Title Participants "Présence de Pseudomonas chez les Culex autogènes et chez les Culex non autogènes" "M Bisoux, J Jadin" "Belgian Culex pipiens pipiens are competent vectors for West Nile virus while Culex modestus are competent vectors for Usutu virus" "Lander De Coninck, Jelle Matthijnssens, Leen Delang" "BACKGROUND: West Nile virus (WNV) and Usutu virus (USUV) are emerging arthropod-borne viruses (arboviruses) in Europe transmitted by Culex mosquitoes. In Belgium, it is currently unknown which Culex species are competent vectors for WNV or USUV and if these mosquitoes carry Wolbachia, an endosymbiotic bacterium that can block arbovirus transmission. The aims of our study were to measure the vector competence of Belgian Culex mosquitoes to WNV and USUV and determine if a naturally acquired Wolbachia infection can influence virus transmission. METHODOLOGY/PRINCIPAL FINDINGS: Female Culex mosquitoes were captured from urban and peri-urban sites in Leuven, Belgium and offered an infectious bloodmeal containing WNV lineage 2, USUV European (EU) lineage 3, or USUV African (AF) lineage 3. Blood-fed females were incubated for 14 days at 25°C after which the body, head, and saliva were collected to measure infection, dissemination, and transmission rates as well as transmission efficiency. Mosquito species were identified by qRT-PCR or Sanger sequencing, the presence of infectious virus in mosquitoes was confirmed by plaque assays, and viral genome copies were quantified by qRT-PCR. Culex pipiens pipiens were able to transmit WNV (4.3% transmission efficiency, n = 2/47) but not USUV (EU lineage: n = 0/56; AF lineage: n = 0/37). In contrast, Culex modestus were able to transmit USUV (AF lineage: 20% transmission efficiency, n = 1/5) but not WNV (n = 0/6). We found that the presence or absence of Wolbachia was species-dependent and did not associate with virus transmission. CONCLUSIONS/SIGNIFICANCE: This is the first report that Belgian Culex mosquitoes can transmit both WNV and USUV, forewarning the risk of human transmission. More research is needed to understand the potential influence of Wolbachia on arbovirus transmission in Culex modestus mosquitoes." "DNA identification and diversity of the vector mosquitoes Culex pipiens s.s. and Culex torrentium in Belgium (Diptera: Culicidae)" "Ann Vanderheyden, Nathalie Smitz, Katrien De Wolf, Isra Deblauwe, Wouter Dekoninck, Kenny Meganck, Sophie Gombeer, Adwine Vanslembrouck, Jacobus De Witte, Anna Schneider, Ingrid Verle, Marc De Meyer, Thierry Backeljau, Ruth Müller, Wim Van Bortel" "This survey reports on the DNA identification and occurrence of Culex torrentium and Cx. pipiens s.s. in Belgium. These native disease-vector mosquito species are morphologically difficult to separate, and the biotypes of Cx. pipiens s.s. are morphologically indistinguishable. Culex torrentium and Cx. pipiens s.s. were identified using the COI and ACE2 loci. We recorded 1248 Cx. pipiens s.s. and 401 Cx. torrentium specimens from 24 locations in Belgium (collected between 2017 and 2019). Culex pipiens biotypes pipiens and molestus, and their hybrids, were differentiated using fragment-size analysis of the CQ11 locus (956 pipiens and 227 molestus biotype specimens, 29 hybrids). Hybrids were observed at 13 out of 16 sympatric sites. These results confirm that both species are widespread in Belgium, but while Cx. torrentium revealed many COI haplotypes, Cx. pipiens s.s. showed only one abundant haplotype. This latter observation may either reflect a recent population-wide demographic or range expansion, or a recent bottleneck, possibly linked to a Wolbachia infection. Finally, new evidence is provided for the asymmetric but limited introgression of the molestus biotype into the pipiens biotype." "Phenotypic insecticide resistance status of the Culex pipiens complex: a European perspective" "Stien Vereecken, Adwine Vanslembrouck, Isabelle Kramer, Ruth Müller" "Background: The common house mosquito Culex pipiens is known to be a major vector for West Nile virus. In order to decrease risks of West Nile virus outbreaks in Europe, insecticides and the bio-larvicide Bacillus thuringiensis israelensis (Bti) are commonly used for vector control. Alarmingly, insecticide resistance has been reported in Cx. pipiens populations from Southern Europe and several countries neighbouring Europe. For Central and Northern Europe, however, the phenotypic insecticide resistance status of Cx. pipiens has not yet been investigated.Methods: A literature review was performed to assess the geographical distribution of insecticide resistance in Cx. pipiens. To fill the gap of knowledge for Central and Northern Europe, WHO susceptibility tests with permethrin, deltamethrin, malathion, bendiocarb and DDT and a larval toxicity test with Bti were performed with a Cx. pipiens population