Cloning and analysis of the esterase genes conferring insecticide resistance in the peach-potato aphid, Myzus persicae (Sulzer).
Ontology highlight
ABSTRACT: Full-length cDNA clones encoding the esterases (E4 and FE4) that confer insecticide resistance in the peach-potato aphid [Myzus persicae (Sulzer)] were isolated and characterized. The E4 cDNA contained an open reading frame of 1656 nucleotides, coding for a protein of 552 amino acids. The FE4 cDNA shared 99% identity with E4 over this region, the most important difference being a single nucleotide substitution resulting in the FE4 mRNA having an extra 36 nucleotides at the 3' end. The derived amino acid sequences for the N-terminus of E4 and FE4 were identical, with the first 23 residues being characteristic of a signal peptide and the next 40 residues being an exact match to the N-terminal sequence determined by Edman degradation of both purified proteins. The predicted molecular masses of 58.8 and 60.2 kDa for the E4 and FE4 polypeptides were consistent with those previously observed by in vitro translation of mRNA. Five potential N-linked glycosylation sites were present in both polypeptides, in accordance with earlier evidence that the native esterases are glycoproteins. Comparison of the aphid esterase protein sequences with other serine hydrolases provided evidence that their activity involves a charge-relay system with a catalytic triad the same as that found in acetylcholinesterase. Restriction mapping and sequencing of cloned genomic DNA showed that the E4 gene is spread over 4.3 kb with six introns and that the previously reported differences between the 3' ends of the E4 and FE4 genes result from single nucleotide substitutions and not gross differences in the DNA sequences.
Project description:The amplification of genes encoding an insecticide-detoxifying esterase (E4) in the peach-potato aphid Myzus persicae is one of the few examples where this genetic phenomenon has been shown to be involved in the response of an intact higher organism to artificial selection. Here we report quantitative and qualitative studies of the repeat units (amplicons) containing the E4 genes in a highly resistant aphid clone. Initial studies to quantify esterase sequences showed a 5-11-fold increase in resistant aphids compared with susceptible aphids, suggesting the presence of 10-22 gene copies per diploid genome. A more incisive analysis by pulsed-field gel electrophoresis confirmed the presence of about 12 copies of the E4 gene and showed them to be on about 24 kb amplicons, arranged as a tandem array of direct repeats. This, together with previous results from crossing experiments and with recent in situ hybridization studies, confirms that the E4 gene amplification in this aphid clone is heterozygous at a single locus. However, these data show that the gene amplification alone cannot account for the approx. 60 times higher levels of E4 protein and its mRNA present in this aphid clone, and therefore resistance must involve changes in both esterase gene copy number and gene expression.
Project description:The peach potato aphid, Myzus persicae (Sulzer), is a worldwide pest of many crops, and the most important aphid pest of peach and potato crops in Tunisia, mainly due to virus transmission, for which insecticides are frequently applied. We studied the genetic structure of M. persicae populations in Tunisia, in order to further our understanding of the biotic and abiotic factors shaping populations and to predict their evolutionary responses to the present management practices. We monitored peach orchards and seed potato crops in different seasons and regions from 2011-2013 and in 2016 (19 populations), assessing the genetic diversity of M. persicae at six microsatellite loci. Temporal and spatial changes in the frequency and distribution of 397 genotypes in 548 sampled aphids were studied. Only 37 genotypes were found more than once (clonal amplification), as most genotypes were found only once (91.60% in peach; 88.73% in potato crops). A similarly high genetic diversity was observed in aphids sampled from peach (G/N = 0.76; Ho = 0.617) and potato (G/N = 0.70; Ho = 0.641). Only a weak genetic differentiation among populations was found, mainly between geographic locations. Clustering analysis revealed genotypes to be grouped mainly according to host plant. The availability of the primary host, high proportion of unique genotypes, high genetic diversity and lack of structuring suggest that the aphid reproduces mainly through cyclical parthenogenesis in Tunisia. On the other hand, we provide a farm-scale study that shows how easily M. persicae can colonize different areas and hosts, which may have important implications in relation to plant virus vectoring.
