Among the several Asian Aedes species that have established in Europe over recent decades, Aedes koreicus has received comparatively little research attention since its first detection in Belgium in 2008, despite its remarkable ability to colonise montane and peri-alpine localities at elevations beyond the reach of its congener Aedes albopictus. Nearly two decades after its European detection, a systematic synthesis of the available evidence and a structured assessment of research and operational priorities are overdue. This paper presents the results of a scoping review of the scientific literature on Ae. koreicus combined with the outcomes of a multidisciplinary expert workshop held in Trento, Italy, in February 2026, which brought together 41 researchers and practitioners from six European countries with expertise in mosquito surveillance, medical entomology, genomics, ecological modelling and public health practice. Through the literature search we obtained a total of 334 articles, of which 91 were eventually included, all published after 2011. Most of the studies were carried out within Europe, focusing mostly on mosquito surveillance. Building upon the scoping review, the workshop identified key priorities for advancing knowledge and operational responses to Ae. koreicus, including strengthened surveillance in montane and lowland areas, improved characterisation of its vector competence and competitive interactions with co-occurring Aedes species, and more effective communication strategies for engaging the public and citizen scientists in monitoring efforts.

Rapid urbanisation and environmental changes are reshaping mosquito population dynamics and the transmission of vector-borne pathogens. Culex pipiens and Aedes albopictus have successfully adapted to anthropogenic environments, becoming key vectors of emerging and re-emerging arboviruses and filarial parasites. This study conducted a comparative entomological and pathogen surveillance between 2022 and 2023 in central Italy and Crete to assess species composition, population dynamics, and circulation of arboviruses and filarial nematodes across different ecological contexts. In Italy, a total of 22,757 mosquitoes were collected, with Cx. pipiens (∼86%) and Ae. albopictus (∼12%) being the predominant species. Significant temporal variations in mosquito diversity and frequency were observed. In 2022, molecular screening detected Usutu virus (USUV) in Cx. pipiens in central Italy, with the first report of co-circulation of USUV and Setaria nematodes. In 2023, co-circulation of West Nile virus (WNV Lineage 2) and USUV was identified in Cx. pipiens from the Marche region. In Crete, 791 mosquitoes were collected, with Culex species dominating the assemblages. The highest mosquito biodiversity was recorded in peri-urban areas, exhibiting temporal variation. Molecular screening detected Dirofilaria repens in Ae. albopictus. No evidence of WNV or USUV circulation was found. We provide the first integrated, regional-scale assessment of mosquito vectors and vector-borne pathogens across two ecologically distinct Mediterranean regions. By combining complementary surveillance components, including mosquito species composition, biodiversity, seasonal abundance, habitat characteristics, and molecular detection of arboviruses and filarial nematodes, our study provides a comprehensive understanding of vector-pathogen dynamics across contrasting ecological settings. Our findings demonstrate that environmental and ecological drivers strongly influence mosquito community structure and pathogen circulation, highlighting the value of integrated, region-specific surveillance as a foundation for evidence-based vector control and public health decision-making.

Background Fungi associated with mosquito larvae can contribute to host development and may influence oviposition-site selection through the release of volatile organic compounds (VOCs). However, it remains unclear to what extent larval mycobiota reflects passive acquisition from breeding waters versus host-associated filtering. Here, we characterized fungal communities associated with larvae and breeding waters of Culex pipiens , Aedes albopictus , and the invasive Aedes koreicus in Italy, and assessed the behavioural activity and preliminary biosafety of larval habitat-derived yeasts. Results Using ITS metabarcoding coupled with culture-dependent isolation, we identified diverse fungal assemblages across mosquito species and sampling sites, with partial overlap between larval and aquatic communities and a set of recurrent yeast taxa shared among hosts and habitats. Culture-based methods recovered seven yeast species including Barnettozyma californica , Candida parapsilosis , Metschnikowia pulcherrima , Rhodotorula paludigena , Schwanniomyces vanrijiae , Sporobolomyces roseus , and Wickerhamomyces anomalus . In vitro screening on HaCaT keratinocytes, performed by co-culturing human cells with seven fungal isolates from breeding water (one per yeast species), demonstrated that B. californica , M. pulcherrima , R. paludigena , W. anomalus isolates did not induce detectable cytotoxic effects. The yeast volatilomes of the seven isolates were profiled by HS-SPME–GC–MS, revealing species-specific VOC blends dominated by alcohols and esters in Ascomycota and enriched in phenolic compounds and organic acids in Basidiomycota. Based on VOC profiles and absence of detectable cytotoxicity, a core of four selected yeasts were further evaluated in dual-choice oviposition assays with gravid Ae. albopictus . Yeast-treated oviposition substrates significantly altered egg-laying preferences, with all tested yeast species resulting in oviposition percentages greater than 50% in the treatment group. Among the tested species, W. anomalus and B. californica exhibited the strongest attractiveness, followed by R. paludigena , whereas M. pulcherrima showed a comparatively weaker effect. Conclusion Fungal communities associated with mosquito larvae mainly mirror breeding-site ecology but also exhibit signs of host-associated filtering. Notably, in addition to Culex pipiens and Aedes albopictus , this study included Aedes koreicus , for which information on mycobiota composition has so far been limited. Importantly, yeasts isolated from larval habitats emit VOC blends that can modulate oviposition behaviour in gravid Ae. albopictus . Together, these results support further investigation of selected yeast-based cues as components of environmentally compatible, oviposition-targeted mosquito management strategies, alongside expanded safety assessment and field validation.