from Belgium, a country in Central Europe.Results: This research provides the first evidence of widespread phenotypic insecticide resistance in Cx. pipiens. In general, Cx. pipiens developed resistance against multiple insecticides in several countries. Another Cx. pipiens population from Belgium was tested and showed insecticide resistance against deltamethrin, permethrin, DDT and possibly against bendiocarb. The bio-larvicide Bti caused lower mortality than reported for other Cx. pipiens populations in the literature.Conclusions: These results indicate the urgent need for insecticide resistance monitoring against commonly used adulticides and larvicides in Europe, for the translation of knowledge gained regarding the limited efficiency and availability of insecticide into EU legislation and the need for innovative non-chemical vector control tools in order to counter the widespread insecticide resistance in Culex populations." "Bidirectional Interactions between Arboviruses and the Bacterial and Viral Microbiota in Aedes aegypti and Culex quinquefasciatus" "Lander De Coninck, Jeroen Raes, Leen Delang, Jelle Matthijnssens" "Mosquitoes are important vectors for many arboviruses. It is becoming increasingly clear that various symbiotic microorganisms (including bacteria and insect-specific viruses; ISVs) in mosquitoes have the potential to modulate the ability of mosquitoes to transmit arboviruses. In this study, we compared the bacteriome and virome (both eukaryotic viruses and bacteriophages) of female adult Aedes aegypti and Culex quinquefasciatus mosquitoes fed with sucrose/water, blood, or blood spiked with Zika virus (ZIKV) or West Nile virus (WNV), respectively. Furthermore, we investigated associations between the microbiota and vector competence. We show that the influence of arboviruses on the mosquito microbiome-and vice versa-is distinct for each combination of arbovirus/mosquito species. The presence of ZIKV resulted in a temporarily increased Aedes ISV diversity. However, this effect was distinct for different ISVs: some ISVs decreased following the blood meal (Aedes aegypti totivirus), whereas other ISVs increased only when the blood contained ZIKV (Guadeloupe mosquito virus). Also, the diversity of the Aedes bacteriome depended on the diet and the presence of ZIKV, with a lower diversity observed for mosquitoes receiving blood without ZIKV. In Cx. quinquefasciatus, some ISVs increased in WNV-infected mosquitoes (Guadeloupe Culex tymo-like virus). Particularly, the presence of Wenzhou sobemo-like virus 3 (WSLV3) was associated with the absence of infectious WNV in mosquito heads, suggesting that WSLV3 might affect vector competence for WNV. Distinct profiles of bacteriophages were identified in Culex mosquitoes depending on diet, despite the lack of clear changes in the bacteriome. Overall, our data demonstrate a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. A better understanding of these interactions may lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures. IMPORTANCE In this study, we first utilized the single mosquito microbiome analysis, demonstrating a complex three-way interaction among arboviruses, resident microbiota, and the host, which is distinct for different arbovirus-mosquito combinations. Some of the previously described ""core virus"" increased in the mosquitos receiving viral blood meal, like Guadeloupe mosquito virus and Guadeloupe Culex tymo-like virus, suggesting their potential roles in ZIKV and WNV infection. Notably, Wenzhou sobemo-like virus 3 was associated with the absence of infectious WNV in heads of Culex mosquitoes, which might affect vector competence for WNV. A better understanding of these interactions will lead to the identification of microbiota able to suppress the ability of arbovirus transmission to humans, and hence improved arbovirus control measures." "Culex modestus: the overlooked mosquito vector" "Leen Delang" "Culex (Barraudius) modestus (Ficalbi 1889) are found in temperate regions across Europe, Asia, and Northern Africa. These mosquitoes thrive during the summer and prefer to breed in permanent vegetative habitats such as rice paddies and marshes. Culex modestus feed on a wide range of bird species but are highly attracted to humans, which makes them a potential 'bridge' vector for enzootic pathogens. There is compelling evidence that Culex modestus is an efficient vector for West Nile virus, potentially capable of causing epidemics in humans and other mammals. This species is also a likely vector for Usutu virus, avian malaria (Plasmodium spp.), and parasitic heartworms (Dirofilaria spp.). Culex modestus can be morphologically identified at the larval and adult stages, and a distinctive phenotype of this species is their ability to overwinter. Despite the widespread establishment of this