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.
Mosquito saliva was shown to play a key role in arbovirus transmission and pathogenesis. In this study, we explored the presence of microbiota in mosquito saliva and their potential effect on mosquito-borne virus infection in vitro. Culturable fungal and bacterial colonies were isolated and identified from saliva harvested from Aedes aegypti (lab strain) and Culex pipiens (field-collected mosquitoes). For the first time, the fungal species Penicillium crustosum was identified in mosquitoes. Culturable bacteria detected in mosquito saliva included Serratia marcescens, Serratia nematodiphila, Enterobacter spp. and Klebsiella spp., which were previously identified as mosquito or insect endosymbionts in the midgut or other organs. Oral treatment of adult mosquitoes with antibiotics or an antifungal drug resulted in a significant reduction of bacteria or fungi in saliva. Saliva depleted from fungi or bacteria significantly decreased Semliki Forest virus infection in human skin fibroblasts compared to non-treated saliva when the virus was preincubated with saliva or when virus and saliva were added to the cells at the same time. These results suggest that the salivary microbiota may play an essential role in mosquito-borne viral replication in human skin cells. Nevertheless, the potential effect of salivary microbiota warrants further investigation in vivo.
The emergence of West Nile virus and Usutu virus in Europe is 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 mosquito vector. However, the selection pressure caused by exposure to insecticides in vector control or agricultural pest control can lead to the development of insecticide resistance 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 the voltage gated sodium channel and G119S in the 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 in Belgium. Two mutations resulting in L1014F and G119S amino acid substitutions were screened for in individual mosquitoes. Both mutations were observed in Culex pipiens and Culex modestus mosquitoes across the four collection sites, but not in Culex torrentium. Furthermore, multi-resistance or cross-resistance could be a developing threat, since both mutations were observed at low frequencies (resistant allele frequency <30%) and always in heterozygosity. These results provide the first report of kdr L1014F and ace-1 G119S resistance in Culex pipiens and Culex modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance for the development of effective arbovirus control strategies in the event of an outbreak in Belgium.
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