Background : Wolbachia is an endosymbiotic bacterium that naturally infects several arthropod and nematode species. Wolbachia gained significant attention in the last couple of decades, due to its impact on host fitness and the ability of specific Wolbachia strains to interfere with vectorial capacity in mosquitoes or to sterilise insect females. These characteristics have been exploited in pest and vector management and in the control of vector-borne diseases. Wolbachia presence in insect species is variable, with species presenting 100% positive individuals in all sampled populations, and others that present a patchy distribution, with variable prevalences in different populations. Factors that might influence Wolbachia infection rate in insect populations have not yet been investigated in all respects. In addition, there might be limits in the methods used for Wolbachia detection. Results: Through an integrated technical approach, we recently detected and identified Wolbachia in the African bush and forest-associated mosquito Aedes africanus . Investigations on the microbiota of Ae. africanus , and phylogenomics on its Wolbachia , highlighted some peculiarities. First, Wolbachia from Ae. africanus is closer to the wolbachiae of other insects, rather than to those generally found in mosquitoes. Second, metagenomics uncovered a phenomenon of microbial competition between Wolbachia and Pantoea vagans within Ae. africanus . Conclusions: The use of Wolbachia in the fight against MBD cannot ignore the characterization of the bacterium in the specific host vector and the knowledge of any competition mechanisms; this to select both the phenotypes most suitable for suppressing pathogen transmission and those to be used in manipulating host reproduction. The mutual negative interference between Wolbachia and Pantoea for the colonisation of Ae. africanus , underlines how interactions between symbionts in the vector host can influence the effectiveness of control methods based on the use of Wolbachia . Our results pinpoint that the possible use of Wolbachia for the control of MBDs must take into due account both the genetic background of the host mosquito, we know in fact that the same strain of Wolbachia can induce different phenomena in the host, and the architecture of its microbiota that can interact in various ways with Wolbachia .

BackgroundThe increase in the frequency and intensity of heatwaves (HWs) under global warming is expected to have more dramatic biological impacts on insects than mean temperature increases. However, evidence of the impact of HWs on insects in their ecological context is limited. Here, we measured across multiple biological scales (e.g., fitness, physiology, transcriptomic and microbiota) the stage-specific responses of the arboviral vector Aedes albopictus when experiencing an ecologically relevant HW. In arboviral vectors, only females require a blood-meal thus contributing to the transmission cycle, but other stages can also be the target of vector control strategies. As such understanding the responses to a HW during development has both biological and epidemiological relevance.ResultsWe observed HW stage-specific responses across the mosquito life cycle. We saw the rate of larvae hatching from eggs exposed to a HW to hatch decreasing and their emergence time increasing. Larvae showed to be resilient to HW by repurposing their energy resulting in trivial mortality. Adults showed sex-specific responses, with extensive male mortality, and a small (16) number of genes elicited following HW exposure, indicating that males are less heat tolerant than females. In females, we observed a reduced reproductive output, only when HW occurred after a blood meal. Finally, we saw extensive HW-dependent changes in the microbial composition of larvae, but female microbiota remained dominated by Wolbachia regardless of the thermal challenge.ConclusionsAe. albopictus exhibit stage-specific thermal sensitivities to a HW, which have relevant implications for both the understanding of mosquito biology and the implementation of vector control strategies as the climate crisis progresses.