mosquito species and their role as vectors for human pathogens, we lack sufficient knowledge on this species to implement and evaluate targeted vector control measures. Since Culex modestus can be considered a potential public health threat, there is a need for a better understanding of this mosquito species." "First Report of Mutations Associated With Pyrethroid (L1014F) and Organophosphate (G119S) Resistance in Belgian Culex (Diptera: Culicidae) Mosquitoes" "Lander De Coninck, Sam Verwimp, Jelle Matthijnssens, Leen Delang" "The emergence of West Nile virus and Usutu virus in Europe poses a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex (Diptera: Culicidae) mosquito vector. However, the selection pressure caused by exposure to insecticides for vector control or agricultural pest control can lead to insecticide resistance, thereby reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in voltage gated sodium channels and G119S in acetylcholinesterase. In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven, Belgium. Individual mosquitoes were screened for two mutations resulting in L1014F and G119S amino acid substitutions. Both mutations were observed in Cx. pipiens and Cx. modestus but not in Cx. torrentium mosquitoes across the four collection sites. Furthermore, multi-resistance or cross-resistance in Cx. pipiens could be a threat in these areas, as both mutations were observed at low frequencies. These results provide the first report of kdr L1014F and ace-1 G119S resistance mutations in Cx. pipiens and Cx. modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance to design effective arbovirus outbreak control strategies." "Multistressor ecotoxicological study on Culex mosquitoes: from toxicity mechanisms to biotic interactions" "Vienna Delnat" "Natural populations are increasingly facing multiple stressors. Their ability to deal with interacting stressor combinations will be crucial for their local persistence. Populations of target species are typically exposed to pesticide mixtures and natural stressors, and are increasingly developing resistance to single pesticides. Nevertheless, we have poor knowledge whether natural stressors and the presence of pesticide resistance shape mixture toxicity. To assess the pesticide control efficiency and to reduce ecological damage to non-target species, it is important to quantify the effect of pesticide mixtures and compare them with the effect of their single pesticides on pest species, non-target species and their predator-prey interactions. Natural stressors that are widespread in freshwater systems are predation risk and warming; both may magnify the toxicity of single pesticides. The latter pattern has been captured in the 'Climate-induced toxicant sensitivity' concept (CITS). Nevertheless, deviations from this general pattern have been reported. To advance insights, there is a pressing need to identify the underlying molecular stress mechanisms of the CITS patterns. Moreover, while many studies on the toxicity of pesticides looked at the effects of a higher mean temperature, effects of the realistic scenario of daily temperature variation (DTV, which is a key component of global warming) are understudied. In this context, developmental stages and consequences for biotic interactions such as competition have also been largely ignored. In Chapters 1-2 (Part I), I tested the single and combined effects of the pesticide chlorpyrifos and the biopesticide Bacillus thuringiensis israelensis (Bti) on the survival of a chlorpyrifos-resistant and non-resistant strain of the Southern house mosquito Culex quinquefasciatus. In Chapter 1, I investigated whether these effects of chlorpyrifos and/or Bti were magnified by synthetic predator cues of Notonecta water bugs. Single exposure to Bti caused mortality in both strains and single exposure to chlorpyrifos caused only mortality in the non-resistant strain, while predator cues did not induce mortality. The chlorpyrifos-resistant strain was more sensitive to Bti, indicating a cost of resistance. The interaction types between chlorpyrifos and Bti (additive), between chlorpyrifos and predator cues (additive), and between Bti and predator cues (additive) were consistent in both strains. In Chapter 2, I tested the effects of chlorpyrifos and/or Bti on the mortality by predation by the pygmy backswimmer Plea minutissima. After exposure to the mixture, equal mortality by predation was caused in both mosquito strains. As expected, less mortality by predation in the chlorpyrifos-resistant mosquito strain compared to the non-resistant strain occurred after the chlorpyrifos exposure. Notably, the predator killed more mosquito larvae of the resistant strain compared to the non-resistant strain after Bti exposure indicating a predation cost of resistance in the presence of a