Vincenzo Puggioni

Presence and impact of allelic variations of two alternative s-kdr mutations, M918T and M918L, in the voltage-gated sodium channel of the green peach aphid Myzus persicae

BACKGROUND Pyrethroids have been widely employed in order to control several agricultural pests, including Myzus persicae. Target-site resistance is the main mechanism that confers insensitivity to this class of compounds, and the most common amino acid substitutions are kdr (L1014F) and s-kdr (M918T), but recently another mutation in the s-kdr locus (M918L) has been described in French and Korean populations of M. persicae. RESULTS Molecular analysis of several Italian populations of M. persicae by pyrosequencing revealed the presence of the new s-kdr mutation (M918L) in different forms. It was found in two different nucleotide polymorphisms (a/t or a/c substitution), in heterozygous or homozygous status, and also in combination with the classic kdr and s-kdr. Bioassays on populations carrying the M918L mutation show that it strongly affects pyrethroid efficacy, particularly of type II pyrethroids such as lambda-cyhalothrin, while it has no effect against DDT. CONCLUSION This work provides more information about the new s-kdr M918L mutation in M. persicae, describing a more complicated situation arising from the possible combination with the classic L1014F and M918T. Our data open new questions concerning the origin of these new genotypes with different combinations of target-site mutations, and also their possible influence on control strategies.

Qualitative Sybr Green real-time detection of single nucleotide polymorphisms responsible for target-site resistance in insect pests: the example of Myzus persicae and Musca domestica.

Chemical insecticides have been widely used to control insect pests, leading to the selection of resistant populations. To date, several single nucleotide polymorphisms (SNPs) have already been associated with insecticide resistance, causing reduced sensitivity to many classes of products. Monitoring and detection of target-site resistance is currently one of the most important factors for insect pest management strategies. Several methods are available for this purpose: automated and high-throughput techniques (i.e. TaqMan or pyrosequencing) are very costly; cheaper alternatives (i.e. RFLP or PASA-PCRs) are time-consuming and limited by the necessity of a final visualization step. This work presents a new approach (QSGG, Qualitative Sybr Green Genotyping) which combines the specificity of PASA-PCR with the rapidity of real-time PCR analysis. The specific real-time detection of Cq values of wild-type or mutant alleles (amplified used allele-specific primers) allows the calculation of ΔCqW-M values and the consequent identification of the genotypes of unknown samples, on the basis of ranges previously defined with reference clones. The methodology is applied here to characterize mutations described in Myzus persicae and Musca domestica and we demonstrate it represents a valid, rapid and cost-effective technique that can be adopted for monitoring target-site resistance in field populations of these and other insect species.