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Culex mosquitoes particularly Culex quinquefasciatus are important arboviral and filariasis vectors, however despite this important epidemiological role, there is still a paucity of data on their bionomics. The present study was undertaken to assess the insecticide resistance status of Cx. quinquefasciatus populations from four districts of Yaoundé (Cameroon). All Culex quinquefasciatus populations except one displayed high resistance to bendiocarb and malathion with mortalities ranging from 0 to 89% while high resistance intensity against both permethrin and deltamethrin was recorded. Molecular analyses revealed high frequencies of the ACE-1 G119S mutation (ranging from 0 to 33%) and kdr L1014F allele (ranging from 55 to 74%) in all Cx. quinquefasciatus populations. Significant overexpression was detected for cytochrome P450s genes CYP6AA7 and CYP6Z10, as well as for Esterase A and Esterase B genes. The total cuticular hydrocarbon content, a proxy of cuticular resistance, was significantly increased (compared to the S-lab strain) in one population. The study confirms strong insecticide resistance mediated by different mechanisms in Cx. quinquefasciatus populations from the city of Yaoundé. The expansion of insecticide resistance in Culex populations could affect the effectiveness of current vector control measures and stress the need for the implementation of integrated vector control strategies in urban settings.

Background Phlebotomine sand flies are prominent vectors of Leishmania parasites that cause leishmaniasis, which comes second to malaria in terms of parasitic causative fatalities globally. In the absence of human vaccines, sand fly chemical-based vector control is a key component of leishmaniasis control efforts. Methods and findings We performed a literature review on the current interventions, primarily, insecticide-based used for sand fly control, as well as the global insecticide resistance (IR) status of the main sand fly vector species. Indoor insecticidal interventions, such as residual spraying and treated bed nets are the most widely deployed, while several alternative control strategies are also used in certain settings and/or are under evaluation. IR has been sporadically detected in sand flies in India and other regions, using non-standardized diagnostic bioassays. Molecular studies are limited to monitoring of known pyrethroid resistance mutations (kdr), which are present at high frequencies in certain regions. Conclusions As the leishmaniasis burden remains a major problem at a global scale, evidence-based rational use of insecticidal interventions is required to meet public health demands. Standardized bioassays and molecular markers are a prerequisite for this task, albeit are lagging behind. Experiences from other disease vectors underscore the need for the implementation of appropriate IR management (IRM) programs, in the framework of integrated vector management (IVM). The implementation of alternative strategies seems context- and case-specific, with key eco-epidemiological parameters yet to be investigated. New biotechnology-based control approaches might also come into play in the near future to further reinforce sand fly/leishmaniasis control efforts., Author summary Phlebotomine sand flies, mainly aggregated in the tropics and subtropics, are prominent vectors Leishmania parasites that cause leishmaniasis, which comes second to malaria in terms of parasitic causative fatalities globally. In the absence of human vaccines, sand fly vector control is a key component of leishmaniasis prevention and control efforts. Here, we review the current methods and active ingredients applied for sand fly chemical control, as well as the IR data (bioassay and underlying mechanisms) available for dominant sand fly vector species worldwide. Future perspectives for improving the efficiency and sustainability of sand fly control applications, in the genomics era, including the use of molecular diagnostics for evidence-based IRM and the introduction of alternative biotechnology-based approaches, are discussed.

High infection rates of Leishmania donovani and L. tropica were detected in Phlebotomus spp. sand flies collected from refugee camps in Greece, indicating increased risk of infection among local populations. Detection and treatment of leishmaniasis, community education, and vector control are essential measures to prevent pathogen transmission and protect public health.

Background: Aedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. Chikungunya). Development of insecticide resistance in the vector is a major concern as its control mainly relies on the use of biocides. Data on the species’ resistance status is essential for efficient and sustainable control. To date the insecticide resistance status of Ae. albopictus populations from Greece against major insecticides used in vector control remains largely unknown.Methods: We investigated the insecticide resistance status of nineteen Ae. albopictus populations from eleven regions of Greece. Bioassays were performed against diflubenzuron (DFB), B. thuringiensis var. israelensis (Bti), deltamethrin and malathion. Known insecticide resistance loci were molecularly analysed, i.e. voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance; presence and frequency of carboxylesterases 3 (CCEae3a) and 6 (CCEae6a) gene amplification associated with organophosphate (OP) resistance and; chitin synthase-1 (CHS-1) for the possible presence of DFB resistance mutations. Results: Bioassays showed full susceptibility to DFB, Bti and deltamethrin, but resistance against the OP malathion (range of mortality: 55.30-91.40%). VGSC analysis revealed a widespread distribution of the mutations F1534C (in all populations, with allelic frequencies between 6.6% - 68.3%), and I1532T (in 6 populations; allelic frequencies below 22.70%), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations (overall frequency: 33%). Co-presence of the F1534C mutation and CCEae3a amplification was reported in 39 of the 156 samples analysed by both assays. No mutations at the CHS-1 I1043 locus were detected.Conclusions: The results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece, (ii) possible incipient pyrethroid resistance due to the presence of kdr mutations and (iii) possible reduced efficacy of OPs, in a scenario of re-introducing them for vector control. The study highlights the need for systematic resistance monitoring for developing and implementing appropriate evidence-based control programs.Key words: diagnostic, bioassay, arbovirus, vector control, mosquito tiger, insecticide resistance, kdr, Bti, gene amplification