Project description:Carboxylesterases from different strains of Myzus persicae were examined to try to understand their contribution to insecticide resistance. Preliminary evidence that they are involved comes from the good correlation between the degree of resistance and the carboxylesterase and paraoxon-degrading activity in aphid homogenates. Furthermore the carboxylesterase associated with resistance could not be separated from the insecticide-degrading enzyme by electrophoresis or ion-exchange chromatography. Homogenates of resistant aphids hydrolysed paraoxon 60 times faster than did those of susceptible aphids, yet the purified enzymes from both sources had identical catalytic-centre activities towards this substrate and also towards naphth-1-yl acetate, the latter being hydrolysed by both 2x10(6) times faster than paraoxon. These observations provide evidence that the enzyme from both sources is identical, and that one enzyme hydrolyses both substrates. This was confirmed by relating the rate of paraoxon hydrolysis to the rate at which paraoxon-inhibited carboxylesterase re-activated. Both had the same first-order rate constant (0.01min(-1)), showing clearly that the hydrolysis of both substrates is brought about by the same enzyme. Its K(m) for naphth-1-yl acetate was 0.131mm, and for paraoxon 75pm. The latter very small value could not be measured directly, but was calculated from substrate-competition studies coupled with measurements of re-activation of the diethyl phosphorylated enzyme. Since the purified enzymes from resistant and susceptible aphids had the same catalytic-centre activity, the 60-fold difference between strains must be caused by different amounts of the same enzyme resulting from mutations of the regulator gene(s) rather than of the structural gene.
Project description:The green peach aphid/peach-potato aphid Myzus persicae can colonize hundreds of plant species, an ability that is in part due to the delivery of saliva proteins – often referred to as effectors – into the host plant that suppress plant defence. As a generalist herbivore with a remarkable ability to colonize new host plants M. persicae represents an outstanding model system for studying the molecular mechanisms underlying plant-insect interactions. Recent advancements in mass spectrometry instrumentation and database search software along with a new high-quality reference genome assembly for M. persicae and a simplified method for improved aphid saliva recovery, collectively enhance the detection of saliva proteins with unprecedented sensitivity and specificity.
Project description:Green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)] is a significant pest with a known history of insecticide resistance. Neonicotinoids could manage this pest; however, their frequent use led to the evolution of resistance in field populations of M. persicae. Toxicity data for neonicotinoid insecticides synergized with pipernyl butoxide (PBO) in a field population (FP) were collected and compared to a laboratory susceptible clone (SC) of aphids. The enhanced expression of metabolic resistance-related cytochrome P450 gene CYP6CY3 and an arginine-threonine substitution were detected in FP, causing a single point mutation (R81T) at β1 subunit of nicotinic acetylcholine receptor (nAChR) within D loop. High level of resistance to imidacloprid was developed in FP with 101-fold resistance ratio and moderate resistance level (10.9-fold) to acetamiprid. The results of PBO synergized bioassay suggested that cytochrome P450 enzymes were involved in the resistance to neonicotinoids. The mRNA transcriptional level of CYP6CY3 gene was significantly higher (3.74 fold) in FP compared to SC. The R81T mutation associated with neonicotinoid resistance had 26% resistant allele frequency in FP. Both P450 enzymes and R81T mutation of nAChR were found in field-evolved neonicotinoid resistance. It is concluded that field-evolved resistance in green peach aphid could be managed by using appropriate synergists such as PBO.
Project description:Insecticide resistance in the aphid Myzus persicae results primarily from the amplification of genes encoding the insecticide-detoxifying esterase, E4. Here we report the analysis of flanking DNA co-amplified with the E4 gene. The 5' end of this gene has an untranslated leader sequence interspersed by two introns, and the promoter region lacks TATA and CAAT boxes. The DNA breakpoint involved in the generation of the amplification is just upstream (approx. 250 bp) of the putative E4 transcription start site; thus the E4 gene is very close to the 5' end of the approx. 24 kb amplicon. PCR primers specific to the 'novel joint' generated during the amplification have been used to show that a wide range of aphid clones have the same amplicons, arranged as a series of head-to-tail direct repeats. Long-distance mapping has revealed the structure of these repeats. This has important implications for understanding both the generation of the amplified genes and the origin and spread of insecticide resistance in M. persicae.