biopesticide. Part I highlights the integrated use of Bti and natural control by predators as a promising strategy to counter the build-up of resistance and to keep vector mosquito populations under control. In chapters 3-5 (Part II), I tested whether daily temperature variation (DTV) magnifies the toxicity of chlorpyrifos in the Northern house mosquito Culex pipiens. In Chapter 3, I also studied whether DTV magnifies the single toxicity of Bti and/or its combined toxicity with chlorpyrifos. DTV was not lethal and did not change the toxicity of the individual pesticides. Yet, a key novel finding was that high DTV increased the mortality of the mixture by changing the interaction between both pesticides from additive to synergistic. In Chapter 4, I tested whether the effect of DTV and its interaction with chlorpyrifos was dependent on the developmental stage. DTV had again no direct negative effects and did not change the toxicity of the individual pesticides. Exposure to chlorpyrifos at a constant temperature-imposed mortality and reduced the heat tolerance in both larvae and adult males, but not in adult females. This pesticide-induced decrease of heat tolerance is in line with the TICS (""toxicant-induced climate change sensitivity"") concept whereby the first evidence was provided that the TICS can be sex-specific. Notably, DTV interacted synergistically with chlorpyrifos for heat tolerance of the larvae and adult females. DTV increased the chlorpyrifos-induced decrease (CITS) of heat tolerance (TICS), providing support for the reciprocal effects between DTV and contaminants, hence the coupling of the TICS and CITS concepts (CITS --> TICS). This coupling of CITS and TICS was developmental stage specific as the interaction effect between DTV and chlorpyrifos was additive for adult males. In Chapter 5, I also studied the effect of competition with the water flea Daphnia magna on the combined effects of DTV and chlorpyrifos on the mosquito larvae. In this experiment, no pesticide-induced mortality was observed in the presence of DTV due to an accelerated pesticide breakdown under DTV, which contrasts with the general pattern of the CITS concept. There was neither a significant effect of competition on mortality, nor did competition influence the interaction between the single pesticide and DTV. Together these chapters underscore the importance to consider the effects of DTV on pesticide toxicity and on pesticide exposure in risk assessment of toxicants. In Chapters 6-7 (Part III), I tested three concentrations of the pesticide chlorpyrifos (absence, low-effect and high-effect) in the absence and presence of 4 °C warming on larvae of the Northern house mosquito Culex pipiens. In Chapter 6, I showed that both the low-effect and high-effect chlorpyrifos concentrations were lethal and generated mostly negative sublethal effects: activity of acetylcholinesterase (AChE) and total fat content decreased, and oxidative damage to lipids increased, yet growth rate increased. Warming was slightly lethal, yet had positive sublethal effects: growth rate, total fat content and metabolic rate increased, and oxidative damage decreased. The independent action model identified the expected synergistic interaction between chlorpyrifos and warming in four out of seven response variables. Notably, three variables (survival, AChE, and fat content) were strongly dependent on the chlorpyrifos concentration, and two of these (AChE and fat content) were not associated with a significant interaction in the general(ised) linear models. In Chapter 7, I tested the effects of chlorpyrifos under warming at the gene expression level. By applying the independent action model on RNA-seq data I confirmed my hypothesis that synergistic interactions between both stressors at the phenotypic level are underpinned by a higher frequency or strength of antagonistic upregulations (less upregulated than expected based on responses to the single stressors) and of synergistic downregulations (more downregulated than expected) of general stress defence response genes (protection of macromolecules, antioxidant processes, detoxification, and energy metabolism/allocation). These results are relevant to improve vector mosquito control strategies, resistance management and ecological risk assessment of pesticides. I identified a novel cost of resistance to a chemical pesticide in terms of increased vulnerability to a biopesticide, and to predation after exposure to the biopesticide. In addition, the finding of a higher toxicity of the mixture at high DTV compared to the typically used constant test temperatures in the laboratory urges caution when evaluating the environmental impact of pesticide mixtures. Not only the effects of DTV on pesticide toxicity, but also on pesticide degradation (hence exposure) should be considered