Background: Aedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. Chikungunya). Development of insecticide resistance in the vector is a major concern as it’s control mainly relies on the use of biocides. Data on the specie’s resistance status is essential for efficient and sustainable control. Methods: We investigated the insecticide resistance status of several Ae. albopictus populations from Greece. Bioassays were performed against diflubenzuron (DFB), B. thuringiensis var. israelensis (Bti), deltamethrin and malathion. Molecular analysis of known insecticide resistance loci was performed, i.e. voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance; presence and frequency of carboxylesterases 3 (CCEae3a) and 6 (CCEae6a) gene amplification associated with organophosphate (OP) resistance and; chitin synthase-1 (CHS-1) for the possible presence of DFB resistance mutations. Results: Bioassays showed full susceptibility to DFB, Bti and deltamethrin, but resistance against the OP malathion. VGSC analysis revealed a widespread distribution of mutations F1534C (in all populations, with allelic frequencies between 6.6% - 68.3%), and I1532T (in 6 populations), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations. Co-presence of mutation F1534C and CCEae3a amplification was reported in a subgroup of samples. No mutations at the CHS locus I1043 were detected. Conclusions: The results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece, (ii) a possible incipient pyrethroid resistance due to the presence of kdr mutations and (iii) a possible reduced efficacy of OPs, in a scenario of re-introducing them for vector control. The study highlights the need for systematic resistance monitoring for developing and implementing appropriate evidence-based control programs. Key words: diagnostic, arbovirus, mosquito tiger, insecticide resistance, vector control, Europe

Background As of 2015 thousands of refugees are being hosted in temporary refugee camps in Greece. Displaced populations, travelling and living under poor conditions with limited access to healthcare are at a high risk of exposure to vector borne disease (VBD). This study sought to evaluate the risk for VBD transmission within refugee camps in Greece by analyzing the mosquito and sand fly populations present, in light of designing effective and efficient context specific vector and disease control programs. Methods A vector/pathogen surveillance network targeting mosquitoes and sand flies was deployed in four temporary refugee camps in Greece. Sample collections were conducted bi-weekly during June–September 2017 with the use of Centers for Disease Control (CDC) light traps and oviposition traps. Using conventional and molecular diagnostic tools we investigated the mosquito/sand fly species composition, population dynamics, pathogen infection rates, and insecticide resistance status in the major vector species. Results Important disease vectors including Anopheles sacharovi, Culex pipiens, Aedes albopictus and the Leishmania vectors Phlebotomus neglectus, P. perfiliewi and P. tobbi were recorded in the study refugee camps. No mosquito pathogens (Plasmodium parasites, flaviviruses) were detected in the analysed samples yet high sand fly Leishmania infection rates are reported. Culex pipiens mosquitoes displayed relatively high knock down resistance (kdr) mutation allelic frequencies (ranging from 41.0 to 63.3%) while kdr mutations were also detected in Ae. albopictus populations, but not in Anopheles and sand fly specimens. No diflubenzuron (DFB) mutations were detected in any of the mosquito species analysed. Conclusions Important disease vectors and pathogens in vectors (Leishmania spp.) were recorded in the refugee camps indicating a situational risk factor for disease transmission. The Cx. pipiens and Ae. albopictus kdr mutation frequencies recorded pose a potential threat against the effectiveness of pyrethroid insecticides in these settings. In contrast, pyrethroids appear suitable for the control of Anopheles mosquitoes and sand flies and DFB for Cx. pipiens and Ae. albopictus larvicide applications. Targeted actions ensuring adequate living conditions and the establishment of integrated vector-borne disease surveillance programs in refugee settlements are essential for protecting refugee populations against VBDs.

