VectorMap-GR: A local scale operational management tool for entomological monitoring, to support vector control activities in Greece and the Mediterranean Basin.

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 tool's key components are a set of controlled vocabularies (ontologies) running throughout the system, the system's 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 programme's 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.

Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control.

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 are 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 19 Ae. albopictus populations from 11 regions of Greece. Bioassays were performed against diflubenzuron (DFB), Bacillus 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 programmes.

Identification and geographical distribution of pyrethroid resistance mutations in the poultry red mite Dermanyssus gallinae.

BACKGROUND: The poultry red mite (PRM) Dermanyssus gallinae is the most common ectoparasite on poultry and causes high economic losses in poultry farming worldwide. Pyrethroid acaricides have been widely used for its control and, consequently, pyrethroid resistance has arisen. In this study we aim to investigate the occurrence of resistance and study the geographical distribution of pyrethroid resistance mutations across PRM populations in Europe. RESULTS: Full dose-response contact bioassays revealed very high levels of resistance against several pyrethroids (α-cypermethrin, fluvalinate, and cyfluthrin) in two PRM populations from Greece, compared to a susceptible reference strain. Resistance was associated with mutations in the gene encoding the target site of pyrethroids, the voltage-gated sodium channel (VGSC). Mutations, M918L and L925V in domain IIS4-S5 and F1534L in domain IIIS6, were found at positions known to play a role in pyrethroid resistance in other arthropod species. Subsequent screening by sequencing VGSC gene fragments IIS4-S5 and IIIS6 revealed the presence and distribution of these mutations in many European populations. In some populations, we identified additional or different mutations including M918V/T, L925M, T929I, I936F, and F1538L. The latter mutation is a possible alternative for F1538I that has been previously associated with pyrethroid resistance in other Acari species. CONCLUSION: We report very high levels of pyrethroid resistance in PRM populations from Greece, as well as the identification and geographical distribution of 10 pyrethroid resistance mutations in PRM populations across Europe. Our results draw attention to the need for an evidence-based implementation of PRM control, taking acaricide resistance management into consideration. © 2019 Society of Chemical Industry.

Detection of West Nile Virus - Lineage 2 in Culex pipiens mosquitoes, associated with disease outbreak in Greece, 2017.

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.

First evidence of resistance to pyrethroid insecticides in Italian Aedes albopictus populations 26 years after invasion.

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 (<90% mortality) of some Italian populations to permethrin and α-cypermethrin, but not to deltamethrin. CONCLUSION: This study reports the first evidence of resistance to pyrethroids in adult Italian Ae. albopictus populations. Results refer to the season preceding the Chikungunya 2017 outbreak in central Italy and highlight the need to increase efforts to monitor the spread of insecticide resistance and the need to develop strategies to limit the spread of insecticide resistance, particularly in areas where extensive treatments have been carried out to contain disease outbreaks. © 2017 Society of Chemical Industry.

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus.

BACKGROUND: The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. PRINCIPAL FINDINGS: Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. SIGNIFICANCE: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.

Insecticide resistance status in the major West Nile virus vector Culex pipiens from Greece.

BACKGROUND: One of the largest West Nile virus outbreaks in Europe occurred in Greece in 2010. Use of insecticides against Culex pipiens was substantially scaled up, as an emergency tool. Although mosquito control has been based on insecticides for several decades in Greece, insecticide resistance data are not available. RESULTS: An examination was made of the resistance status of 13 Cx. pipiens populations from five regional units in Greece against four insecticides used for its control over a 3 year period. Bioassays demonstrated susceptibility of most populations to all insecticides, except for temephos in some regions, and deltamethrin and diflubenzuron on one occasion each. The authors also monitored the frequency of the pyrethroid target-site resistance mutations L1014F (kdr), as well as G119S and F290V in the Ace1 gene. Ace1 insensitivity mutations were found at low frequencies and always in heterozygocity. However, the frequency of kdr pyrethroid resistance mutations was higher (up to 63.0% in Thessaloniki). CONCLUSIONS: The high frequency of kdr mutations indicates a risk that needs to be addressed, should the use of pyrethroids be further extended. There was no strong evidence of significant resistance levels against Bacillus thuringiensis var. israelensis and diflubenzuron. Continued monitoring of insecticide resistance is recommended for the application of appropriate management tactics.