Rapid urbanisation and environmental changes are reshaping mosquito population dynamics and the transmission of vector-borne pathogens. Culex pipiens and Aedes albopictus have successfully adapted to anthropogenic environments, becoming key vectors of emerging and re-emerging arboviruses and filarial parasites. This study conducted a comparative entomological and pathogen surveillance between 2022 and 2023 in central Italy and Crete to assess species composition, population dynamics, and circulation of arboviruses and filarial nematodes across different ecological contexts.In Italy, a total of 22,757 mosquitoes were collected, with Cx. pipiens (~86%) and Ae. albopictus (~12%) being the predominant species. Significant temporal variations in mosquito diversity and frequency were observed. In 2022, molecular screening detected Usutu Virus (USUV) in Cx. pipiens in Central Italy, with the first report of co-circulation of USUV and Setaria nematodes. In 2023, co-circulation of West Nile Virus (WNV lineage 2) and USUV was identified in Cx. pipiens from the Marche region.In Crete, 791 mosquitoes were collected, with Culex species dominating the assemblages. The highest mosquito biodiversity was recorded in peri-urban areas, exhibiting temporal variation. Molecular screening detected Dirofilaria repens in Ae. albopictus. No evidence of WNV or USUV circulation was found.We provide a novel regional-scale approach by comparing two ecologically distinct Mediterranean regions. The findings underscore how environmental and ecological factors shape vector-pathogen interactions, and in particular biodiversity distribution, highlighting the need for targeted, region-specific mosquito surveillance and control strategies.

BACKGROUND: The global decline of honey bee health, driven by multiple stressors impairing immunity, threatens both natural and managed ecosystems. Deformed Wing Virus (DWV) is often found at high loads in collapsing colonies, associated with an immunosuppressive syndrome still poorly understood. This study investigates gut microbiota community patterns associated with different levels of DWV infection, an overlooked aspect which can contribute to the reduction of immunocompetence in infected honey bees. RESULTS: We quantified DWV in nurse bees from the field and compared gut microbiota in individuals with high vs. low viral loads. We also examined microbiota changes in bees experimentally infected with DWV. In both cases, highly infected bees showed a decreased transcription level of dorsal 1-A, a consolidated molecular proxy of reduced immunocompetence in honey bees. Host immunosuppression was associated with a consistent gut dysbiosis, marked by a reduced relative abundance of beneficial genera such as Fructobacillus, Lactobacillus, and Apilactobacillus (Firmicutes, Bacilli), and a relative enrichment of Bartonella (Proteobacteria). CONCLUSIONS: These findings shed new light on host-pathogen interactions and will allow a targeted manipulation of honey bee gut microbiota to limit DWV infections, which strongly contribute to colony losses.

[This corrects the article DOI: 10.1371/journal.pone.0305207.].

The increase in the frequency and intensity of heatwaves (HWs) under global warming is expected to have more dramatic biological impacts on insects than mean temperature increases by challenging their thermal thresholds. However, experimental evidence that quantifies the impact of HWs on insects in their ecological context is limited. Here, we measured across multiple biological scales (e.g. fitness, physiology, transcriptomic and microbiota) the stage-specific responses of the arboviral vector Aedes albopictus when experiencing an ecologically relevant HW. In arboviral vectors, only females require a blood-meal thus contributing to the transmission cycle, but males, eggs and larvae can also be the target of control strategies to reduce population size. As such the understanding of the responses to a HW during mosquito development has both biological and epidemiological relevance. We observed stage-specific responses, starting at the egg stage. We saw egg hatching decreasing and delaying after HW exposure. On the contrary, larvae showed to be resilient to HW. Larvae repurposed their energy resulting in trivial mortality, but delayed development. At the adult stage, we observed a marked sex difference, with extensive (>50%) male mortality, accompanied by a small (16) number of genes elicited following HW exposure, which indicates that males have limited coping mechanisms against hot events. In females, we observed a reduced reproductive output, but only when HW occurred after a blood-meal. Finally, we saw extensive HW-dependent changes in the microbial composition of larvae, but female microbiota remained dominated by Wolbachia regardless of the thermal challenge. Our results have relevant implications for both the understanding of mosquito biology and the implementation of vector control strategies as the climate crisis progresses. ### Competing Interest Statement The authors have declared no competing interest. Ministero dell’Università e della Ricerca, Italia, 2022J45MLL EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases, PE00000007

