reduced bee survival but did not result in complete mortality, with some individuals surviving even under severe soil disturbance. There was no evidence that soil disturbance affected the timing of spring emergence, suggesting that disturbance is unlikely to alter the timing of nest construction and, consequently, the production of the next generation. Our results indicate that translocating soil containing overwintering bees could be a promising conservation measure to compensate for habitat loss caused by land conversion.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

The bean thrips, Caliothrips phaseoli, is an emergent pest affecting peanut production. Thus, gaining a deeper understanding of the naturally occurring viral populations associated with this insect is of major importance, as such knowledge could support the design of novel pest management strategies that are both ecologically responsible and sustainable over the long term. In this study, we performed the first RNA high-throughput sequencing (HTS) analysis of C. phaseoli to unravel its virome, discovering a wide number of novel RNA viruses. Our HTS analysis revealed the presence of 31 viral segments, corresponding to 18 viruses, including double-stranded RNA viruses as well as positive- and negative-sense single-stranded RNA viruses. All these are novel viruses and cover multiple viral lineages belonging to diverse families. Overall, these results indicates that C. phaseoli hides a complex virome. The findings presented here provide an important framework for subsequent studies aimed at understanding the ecological significance, evolutionary patterns, and possible use of these viruses as components of biologically based management programs for this emerging peanut pest.

Tsetse flies are strictly found is sub-Saharan Africa where they are responsible for the transmission and maintenance of African trypanosomiases in humans (HAT) and animals (AAT). Vector control has been recognized as an essential tool to fight against these diseases. Nevertheless, it requires the best possible knowledge of the biology of the targeted population. Population genetics tools can prove very useful to obtain such information but require the use of polymorphic and reliable genetic markers. In this paper we present the development of microsatellite markers using a new high-throughput sequencing based technology (SSRseq). We applied it on two species of tsetse flies from three HAT foci and obtained more accurate results as compared to microsatellite loci developed with classic methods. We could indeed use 9 to 14 SSRseq loci without the several problems generally met with classic microsatellites, as all were located in autosomes, without short allele dominance or stuttering and very few null alleles. SSRseq loci appeared much more polymorphic in tsetse flies as compared to other species (fungi, trees, bees, or fishes), which suggested much higher effective population sizes, much higher mutation rates of the genome or both, suggesting high capacities for evolutionary adaptation. With the 9-14 loci kept, we confirmed the propensity of these flies to move almost freely in the whole zones investigated, and also highlighted the possible evolutionary response of one of the unkept loci regarding vector control devices used in these HAT foci, which will require further studies. We suggest for further population structure studies to use only loci with less than 1% missing data, which proved being a good and fast selection strategy to get the most reliable results.

Traditional veterinary pathology investigations on wild animals generally conclude a primary cause of death based on a combination of observed clinical signs, gross and microscopic lesions, microbial culture and PCR. However, these methods may fail to capture the broader microbial community of the host. We employed metatranscriptomic sequencing to characterise the potential pathobiome of necropsied marine turtles, comparing these findings with corresponding veterinary pathology reports. Tissue samples were collected from 76 individuals spanning three species of marine turtles -- the green turtle (Chelonia mydas), the hawksbill turtle (Eretmochelys imbricata), and the loggerhead turtle (Caretta caretta) -- across the coastline of New South Wales, Australia. Reported causes of mortality ranged from infectious disease to trauma. We identified a diverse range of parasites within the genera Neospirochis, Hapalotrema, Paraorgymatobothrium, Eimeria and Callitetrarhynchus, and bacteria including the genera Aeromonas, Clostridium, and Vibrio. Novel viruses of the families Astroviridae, Caliciviridae, Hepaciviridae, Hepeviridae, Nyamivirdae, Orthomyxoviridae, Picobirnaviridae and Rhabdoviridae were also identified. In cases where infectious disease was noted in the pathology report, we frequently observed high pathogen read counts consistent with the listed primary diagnoses. However, we also detected a range of incidental infections involving eukaryotic parasites, bacteria, and viruses. The diversity of infectious agents detected in marine turtles suggests that the observed disease outcomes are unlikely to be driven by a single causative agent, but rather by complex interactions between host health, the pathobiome, and environmental stressors. We discuss the potential implications for turtle health and conservation.

Abstract Habitat configuration influences population connectivity and, consequently, genetic structure. River networks provide heterogeneous, hierarchically arranged environments that shift drastically from upstream to downstream. We compared congeneric species across Ephemeroptera, Plecoptera, and Trichoptera, emphasizing longitudinal replacement (upstream to downstream) and the rarely studied wet rock (hygropetric) habitats. We surveyed six rivers on the Muroto Peninsula, Japan, qualitatively sampling aquatic insects at 63 sites. Cluster analysis based on the environmental data classified surveyed sites into four clusters. We analyzed a total of 10 species from four genera that exhibit longitudinal replacement patterns within genera. Genetic analyses based on the mitochondrial cytochrome c oxidase subunit I region revealed that upstream species showed higher genetic diversity than downstream species. In contrast, species adapted to hygropetric exhibited the lowest genetic differentiation among all habitat types. A novel contribution of this study is the inclusion of hygropetric species. The surprisingly low differentiation in hygropetric species suggests high connectivity, similar to lentic species. By comparing congeneric taxa across orders within environmentally similar rivers, we reduce phylogenetic and environmental confounds, strengthening inference that habitat configuration and dispersal traits jointly shape genetic structure. These findings provide a new perspective on riverine spatial ecology and underscore the importance of microhabitat-aware comparisons for evolutionary inference.

