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Culex pipiens (Linnaeus), one of the most abundant mosquito species in Europe, plays a crucial role in the endemic transmission of West Nile virus and caused the large outbreak with1600 human cases in 2018. Although evidence of resistance to pyrethroids has been reported for Cx. pipiens populations from Spain and Greece, resistance monitoring has been largely neglected in Italy. Herein, we investigate susceptibility of Italian Cx. pipiens populations to the pyrethroids permethrin and deltamethrin. Results from WHO-tube-bioassays revealed mortalities ranging from 14-54%, indicating high levels of resistance, in four out of 10 populations exposed to permethrin (0.75%) and of 63% in one of three populations exposed to deltamethrin (0.05%). Reduced susceptibility (mortality98%) was detected in almost all other populations. A clear association is shown between the resistant phenotype and the presence of kdr-alleles in position 1014 of the VSSC, strongly suggesting its role in reducing susceptibility. The study provides the first evidence of pyrethroid-resistance in Italian Cx. pipiens populations and reports levels of resistance paralleled in the European region only in Turkey. This highlights the urgent need to implement insecticide-resistance management plans to restore the efficacy of the nowadays only chemical weapon available to control arbovirus transmission in Europe.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus&mdash, an Asian species that has invaded and colonized the whole world, including temperate regions&mdash, sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as &gt, 5% in most sampling sites, with peaks of 20&ndash, 45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

BACKGROUND Aedes albopictus has spread during the last few decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. chikungunya, dengue, and Zika) also in temperate areas, as demonstrated by the Chikungunya 2007 and 2017 outbreaks in northeastern and central Italy. Insecticides are an important tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study was to address the gap in current knowledge of pyrethroid insecticide resistance of European Ae. albopictus populations, focusing on populations from Italy, Albania and Greece. RESULTS Bioassays for resistance to permethrin (0.75%), α-cypermethrin (0.05%) or deltamethrin (0.05%) were performed according to World Health Organization (WHO) protocols and showed reduced susceptibility (

The selection of the ideal trap for arbovirus surveillance is an issue of primary importance to increase the sensitivity of virus detection and the cost-effectiveness of the entomological surveillance. During the summer 2011, the effectiveness of five types of mosquito traps (CDC gravid trap, CO2(-)baited trap, BG-Sentinel™ and two experimental prototypes) to attract females potentially infected with West Nile virus were assessed. The study was carried out in three natural wetland sites located in the Emilia-Romagna Region (Northern Italy), using a Latin square scheme. Single night collections of adult females were performed and determination of species and physiological state (gravid, nulliparous or parous) was made upon return to the laboratory. The species most frequently collected in the gravid trap was Culex pipiens sl. L., being gravid females the large majority of the individuals. Species diversity was much higher in CO2(-)baited traps, which may therefore enable a more comprehensive description of the vector species composition and their role in arboviruses circulation. Our findings indicate that gravid traps can be a valid tool and should be integrated in the West Nile virus surveillance system in the Emilia-Romagna region, mainly based on collections made with CO2-baited traps.

Recently, Italy-particularly the Emilia-Romagna region-was the location of consecutive outbreaks of human diseases caused by the arboviruses chikungunya virus and West Nile virus. The two outbreaks, spread by different species of mosquitoes, were not related, but pointed out the lack of an arboviral surveillance program in this region. Beginning in 2007 entomological surveillance was initiated in the Emilia-Romagna region, and in 2008 the program was improved and extended at Lombardia region. Using CO(2)-baited traps, 65,292 mosquitoes were collected; pooled by date of collection, location, and species; macerated manually; and tested by reverse transcription (RT)-polymerase chain reaction for the presence of alphaviruses, orthobunyaviruses, and flaviviruses. Amplicons were sequenced and employed for identification of viral RNA by basic local alignment search tool search in GenBank. Results of these assays showed (1) the presence of West Nile virus in two pools of Culex pipiens mosquitoes, (2) the presence of RNA of two orthobunyaviruses, Tahyna virus in a pool of Ochlerotatus caspius mosquitoes and Batai virus in a pool of Anopheles maculipennis mosquitoes, and (3) the presence of flavivirus RNAs in pools of Oc. caspius, Aedes albopictus, and Aedes vexans mosquitoes; the sequences of these amplicons were most closely related to flaviviruses that have been detected only in mosquitoes and had no recognized vertebrate host (Aedes flavivirus, Culex flavivirus, and Kamiti River virus).

Mating competitiveness trials have been conducted in large net-screened enclosures (8 by 5 by 2.8 m) built in a natural shaded environment, in the summers of 2006, 2007, 2008, and 2009 in northern Italy. Aedes albopictus (Skuse) males were radio-sterilized by applying gamma radiations at doses in the range 30-60 Gy. Gamma radiation was administered to aged pupae at the rate of 2.3 Gy/min. Reared radiated males (originally collected in Rimini, Forli, Bologna, Matera, Pinerolo) and hybrid radiated males were tested against wild fertile males (originated from eggs collected in Rimini and Cesena) and reared fertile males, in multiple comparisons for mating competitiveness with reared or wild females. The ratio was kept constant at 100-100_100 (fertile males-radiated males_virgin females). Mating competitiveness was estimated through the calculation of the hatching rate of the eggs laid in oviposition traps positioned inside enclosures. No clear effect of the strains tested (reared, wild, or hybrid) was found. Results demonstrated that reducing the radiation dose from 60 to 30 Gy increases males' competitiveness. Laboratory investigations conducted after controversial results in the 2006 preliminary trials, showed that radiation induces precociousness in adult male emergence.

The increasing concern about vector-borne diseases such as West Nile disease in northern Italy motivated our analysis of data on the mosquito fauna and the seasonal and daily flight patterns collected in 1998 in the Po Valley. Collections were performed once a week from May to November, with human landing collections and CO(2) traps. Culex pipiens was present from July to October and showed a clearly unimodal nocturnal flight habit. Culex modestus appeared in July-August and showed a bimodal flight pattern, (main peak during the evening and a minor one in the morning). Aedes caspius was present from May to November (highest densities in July-August) and showed a bimodal flight pattern with a major crepuscular peak and a minor dawn peak in the morning. Aedes detritus was the most abundant species in May, with a crepuscular sharply bimodal flight pattern, particularly according to human landing collections. Sunset and sunrise time, in combination with the solar phase (that determines daylight duration and its trend of changing) were the main factors affecting flight behavior. Temperature, relative humidity, and wind speed differently affected the flight behavior of mosquito females according to the species.

BACKGROUND: Aedes albopictus has spread during the last few decades all over the world. This has increased significantly the risk of exotic arbovirus transmission (e.g. chikungunya, dengue, and Zika) also in temperate areas, as demonstrated by the Chikungunya 2007 and 2017 outbreaks in northeastern and central Italy. Insecticides are an important tool for limiting the circulation of these mosquito-borne viruses. The aim of the present study was to address the gap in current knowledge of pyrethroid insecticide resistance of European Ae. albopictus populations, focusing on populations from Italy, Albania and Greece. RESULTS: Bioassays for resistance to permethrin (0.75%), α-cypermethrin (0.05%) or deltamethrin (0.05%) were performed according to World Health Organization (WHO) protocols and showed reduced susceptibility (