in ecotox testing. I identified several factors that may affect the detection of the CITS concept: dependence on concentration, developmental stage, genetic strain and sex, and appropriate null model testing. The identified factors are important to take into account when studying the toxicity of contaminants in a warming world. Finally, I provide guidelines to formally test the stressor interaction type for gene expression data. Following this approach, my results highlight that a quantitative analysis of the frequency and strength of the interaction types of general stress response genes, specifically focusing on antagonistic upregulations and synergistic downregulations, may advance the mechanistic understanding of how other stressors modify the toxicity of contaminants." "Establishment of Culex modestus in Belgium and a Glance into the Virome of Belgian Mosquito Species" "Lander De Coninck, Jelle Matthijnssens, Leen Delang" "Culex modestus mosquitoes are considered potential transmission vectors of West Nile virus and Usutu virus. Their presence has been reported across several European countries, including one larva detected in Belgium in 2018. In this study, mosquitoes were collected in the city of Leuven and surrounding areas in the summers of 2019 and 2020. Species identification was performed based on morphological features and partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The 107 mosquitoes collected in 2019 belonged to eight mosquito species, Culex pipiens (24.3%), Cx. modestus (48.6%), Cx. torrentium (0.9%), Culiseta annulata (0.9%), Culiseta morsitans (0.9%), Aedes sticticus (14.0%), Aedes cinereus (9.3%), and Anopheles plumbeus (0.9%), suggesting the presence of an established Cx. modestus population in Belgium. The collection of Cx. modestus mosquitoes at the same locations in 2020 confirmed their establishment in the region. Haplotype network analysis of the COI sequences for Cx. modestus showed that the Belgian population is rather diverse, suggesting that it may have been established in Belgium for some time. The Belgian Cx. modestus population was most closely related to populations from the United Kingdom and Germany. Characterization of the virome of the collected mosquitoes resulted in the identification of at least 33 eukaryotic viral species. Nine (nearly) complete genomes belonging to 6 viral species were identified, all of which were closely related to known viruses. In conclusion, here, we report the presence of Cx. modestus in the surrounding areas of Leuven, Belgium. As this species is considered to be a vector of several arboviruses, the implementation of vector surveillance programs to monitor this species is recommended.IMPORTANCECulex modestus mosquitoes are considered to be a potential ""bridge"" vector, being able to transmit pathogens between birds as well as from birds to mammals, including humans. In Belgium, this mosquito species was considered absent until the finding of one larva in 2018 and subsequent evidence of a large population in 2019 to 2020 described here. We collected mosquitoes in the summers of 2019 and 2020 in the city of Leuven and surrounding areas. The mosquito species was identified by morphological and molecular methods, demonstrating the presence of Cx. modestus in this region. The ability of mosquitoes to transmit pathogens can depend on several factors, one of them being their natural virus composition. Therefore, we identified the mosquito-specific viruses harbored by Belgian mosquitoes. As Cx. modestus is able to transmit viruses such as West Nile virus and Usutu virus, the establishment of this mosquito species may increase the risk of virus transmission in the region. It is thus advisable to implement mosquito surveillance programs to monitor this species." "First record of the West Nile virus bridge vector Culex modestus Ficalbi (Diptera: Culicidae) in Belgium, validated by DNA barcoding" "Katrien de Wolf, Ann Vanderheyden, Isra Deblauwe, Nathalie Smitz, Sophie Gombeer, Adwine Vanslembrouck, Kenny Meganck, Wouter Dekoninck, Marc de Meyer, Thierry Backeljau, Ruth Mueller, Wim Van Bortel" "A thorough knowledge of the presence and spatio-temporal distribution patterns of vector species are pivotal to assess the risk of mosquito-borne diseases in Europe. In 2018, a Culex larva was collected during routine monitoring activities to intercept exotic Aedes mosquito species in the port of Antwerp (Kallo, Belgium). The larva, collected from a pond in mid-September, was morphologically identified as Culex modestus, and this identification was subsequently confirmed by COI barcoding. It is the first confirmed record of this West Nile virus bridge vector in Belgium. The present study also demonstrates the value of DNA-based identification techniques to validate the presence of potential vector species."