Sustained by urbanization, globalization and climate change, infectious diseases transmitted by arthropod vectors, such as mosquitoes, ticks and sandflies, are emerging or resurging in Europe, including Italy. There are limited therapeutic treatments and vaccines for most arthropod-borne pathogens, thus monitoring and control of vectors remains the most-effective prevention strategy. Supported by a country-wide initiative that aims at providing strategic guidance for preventing vector-borne diseases in Italy, including suirveillance and control initiatives that results in the acquistion of a large number of field samples, we conceived a digital repository of samples from arthropod vectors and their metadata to promote their sharing among the scientific community. We built a relational database called RAV-IT, accessible at https://mosqit.unipv.it/. Currently, RAV-IT aggregates seventeen Italian institutions and hosts nearly two thousand vector samples and their metadata, which can be viewed and requested for research purposes. RAV-IT is interactive and can accept further samples from any users. RAV-IT is a non-profit repository that is expected to enhance resource sharing for research on arthropod vectors.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Dietary fiber has been shown to have multiple health benefits, including a positive effect on longevity and the gut microbiota. In the present study, Drosophila melanogaster has been chosen as an in vivo model organism to study the health effects of dietary fiber supplementation (DFS). DFS extended the mean half-life of male and female flies, but the absolute lifespan only increased in females. To reveal the underlying mechanisms, we examined the effect of DFS on gut microbiota diversity and abundance, local gut immunity, and the brain proteome. A significant difference in the gut microbial community was observed between groups with and without fiber supplementation, which reduced the gut pathogenic bacterial load. We also observed an upregulated expression of dual oxidase and a modulated expression of Attacin and Diptericin genes in the gut of older flies, possibly delaying the gut dysbiosis connected to the age-related gut immune dysfunction. Brain proteome analysis showed that DFS led to the modulation of metabolic processes connected to mitochondrial biogenesis, the RhoV-GTPase cycle, organelle biogenesis and maintenance, membrane trafficking and vesicle-mediated transport, possibly orchestrated through a gut-brain axis interaction. Taken together, our study shows that DFS can prolong the half-life and lifespan of flies, possibly by promoting a healthier gut environment and delaying the physiological dysbiosis that characterizes the ageing process. However, the RhoV-GTPase cycle at the brain level may deserve more attention in future studies.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia–Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control.

Background Knockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented. Methods The present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing. Results An. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote "RS" state in An. arabiensis and An. gambiae in Yangah and Santchou. Conclusions The present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission.

IntroductionMalaria transmission occurs when Plasmodium sporozoites are transferred from the salivary glands of anopheline mosquitoes to a human host through the injection of saliva. The need for better understanding, as well as novel modes of inhibiting, this key event in transmission has driven intense study of the protein and miRNA content of saliva. Until now the possibility that mosquito saliva may also contain bacteria has remained an open question despite the well documented presence of a rich microbiome in salivary glands.MethodsUsing both 16S rRNA sequencing and MALDI-TOF approaches, we characterized the composition of the saliva microbiome of An. gambiae and An. stephensi mosquitoes which respectively represent two of the most important vectors for the major malaria-causing parasites P. falciparum and P. vivax.ResultsTo eliminate the possible detection of non-mosquito-derived bacteria, we used a transgenic, fluorescent strain of one of the identified bacteria, Serratiamarcescens, to infect mosquitoes and detect its presence in mosquito salivary glands as well as its transfer to, and colonization of, mammalian host tissues following a mosquito bite. We also showed that Plasmodium infection modified the mosquito microbiota, increasing the presence of Serratia while diminishing the presence of Elizabethkingia and that both P. berghei and Serratia were transferred to, and colonized mammalian tissues.DiscussionThese data thus document the presence of bacteria in mosquito saliva, their transfer to, and growth in a mammalian host as well as possible interactions with Plasmodium transmission. Together they raise the possible role of mosquitoes as vectors of bacterial infection and the utility of commensal mosquito bacteria for the development of transmission-blocking strategies within a mammalian host.

Recently, two invasive Aedes mosquito species, Ae. japonicus and Ae. koreicus, are circulating in several European countries posing potential health risks to humans and animals. Vector control is the main option to prevent mosquito-borne diseases, and an accurate genome sequence of these mosquitoes is essential to better understand their biology and to develop effective control strategies. Here, we present a de novo genome assembly of the Ae. japonicus (Ajap1) and Ae. koreicus (Akor1) based on a hybrid approach that combines Oxford Nanopore long reads and Illumina short reads data. Their quality was ascertained using various metrics. Masking of repetitive elements, gene prediction and functional annotation was performed. Sequence analysis revealed a very high presence of repetitive DNA and, among others, thermal adaptation genes and insecticide-resistance genes. Through the RNAseq analysis of larvae and adults of Ae. koreicus and Ae. japonicus exposed to different temperatures (15 and 28°C) we also identified genes showing a differential temperature-dependent activation. The assembly of Akor1 and Ajap1 genomes constitutes the first updated collective knowledge of the genomes of both mosquito species, providing the possibility of understanding key mechanisms of their biology such as the ability to adapt to harsh climates and to develop insecticide-resistance mechanisms.