Longitudinal survey of insecticide resistance in a village of central region of Burkina Faso reveals co-occurrence of 1014F, 1014S and 402L mutations in Anopheles coluzzii and Anopheles arabiensis

Pyrethroid resistance is one of the major threats for effectiveness of insecticide-treated bed nets (ITNs) in malaria vector control. Genotyping of mutations in the voltage gated sodium channel (VGSC) gene is widely used to easily assess the evolution and spread of pyrethroid target-site resistance among malaria vectors. L1014F and L1014S substitutions are the most common and best characterized VGSC mutations in major African malaria vector species of the Anopheles gambiae complex. Recently, an additional substitution involved in pyrethroid resistance, i.e. V402L, has been detected in Anopheles coluzzii from West Africa lacking any other resistance alleles at locus 1014. The evolution of target-site resistance mutations L1014F/S and V402L was monitored in An. coluzzii and Anopheles arabiensis specimens from a Burkina Faso village over a 10-year range after the massive ITN scale-up started in 2010. Methods Anopheles coluzzii (N = 300) and An. arabiensis (N = 362) specimens collected both indoors and outdoors by different methods (pyrethrum spray catch, sticky resting box and human landing collections) in 2011, 2015 and 2020 at Goden village were genotyped by TaqMan assays and sequencing for the three target site resistance mutations; allele frequencies were statistically investigated over the years. Results A divergent trend in resistant allele frequencies was observed in the two species: 1014F decreased in An. coluzzii (from 0.76 to 0.52) but increased in An. arabiensis (from 0.18 to 0.70); 1014S occurred only in An. arabiensis and slightly decreased over time (from 0.33 to 0.23); 402L increased in An. coluzzii (from 0.15 to 0.48) and was found for the first time in one An. arabiensis specimen. In 2020 the co-occurrence of different resistance alleles reached 43% in An. coluzzii (alleles 410L and 1014F) and 32% in An. arabiensis (alleles 1014F and 1014S). Conclusions Overall, an increasing level of target-site resistance was observed among the populations with only 1% of the two malaria vector species being wild type at both loci, 1014 and 402, in 2020. This, together with the co-occurrence of different mutations in the same specimens, calls for future investigations on the possible synergism between resistance alleles and their phenotype to implement local tailored intervention strategies

A novel allele specific polymerase chain reaction (As-pcr) assay to detect the v1016g knockdown resistance mutation confirms its widespread presence in aedes albopictus populations from Italy

Funding Information: Funding: The study received support from the project ”Sapienza 2017” and JP received support from FCT-Fundação para a Ciência e a Tecnologia, I.P., through national funds in the framework of the project PTDC/BIA-OUT/29477/2017. Funding Information: The study received support from the project ?Sapienza 2017? and JP received support from FCT-Funda??o para a Ci?ncia e a Tecnologia, I.P., through national funds in the framework of the project PTDC/BIA-OUT/29477/2017. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.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—an Asian species that has invaded and colonized the whole world, including temperate regions—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 >5% in most sampling sites, with peaks of 20–45% 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.publishersversionpublishe

Studying mosquito vector bionomics and behaviour to boost pyrethroid-free invasive mosquito control in urban areas

STUDYING MOSQUITO VECTOR BIONOMICS AND BEHAVIOUR TO BOOST PYRETHROID-FREE INVASIVE MOSQUITO CONTROL IN URBAN AREAS Martina Micocci (a), Nigel Beebe (b), Beniamino Caputo (a), Alessandra della Torre (a) (a) Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy (b) School of Biological Sciences, University of Queensland, Brisbane, Australia Background: Aedes invasive mosquitoes are responsible for the global transmission of tropical arboviruses, such as Dengue and Chikungunya - a major public health problem of increasing importance also in temperate regions due to the establishment and spread of Aedes albopictus. The control of Aedes densities, nuisance and arbovirus transmission is still largely dependent from pyrethroid insecticides, but their environmental impact and the spreading of resistance urge for the need to develop innovative pyrethroid-free control approaches, such as Sterile-Insect Technique (SIT, based on release of irradiated males), Incompatible Insect Technique (IIT, based on the release of males with a different endosymbionts Wolbachia strains from those present in wild females capable of inducing cytoplasmic incompatibility) or “auto-dissemination” of Pyriproxyfen (PPF, an Insect Growth Regulator interfering with adult emergence to be exploited to overcome the difficulty in targeting the multiplicity of larval sources). My PhD thesis is focused on studies devoted to better characterize aspects of mosquito bionomics and behaviour (e.g., dispersal, survival, resistance to insecticides) relevant for the design and implementation of integrated management strategies against Ae. albopictus in urban areas of temperate regions. Here, I describe the experiments and preliminary results achieved during the 5 month-visit at University of Queensland. Methods: First, a Mark-Release-Recapture (MRR) was carried out: >12,000 Aedes aegypti males were reared, marked with 6 fluorescent dusts by a novel marking technique using polystyrene beans, released at daytime and night-time per 3 replicates in about 100-sqm area in Goomeri (QLD), and recaptured daily for 7 days by 40 BG-Sentinel traps. Second, a preliminary experiment was carried out under laboratory conditions to test the hypothesis that feeding males with PPF could bring an increase in population suppression through contamination of females by mating. Results: Preliminary results of the MRR show significant higher longevity and dispersal than previously assumed, as well as disproved previous indications suggesting that releases during night hours could be more effective than daily ones. Results of PPF test are under analysis. Discussion: The acquired skills and the expertise developed during the first half of my PhD studies in the group of medical entomology in DSPMI, at Entostudio and at University of Queensland are at the basis of the development of the research activities to be carried out on Aedes albopictus in Rome during the second part of the PhD

Is Aedes albopictus truly exophagic in Italy? Citizen science data can tell.

In its native range in Asia, the tiger mosquito, Aedes albopictus, bites mostly outdoors. This is a major behavioural difference compared to Aedes aegypti and one of the reason of the Ae. albopictus lower synanthropy and lower efficiency as vector of human arboviruses. However, whether this exophagic behaviour is maintained also by Ae. albopictus invasive populations after adaptation to temperate climates has never been specifically investigated. We here exploited 4,914 photographic records provided by citizen during 2-years implementation of Mosquito Alert app in Italy to investigate indoor/outdoor contacts between Ae. albopictus and humans relatively to Culex pipiens. Aedes albopictus represented 90% and 52% of the total number of mosquitoes photographed outdoors (N=1,087) and indoors (N=1,842), respectively. Despite the total number of mosquitos recorded indoors vs outdoors is likely biased by the higher difficulty of taking a picture outdoors, the observation of such a high proportion of Ae. albopictus among mosquitoes recorded indoors represents an objective evidence of a strong endophagic activity of this species in Italy. This may reflect a change in the species behaviour with a high epidemiological relevance, as it implies a higher contact with humans than that estimated by data obtained by conventional outdoor collection methods. Moreover, it may imply a change in practices to prevent arbovirus transmission, i.e. no longer personal protection only, but also protection of the indoor environment. Finally, the results show the exciting capacity of citizen science in providing original data in revealing biological aspects difficult to be investigated by conventional monitoring approaches