When mating among genetically related individuals results in inbreeding depression, various behaviors and life-history traits evolve to favor outbreeding. Long-range sexual communication following natal dispersal is one such adaptation. In parasitoid wasps with single-locus complementary sex determination (sl-CSD), inbreeding increases the production of unviable or sterile diploid males. Mastrus ridens is such a species, with a genetic load underpinned by sl-CSD, combined with a high potential for sibmating because siblings emerge in close proximity from the same host and become sexually active shortly after emergence. With this context in mind, we investigated the mate-finding strategy of M. ridens using complementary behavioral, chemical, and electrophysiological approaches. Wind-tunnel bioassays and field experiments showed that males orient toward virgin females and, to a lesser extent, mated females. This attraction from a distance appeared to be the sole form of conspecific attraction. Chemical analyses using static and dynamic headspace sampling and solvent extracts, followed by GC-MS, revealed several potential pheromonal candidates, including methyl 6-methylsalicylate. This compound elicited reproducible antennal responses in males in GC-EAD recordings. Altogether, our results suggest that mate finding in M. ridens is guided by a volatile sex pheromone emitted by females, with methyl 6-methylsalicylate as one putative component. Such mate-finding mechanisms promote outbreeding and may explain the low proportion of unfit diploid males observed in natural populations. Surprisingly, chemical analyses suggested that males also emit this compound, whereas neither males nor females showed behavioral responses to males. This apparent discrepancy suggests that attraction may depend on a more complex pheromonal signal and opens further avenues for investigation.

Mosquito-borne orthoflaviviruses pose a significant threat to human and animal health worldwide. In Europe, West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) viruses have expanded their geographic distribution and are detected with increasing frequency. Following the emergence of BAGV in 2021, we investigated the circulation dynamics and genomic diversity of orthoflaviviruses in southern Portugal, where their epidemiology remains poorly understood. Active Orthoflavivirus surveillance was conducted in birds and mosquitoes during 2021-2023 using serological and molecular assays, complemented by viral genome sequencing and phylogenomic analyses. Viral RNA was detected in 3.5% (41/1,171) of birds, while seroneutralization assays identified an overall seroprevalence of 55.3% (503/910). WNV RNA (lineage-1, strains WMed-1.2 and WMed-1.3) was detected annually in resident birds, including Alectoris rufa, Cyanopica cooki and Garrulus glandarius (1.5%, 18/1,171), and in Culex univittatus mosquitoes (0.8%, 2/255). Together with a WNV-specific seroprevalence of 28.5% (259/910) in birds, these findings support sustained local circulation. USUV RNA (Africa 3.1 sub-lineage) was detected in A. rufa in 2021 and 2023 (0.2%, 2/1,171), but seroprevalence was low (1.3%, 8/623), consistent with intermittent circulation. BAGV RNA (genotype-2) was detected only during the 2021 outbreak in A. rufa and Emberiza calandra (1.8%, 21/1,171), although BAGV-specific antibodies (6.6%, 60/910) were detected for up to two years after the outbreak, suggesting continued exposure or low-level circulation. Overall, our findings reveal contrasting circulation dynamics of orthoflaviviruses in southern Portugal. Sustained WNV circulation implies the need to strengthen avian and human surveillance in Portugal to better assess its public health risk, while the sporadic detection of USUV and BAGV underscores the value of long-term, integrated One Health surveillance for improving early warning and preparedness for emerging mosquito-borne diseases.

Abstract Background: Cyprus has transitioned into a high-alert zone for mosquito-borne viruses. This necessitates a proactive assessment of its future physicians. Following the 2022 re-establishment of Aedes aegypti in Larnaca and Aedes albopictus in Limassol, Cyprus, the island is now biologically primed for local outbreaks of dengue, Zika, and chikungunya, alongside Culex-borne West Nile virus. The study assessed medical students' knowledge and risk perception. Methods: A cross-sectional online survey administered the validated MosquitoWise Health Belief Model instrument to undergraduate-entry and graduate-entry students at a university in Cyprus in mid-2026. Results: Of 1023 students, 119 responded (11.6%), with pre-clinical students overrepresented (p<0.001). Mean knowledge was 5.43/9, stronger on mosquito biology (72.5%) than virus knowledge (54.2%). Median severity was 6.00/7 against susceptibility 4.25/7. Knowledge differed by study stage (p<0.001), correlated with severity (rs = 0.399) but not susceptibility. Conclusions: The study establishes a critical baseline for pre-clinical medical students in Cyprus. Overall, participants performed better on mosquito biology when compared to mosquito-borne viruses and how they spread. Our findings highlight the value of integrating theoretical microbiology with clinical practice in medical education to enhance physicians' preparedness for the changing landscape of newly emerging infections in Cyprus and across Europe as a whole. Key Words: Arboviruses, Cross-Sectional Studies, Cyprus, Health Belief Model, Medical Students, Risk Perception.