Phd in ENVIRONMENTAL AND EVOLUTIONARY BIOLOGY

Thesis title: Evolution of resistance to the insecticide diflubenzuron in populations of the mosquito Culex pipiens: origin, selective pressures, and phenotypic effects

The evolution of insecticide resistance is one of the most serious obstacles in controlling populations of harmful species and reducing the risks associated with them. In mosquitoes this problem is particularly relevant because, as vectors of many viruses and pathogens, they are of public health concern. Since resistance is an adaptive process resulting from the interplay of several genetic, biological, and ecological factors, both molecular and evolutionary insights are essential to get a complete picture of the phenomenon and provide the basis for its correct management. Moreover, insecticide resistance is an ideal model to investigate the origin of new traits and adaptation to changing environments. This research aims to study the evolution of resistance to the insecticide diflubenzuron (DFB), a chitin synthesis inhibitor, in the mosquito vector Culex pipiens. The genetic mechanism conferring DFB resistance in this species has been characterized and is due to the presence of three different resistant alleles (I1043M, I1043L, and I1043F) resulting from amino acid substitutions at position 1043 of the chitin-synthase gene (chs-1). Less is known, instead, on the evolutionary factors that determined the emergence and spread of DFB resistance, nor on its phenotypic effects. Here, we addressed these issues by investigating: (i) the phylogenetic origin of resistant mutations; (ii) the involved selective pressures; (iii) the characteristic of the resistant phenotype. First, we obtained interesting results from phylogenetic analyses conducted on Italian and Turkish populations of Cx. pipiens, where the presence of the resistant alleles was previously documented, which strongly suggest the occurrence of multiple origins of the three mutations and an independent evolution of DFB resistance in Italy and Turkey. Second, by analyzing DNA sequences of current and historical samples of Cx. pipiens from Northern Italy, we showed a role of agricultural applications in selecting and maintaining resistant alleles in Cx. pipiens populations; mutations conferring resistance to DFB, indeed, were already present before the use of DFB in mosquito control, which, from 2000 onwards, enhanced the frequency and distribution of resistant alleles. Third, by comparing susceptible and resistant laboratory colonies established from field samples, we found that resistant mosquitoes have a constitutive over-expression of the chs-1 gene, the target of DFB, and a modified cuticle characterized by a higher thickness and chitin content. These findings, taken together, provide us a wider comprehension of resistance evolutionary dynamics and have important implications for application purposes, which are discussed in this thesis.