Chemical insecticides are the main tool for vector control worldwide with pyrethroids and the larvicide diflubenzuron (DFB) used extensively against the primary West Nile virus vector Culex pipiens. In this study we monitored the presence, frequency and geographical distribution of the Chitin synthase 1 gene mutations I1043L, I1043M associated with striking diflubenzuron resistance and the knock down resistance mutation L1014F associated with pyrehtroid resistance, in Cx. pipiens populations from Turkey. DFB resistance mutations I1043L, I1043M were found at high frequencies ranging from 15.7% to 37.5% and 25% to 52.7% in all study sites (first report for the presence of DFB resistant mutations, anywhere outside Italy). The L1014F pyrethroid resistance mutation was also present at relatively high frequencies, ranging from 40% to 50%. The high frequency of both DFB and pyrethroid resistance mutations are possibly associated with the intense use of these insecticides in agricultural and mosquito control applications in the region. Given the limited number of available insecticides for vector control these findings are of major importance for public health in Turkey and neighboring countries. © 2019 Elsevier B.V., 532 General Secretariat for Research and Technology, GSRT Hellenic Foundation for Research and Innovation, HFRI, We thank Mu?la Metropolitan Municipality for their support and for providing mosquito samples used in this study. E.A. F. was supported by the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI) in the context of the action ?1st Proclamation of Scholarships from ELIDEK for PhD Candidates? (Scholarship Code: 532)., We thank Muğla Metropolitan Municipality for their support and for providing mosquito samples used in this study. E.A. F., was supported by the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI) in the context of the action “1st Proclamation of Scholarships from ELIDEK for PhD Candidates” (Scholarship Code: 532).

International audience; Insecticide resistance is a major threat for vector control and prevention of mosquito borne diseases. In the Culex pipiens mosquitoes, resistance against diflubenzuron (DFB) was firstly detected in Ravenna (Emilia-Romagna region, Northern Italy), in 2015. The resistant phenotypes were associated with two mutations, I1043 M and I1043 L, at the amino acid 1043 of the chitin synthase gene. In this study, we monitored the presence, frequency and geographical distribution of the DFB resistant mutations in Cx. pipiens populations from Northern Italy, and in populations from Greece and France. In the Emilia-Romagna region, the resistant mutations were detected in 20 out of the 30 populations analysed, reaching allelic frequencies over 70%. The presence and distribution of the resistance mutations was highly focal, with a clear pattern of increasing resistant allelic frequencies moving from the Western towards the Eastern provinces of Emilia-Romagna. Contrary to Italy, DFB resistant alleles were not detected in the Cx. pipiens mosquitoes sampled from Greece and France. Following statistical, literature and bibliographical database analyses on the history of DFB insecticide use in the study areas, we suggest that the selection pressures from the intense agricultural DFB applications occurring throughout the' 80-'90 s against orchard pests, followed, from 2000s onwards by mosquito control DFB applications, may account for the high mutation frequencies observed in the Cx. pipiens populations of the Eastern provinces of Emilia-Romagna. The findings are of major concern for public health in Italy and Europe, as DFB remains a very important insecticide used for controlling arbovirus mosquito vectors, where alternative larvicides are extremely limited.

Over the past decade, Greece and other Mediterranean countries have witnessed the emergence and resurgence of several vector-borne diseases (VBDs), posing important public health challenges and threatening the tourist industry. An essential prerequisite for the design and execution of efficient and sustainable context-specific VBD control programmes is the establishment of integrative entomological and epidemiological surveillance systems. However, the monitoring and management of surveillance datasets (often chronologically fragmented, scattered in regional health district offices and partially accessible upon requisition), as well as their transformation into actionable information, is a complex undertaking. In light of aiding and optimizing vector control efforts in the Mediterranean Basin, we developed VectorMap-GR, an online, open access, operational management tool for entomological and complementary epidemiological monitoring data. The toolʼs key components are a set of controlled vocabularies (ontologies) running throughout the system, the systemʼs database and a map interface for data querying and display. The tool supports transformation of raw data into operationally relevant information (i.e. customized maps, charts, tables and reports) in a highly interactive fashion achieved through query filters and the ArcGIS technology embedded in the system. End-users may search for and obtain information on (i) the mosquito fauna composition, abundance and spatiotemporal dynamics; (ii) the mosquito insecticide resistance status and underlying resistance mechanisms; (iii) the occurrence of VBD pathogens and infections in vectors, animals and humans; and (iv) operationally relevant physical feature georeferenced datasets (e.g. mosquito breeding sites). VectorMap-GR was pilot implemented during 2018–2020 in a mosquito control programme in the Region of Crete (southern Greece). The programmeʼs control efforts coupled with VectorMap-GR pilot implementation phase, very likely contributed to the reduction of vector population numbers and the prevention of human VBD occurrences, recorded in this period.

