Michela Panini

Detecting the presence of target-site resistance to neonicotinoids and pyrethroids in Italian populations of Myzus persicae

BACKGROUND Myzus persicae is a key pest of peach, which in commercial orchards is mainly controlled by chemical treatments. Neonicotinoids represent the main control strategy, but resistance monitoring programmes in Southern Europe have shown the widespread presence of populations highly resistant to this insecticide class in peach orchards. Moreover, in Italy reports of neonicotinoid application failures are increasing. This work describes the status of the main target-site mutations associated with neonicotinoid and pyrethroid resistance in Italian populations collected in 2012. RESULTS R81T mutation linked with neonicotinoid resistance was found in 65% of analysed aphids (35.5% with a homozygous resistant genotype). For the first time, R81T was found in samples collected from herbaceous hosts. Bioassays on a few genotyped populations also revealed the involvement of P450-based metabolic resistance. Only a few individuals without kdr (L1014F) and s-kdr (M918T) target-site mutations were collected. A new single nucleotide polymorphism in the s-kdr locus producing M918L substitution was found. CONCLUSION Target-site resistance to neonicotinoids is common in specialised peach-growing areas, and it is spreading in other Italian regions and on herbaceous hosts. The high frequency of target-site mutations and data obtained from bioassays confirm the presence of multiple resistance mechanisms and suggest the importance of coordinated control strategies.

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.

A1-3 chromosomal translocations in Italian populations of the peach potato aphid Myzus persicae (Sulzer) not linked to esterase-based insecticide resistance

Esterase-based resistance in the peach-potato aphid, Myzus persicae (Sulzer), is generally due to one of two alternative amplified carboxylesterase genes, E4 or FE4 (fast E4). The E4 amplified form is distributed worldwide and it is correlated with a particular translocation between autosomes 1 and 3, whereas the FE4 form, which has hitherto not been found to be associated with chromosomal rearrangements, is typical of the Mediterranean regions. In this study, we present for the first time cytogenetic and molecular data on some M. persicae parthenogenetic lineages, which clearly show a chromosomal A1-3 translocation associated with esterase FE4 genes and unrelated to high levels of esterase-based resistance.

Karyotype variations in Italian populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae)

In this study, we present cytogenetic data regarding 66 Myzus persicae strains collected in different regions of Italy. Together with the most common 2n = 12 karyotype, the results showed different chromosomal rearrangements: 2n = 12 with A1-3 reciprocal translocation, 2n = 13 with A1-3 reciprocal translocation and A3 fission, 2n = 13 with A3 fission, 2n = 13 with A4 fission, 2n = 14 with X and A3 fissions. A 2n = 12-13 chromosomal mosaicism has also been observed. Chromosomal aberrations (and in particular all strains showing A1-3 reciprocal translocation) are especially frequent in strains collected on tobacco plants, and we suggest that a clastogenic effect of nicotine, further benefited by the holocentric nature of aphid chromosomes, could be at the basis of the observed phenomenon.

Use of the synergist piperonyl butoxide can slow the development of alpha-cypermethrin resistance in the whitefly Bemisia tabaci

The development of insecticide resistance in insect pests of crops is a growing threat to sustainable food production, and strategies that slow the development of resistance are therefore urgently required. The insecticide synergist piperonyl butoxide (PBO) inhibits certain insect detoxification systems and so may delay the evolution of metabolic resistance. In the current study we characterized resistance development in the silverleaf whitefly, Bemisia tabaci, after selection with either a neonicotinoid (thiacloprid) or pyrethroid (alpha‐cypermethrin) insecticide alone or in combination with PBO. Resistance development was significantly suppressed (> 60%) in the line selected with alpha‐cypermethrin + PBO compared to the line selected with alpha‐cypermethrin alone. RNA sequencing (RNAseq) analyses revealed an increase in frequency of a knock‐down resistance mutation but no differentially expressed genes were identified that could explain the sensitivity shift. No significant difference was observed in the level of resistance between the thiacloprid and thiacloprid + PBO selected lines, and RNA sequencing (RNAseq) analyses revealed that the cytochrome P450 monooxygenase CYP6CM1, known to metabolize neonicotinoids, was significantly upregulated (>10‐fold) in both lines. The findings of this study demonstrate that PBO used in combination with certain insecticides can suppress the development of resistance in a laboratory setting; however, the mechanism by which PBO supresses resistance development remains unclear.

Ecological genetics of Italian peach-potato aphid (Myzus persicae) populations in relation to geography, dispersal and insecticide resistance as studied using microsatellite and resistance markers

Eight polymorphic microsatellites and two insecticide resistance markers (kdr and R81T) were used to investigate the population genetics and demography of resistance mechanisms in field populations of the peach‐potato aphid, Myzus persicae (Sulzer), a global crop pest, in several areas of Italy, notably the north‐east, aiming to determine whether these parameters are linked to population structuring resulting from insecticide selective sweeps. Individuals collected directly from peach orchards (main primary host) in the spring, especially after control failure, revealed 90 microsatellite alleles, of which approximately one‐third were rare. Many populations deviated from Hardy–Weinberg expectations, in a few instances as a result of null alleles. FIS values were on average negative (indicating heterozygote excess), whereas FST values (mean = 0.137) reflected moderate interpopulation gene flow, with nonstructured populations having an estimated higher level than insecticide‐treated ones. Population assignment and Mantel testing showed a non‐homogenous pattern, with some populations isolated genetically. Linkage disequilibrium was detected in four of the microsatellites and both resistance markers. Contrasting FST patterns with resistance status supported the view that some populations were indeed structured. Neighbour‐joining trees revealed some populations to have similar insecticide resistance profiles, independent of geographical origin. Overall, the findings reveal that the natural M. persicae populations investigated in the present study, living within an intensively cultivated agro‐ecosystem, bear the signature of such management in terms of population structuring, as well as possibly also dynamics.

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.