Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae.

The evolution of resistance to insecticides threatens the sustainable control of many of the world's most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

Survey of target site mutations linked with insecticide resistance in Italian populations of Aphis gossypii.

BACKGROUND: Aphis gossypii is a worldwide agricultural pest that causes high levels of economic losses by feeding and transmitting virus diseases. It is usually controlled by chemical insecticides, but this could lead to the selection of resistant populations. Several single nucleotide polymorphisms (SNPs) have been identified associated with insecticide resistance. Monitoring activities to detect the presence of such mutations in field populations can have an important role in insect pest management but, currently, no information on Italian strains is available. RESULTS: The presence of target site mutations conferring resistance to different insecticides was analysed in Italian field collected populations of A. gossypii with an allele specific approach (QSGG, Qualitative Sybr-Green Genotyping). Primers were designed to detect mutations in genes coding acetylcholinesterase (S431F), nicotinic acetylcholine receptor (R81T) and voltage-gated sodium channel (M918L and L1014F). S431F was widespread but with high variability across populations. R81T was detected for the first time in Italy but only in two populations. The L1014F mutation (kdr) was not found, while in the samples showing the M918L two different nucleotidic substitutions were detected. Mutant allele frequencies were, respectively, 0.70 (S431), 0.31 (M918) and 0.02 (R81). Further analysis on the voltage-gated sodium channel gene showed the presence of eight haplotypes and one non-synonymous mutation in the gene coding region. CONCLUSION: Multiple target-site mutations were detected within Italian populations. The combinations of genotypes observed in certain locations could affect negatively the control of this pest. Preliminary insights on the genetic structure in the Italian populations of A. gossypii were acquired. © 2024 Society of Chemical Industry.

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.

Target site mutations underlying insecticide resistance in Tunisian populations of Myzus persicae (Sulzer) on peach orchards and potato crops.

BACKGROUND: The massive use of synthetic insecticides strongly affects the level of insecticide resistance in populations of Myzus persicae worldwide. The selection of target site insensitivity-mutations is particularly worrying in areas where agro-industrial crops are vulnerable to the attacks of aphids that vector viruses, as in the case of Tunisia. Knowledge of the resistance mechanisms evolved locally in this aphid pest is a prerequisite to improving and retaining the sustainability of integrated pest management strategies. RESULTS: Target site mutations were surveyed in several populations of M. persicae collected from peach and potato crops between 2011 and 2017 in three Tunisian regions using real-time allele-specific PCR. The L1014F mutation (kdr locus) was found at a moderate frequency mostly in the heterozygous state and the homozygous resistant genotype was very uncommon. The M918T mutation (super-kdr locus) was present in a few heterozygous individuals, whereas the M918L mutation was detected for the first time in Tunisia and extreme North Africa. This latter mutation was shown to be widespread and well-established in Tunisia mainly as homozygous individuals, and was more abundant on peach than on potato crops. The S431F mutation (MACE) was found in a few heterozygous individuals. No individuals carrying the R81T mutation linked to neonicotinoid resistance were detected. CONCLUSION: This study points out a critical situation for the efficacy of pyrethroid insecticides to control M. persicae populations in Tunisia. It also confirms the rapid spread of the M918L mutation which has been detected in many different areas of the Mediterranean basin. © 2022 Society of Chemical Industry.

Do It by Yourself: Larval Locomotion in the Black Soldier Fly Hermetia illucens, with a Novel "Self-Harvesting" Method to Separate Prepupae.

The neotropical insect Hermetia illucens has become a cosmopolite species, and it is considered a highly promising insect in circular and sustainable economic processes. Being able to feed on a wide variety of organic substrates, it represents a source of lipids and proteins for many uses and produces recyclable waste. We investigated the characteristics and differences in the poorly-known locomotory behaviour of larvae of different instars, paying particular attention to the unique characteristics of the prepupal stage, key to farming and industrial processes. Moreover, we attempted to develop a "self-harvesting" system relying on the behavioural traits of prepupae to obtain their separation from younger larvae under rearing condition with minimum effort. Prepupae differ from younger larvae in their response to physical disturbance in the form of tonic immobility and significantly differ in their locomotory movements. Both prepupae and younger larvae reacted similarly to heat or light-induced stress, but low light and high moisture induced only prepupae to migrate away, which resulted in the development of a highly efficient separation methodology. The new data on the behaviour of H. illucens not only shed new light on some unexplored aspects of its biology, but also led to develop an inexpensive self-harvesting system that can be implemented in small-scale and industrial farming.

Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.

The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host-plant associations, uncovering the widespread co-option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.

Pest Management and Ochratoxin A Contamination in Grapes: A Review.

Ochratoxin A (OTA) is the most toxic member of ochratoxins, a group of toxic secondary metabolites produced by fungi. The most relevant species involved in OTA production in grapes is Aspergillus carbonarius. Berry infection by A. carbonarius is enhanced by damage to the skin caused by abiotic and biotic factors. Insect pests play a major role in European vineyards, and Lepidopteran species such as the European grapevine moth Lobesia botrana are undoubtedly crucial. New scenarios are also emerging due to the introduction and spread of allochthonous pests as well as climate change. Such pests may be involved in the dissemination of OTA producing fungi even if confirmation is still lacking and further studies are needed. An OTA predicting model is available, but it should be integrated with models aimed at forecasting L. botrana phenology and demography in order to improve model reliability.

Slug Monitoring and Impacts on the Ground Beetle Community in the Frame of Sustainable Pest Control in Conventional and Conservation Agroecosystems.

In conservation agriculture, slugs are considered significant pests and their monitoring is a key option in the integrated pest management framework. Together with molluscicide applications, predators such as ground beetles can offer a tool for slug control in the field. Through the evaluation of slug and ground beetle monitoring strategies, this work compared their presence in conventional and conservation agricultural plots. The invasive Deroceras invadens was the dominant slug species to occur in all sampling periods. Among Carabidae, Poecilus cupreus and Pterostichus melas were the most abundant species, and Bembidion spp., Brachinus spp., and Harpalus spp. were also common. Beer-baited pitfall traps, whatever their alcoholic content, caught more slugs and ground beetles than wooden boards used as shelters. Slugs were more abundant in conventional plots than in conservation plots, possibly due to the lower presence of natural enemies such as ground beetles. Despite possible impacts on Carabidae, beer-baited pitfall traps should be considered a useful tool for slug monitoring and for the planning of molluscicide applications. Soil management such as minimum- or no-tillage and the presence of cover crops are important elements influencing both slug and ground beetle presence, possibly playing a key role in the maintenance of natural enemy populations.

The genetic architecture of a host shift: An adaptive walk protected an aphid and its endosymbiont from plant chemical defenses.

Host shifts can lead to ecological speciation and the emergence of new pests and pathogens. However, the mutational events that facilitate the exploitation of novel hosts are poorly understood. Here, we characterize an adaptive walk underpinning the host shift of the aphid Myzus persicae to tobacco, including evolution of mechanisms that overcame tobacco chemical defenses. A series of mutational events added as many as 1.5 million nucleotides to the genome of the tobacco-adapted subspecies, M. p. nicotianae, and yielded profound increases in expression of an enzyme that efficiently detoxifies nicotine, both in aphid gut tissue and in the bacteriocytes housing the obligate aphid symbiont Buchnera aphidicola. This dual evolutionary solution overcame the challenge of preserving fitness of a mutualistic symbiosis during adaptation to a toxic novel host. Our results reveal the intricate processes by which genetic novelty can arise and drive the evolution of key innovations required for ecological adaptation.

Analysis of the extent of synteny and conservation in the gene order in aphids: A first glimpse from the Aphis glycines genome.

In the last decade several insect genomes have been sequenced, but for most the chromosomal mapping of the identified scaffolds/annotated genes is not available. The lack of this information makes it difficult to analyse various genetic aspects, including the presence of genome rearrangements and the extent of synteny within and across species. We mapped five multigenic DNA families (major and minor rDNAs, histone gene cluster, esterases and carotenoid desaturases) and seven scaffolds corresponding to 9 Mb of the soybean aphid, Aphis glycines, genome and identified loci spanning the four soybean aphid chromosomes. A comparative analysis of the localization of the annotated A. glycines genes with respect to the peach potato aphid, Myzus persicae, and the fly, Drosophila melanogaster, evidenced a lower degree of synteny between the two aphid species than in the aphid-fly comparison. Only 1.4 genes per syntenic block were observed in aphids in contrast to 2.3 genes per block in flies. This higher chromosomal rearrangement rate in aphids could be explained considering that they possess holocentric chromosomes that can favour the stabilization and inheritance of chromosomal rearrangements. Lastly, our experiments did not detect the presence of chimeric assemblies in the newly available A. glycines biotype 1 genome, differently from what reported in assembled genome of other aphid species, suggesting that chromosomal mapping can be used to ascertain the quality of assembled genomes.

Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).

BACKGROUND: Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase-based mechanism has been attributed to pyrethroid resistance and over-expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. RESULTS: In vitro interactions between putative synergists and CYP6CM1, B and Q-type esterases were investigated, and structure-activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B-type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q-esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. CONCLUSIONS: Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industry.

The interactions of piperonyl butoxide and analogues with the metabolic enzymes FE4 and CYP6CY3 of the green peach aphid Myzus persicae (Hemiptera: Aphididae).

BACKGROUND: Piperonyl butoxide (PBO) is a well-known insecticide synergist capable of interacting with phase 1 metabolic enzymes, specifically esterases and cytochrome P450s. In this study, structure-activity relationship analyses were used to characterise the interaction of around 30 analogues of PBO with the esterase FE4 and the P450 CYP6CY3 from insecticide-resistant Myzus persicae (Sulzer), in order to predict the synthesis of more potent inhibitors. RESULTS: Enzyme inhibition studies were performed against esterase and oxidase activities and, together with in silico modelling, key activity determinants of the analogues were identified and optimised. Novel analogues were then designed and synthesised, some of which showed greater inhibition against both enzymatic systems: specifically, dihydrobenzofuran moieties containing an alkynyl side chain and a butyl side chain against FE4, and benzodioxole derivatives with a propyl/butyl side chain and an alkynyl ether moiety for CYP6CY3. CONCLUSIONS: In vitro assays identified potential candidate synergists with high inhibitory potency. The in vivo confirmation of such results will allow consideration for a possible use in agriculture. © 2016 Society of Chemical Industry.

Use of the RFLP-PCR diagnostic test for characterizing MACE and kdr insecticide resistance in the peach potato aphid Myzus persicae.

The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids.

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.

Analysis of insecticide-resistant Myzus persicae (Sulzer) populations collected in Italian peach orchards.

Several populations of the green peach aphid, Myzus persicae (Sulzer), were collected in the most important Italian peach-growing areas. In these populations, collected as primary colonies or as secondary ones, total esterase content and acetylcholinesterase sensitivity towards pirimicarb was assessed using biochemical assays. Most of the individuals analysed generally exhibited an elevated esterase content, and in some cases acetylcholinesterase insensitivity. The implications of resistance levels and distribution on M persicae control strategies are discussed.

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.

Field control of Fusarium ear rot, Ostrinia nubilalis (Hübner), and fumonisins in maize kernels.

BACKGROUND: A 6 year study was conducted to evaluate the possible support of pesticides in mitigating mycotoxin contamination in maize grown in northern Italy. Different pesticides to control Ostrinia nubilalis (Hübner) (ECB), Fusarium verticillioides (Sacc.) Nirenb. and Aspergillus flavus Link were considered to check their efficacy and the best schedule to reduce fumonisin and aflatoxin contamination. RESULTS: Fumonisin B1 (FB1) contamination at harvest has been reduced by ECB control, while the addition of ingredients targeted to F. verticillioides gave a limited advantage, mainly with low FB1 contamination; nevertheless, fusaria control is relevant in high-risk conditions, when FB1 is likely to exceed the legal limit. The combination of fungal and pest control in a single spray at silk browning stage (BBCH 67) is the best way of application. These results are robust because they were obtained in a 6 year trial, with relevant differences in meteorological conditions, ECB and fungal presence and mycotoxin contamination. CONCLUSION: Insecticides have been shown to give advantages in their application, while the low FB1 contamination in the final years of the study did not lead to conclusive data on the advantage of adding a fungicide. Chemicals applied did not modify A. flavus incidence or aflatoxin contamination (only detected in trace amounts).