Phlebotomine sand flies (Diptera: Psychodidae) are primary vectors of leishmaniasis. Greece and Turkey are both endemic for visceral and cutaneous leishmaniasis and are widely affected by the disease. Measures commonly applied for controlling sand flies rely on the use of insecticides, predominantly pyrethroids. A worldwide problem associated with the intensive use of insecticides is the development of resistance. Scarce information is available regarding the resistance status in sand fly populations. Sand flies were collected from Greece (Thessaloniki, Peloponnese, Chios island) and Turkey (Sanliurfa) and analyzed for the presence and frequency of target-site knockdown resistance mutations on the voltage-gated sodium channel (Vgsc) gene. Five sand fly species were included in the analysis: Phlebotomus perfiliewi Parrot, Phlebotomus neglectus Tonnoir, Phlebotomus simici Nitzulescu, Phlebotomus tobbi Adler and Theodor, and Phlebotomus papatasi Scopoli. Their Vgsc gene-domain II was analyzed for the presence of known pyrethroid resistance mutations. The mutation 1014F, associated with pyrethroid-resistant phenotypes, was detected in P. papatasi sand flies from Sanliurfa at an allele frequency of 48%. Homozygotes for the wild type allele 1014L (Leu/Leu) represented 36% of the population, while homozygotes for the resistant allele 1014F (Phe/Phe) and heterozygotes encompassing both alleles (Leu/Phe) each had a frequency of 32%. In all other sand fly species, only the wild type allele 1014L was detected. This is the first report for the detection of resistance mutations in the major leishmaniasis vector P. papatasi and is of major concern regarding leishmaniasis control.

During July-October 2017 a WNV outbreak took place in the Peloponnese, Southern Greece with five confirmed deaths. During routine monitoring survey in the Peloponnese, supported by the local Prefecture, we have confirmed the presence of all three Culex pipiens biotypes in the region, with a high percentage of Culex pipiens/molestus hybrids (37.0%) which are considered a highly competent vector of WNV. Kdr mutations related to pyrethroid resistance were found at relatively low levels (14.3% homozygosity) while no mosquitoes harboring the recently identified chitin synthase diflubenzuron-resistance mutations were detected in the region. As an immediate action, following the disease outbreak (within days), we collected a large number of mosquitoes using CO2 CDC traps from the villages in the Argolis area of the Peloponnese, where high incidence of WNV human infections were reported. WNV lineage 2 was detected in 3 out of 47 Cx. pipiens mosquito pools (detection rate = 6.38%). The virus was not detected in any other mosquito species, such as Aedes albopictus, sampled from the region at the time of the disease outbreak. Our results show that detection of WNV lineage 2 in Cx. pipiens pools is spatially and chronologically associated with human clinical cases, thus implicating Cx. pipiens mosquitoes as the most likely WNV vector. The absence of diflubenzuron resistance mutations and the low frequency of pyrethroid (kdr) resistance mutations indicates the suitability of these insecticides for Cx. pipiens control, in the format of larvicides and/or residual spraying applications respectively, which was indeed the main (evidence based) response, following the disease outbreak.

We assessed window screens and eave baffles (WSEBs), which enable mosquitoes to enter but not exit houses, as an alternative to indoor residual spraying (IRS) for malaria vector control. WSEBs treated with water, the pyrethroid lambda-cyhalothrin, or the organophosphate pirimiphos-methyl, with and without a binding agent for increasing insecticide persistence on netting, were compared with IRS in experimental huts. Compared with IRS containing the same insecticide, WSEBs killed similar proportions of Anopheles funestus mosquitoes that were resistant to pyrethroids, carbamates and organochlorines and greater proportions of pyrethroid-resistant, early exiting An. arabiensis mosquitoes. WSEBs with pirimiphos-methyl killed greater proportions of both vectors than lambda-cyhalothrin or lambda-cyhalothrin plus pirimiphos-methyl and were equally efficacious when combined with binding agent. WSEBs required far less insecticide than IRS, and binding agents might enhance durability. WSEBs might enable affordable deployment of insecticide combinations to mitigate against physiologic insecticide resistance and improve control of behaviorally resistant, early exiting vectors.