Project description:BACKGROUND: Insecticide resistance is one of the best examples of rapid micro-evolution found in nature. Since the development of the first synthetic insecticide in 1939, humans have invested considerable effort to stay ahead of resistance phenotypes that repeatedly develop in insects. Aphids are a group of insects that have become global pests in agriculture and frequently exhibit insecticide resistance. The green peach aphid, Myzus persicae, has developed resistance to at least seventy different synthetic compounds, and different insecticide resistance mechanisms have been reported worldwide. METHODOLOGY/PRINCIPAL FINDINGS: To further characterize this resistance, we analyzed genome-wide transcriptional responses in three genotypes of M. persicae, each exhibiting different resistance mechanisms, in response to an anti-cholinesterase insecticide. The sensitive genotype (exhibiting no resistance mechanism) responded to the insecticide by up-regulating 183 genes primarily ones related to energy metabolism, detoxifying enzymes, proteins of extracellular transport, peptidases and cuticular proteins. The second genotype (resistant through a kdr sodium channel mutation), up-regulated 17 genes coding for detoxifying enzymes, peptidase and cuticular proteins. Finally, a multiply resistant genotype (carrying kdr and a modified acetylcholinesterase), up-regulated only 7 genes, appears not to require induced insecticide detoxification, and instead down-regulated many genes. CONCLUSIONS/SIGNIFICANCE: This study suggests strongly that insecticide resistance in M. persicae is more complex that has been described, with the participation of a broad array of resistance mechanisms. The sensitive genotype exhibited the highest transcriptional plasticity, accounting for the wide range of potential adaptations to insecticides that this species can evolve. In contrast, the multiply resistant genotype exhibited a low transcriptional plasticity, even for the expression of genes encoding enzymes involved in insecticide detoxification. Our results emphasize the value of microarray studies to search for regulated genes in insects, but also highlights the many ways those different genotypes can assemble resistant phenotypes depending on the environmental pressure.
Project description:BackgroundAmong herbivorous insects that have exploited agro-ecosystems, the peach-potato aphid, Myzus persicae, is recognized as one of the most important agricultural pests worldwide. Uses over 400 plant species and has evolved different insecticides resistance mechanisms. As M. persicae feeds upon a huge diversity of hosts, it has been exposed to a wide variety of plant allelochemicals, which probably have promoted a wide range of detoxification systems.Methodology/principal findingsIn this work we (i) evaluated whether insecticide resistance mutations (IRM) in M. persicae can give an advantage in terms of reproductive fitness when aphids face two hosts, pepper (Capsicum annuum) a suitable host and radish (Raphanus sativus) the unfavorable host and (ii) examined the transcriptional expression of six genes that are known to be up-regulated in response to insecticides. Our results show a significant interaction between host and IRM on the intrinsic rate of increase (r(m)). Susceptible genotypes (not carrying insensitivity mutations) had a higher r(m) on pepper, and the transcriptional levels of five genes increased on radish. The r(m) relationship was reversed on the unfavorable host; genotypes with multiple IRM exhibited higher r(m), without altering the transcriptional levels of the studied genes. Genotypes with one IRM kept a similar r(m) on both hosts, but they increased the transcriptional levels of two genes.Conclusions/significanceAlthough we have studied only nine genotypes, overall our results are in agreement with the general idea that allelochemical detoxification systems could constitute a pre-adaptation for the development of insecticide resistance. Genotypes carrying IRM exhibited a higher r(m) than susceptible genotypes on radish, the more unfavorable host. Susceptible genotypes should be able to tolerate the defended host by up-regulating some metabolic genes that are also responding to insecticides. Hence, our results suggest that the trade-off among resistance mechanisms might be quite complex, with a multiplicity of costs and benefits depending on the environment.
Project description:cDNA clones for the esterase (E4) responsible for broad insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) were isolated and used to study the molecular basis of resistance. Increased esterase synthesis by resistant aphids was found to be associated with amplification of the structural gene for the esterase (E4 or its closely related variant, FE4), the degree of amplification being correlated with the activity of the esterase and the level of resistance. Hybridization of the cDNA clones to genomic Southern blots showed that only some of the esterase-related restriction fragments are amplified. Qualitative differences between restriction patterns in different clones of resistant aphids correlated with the presence or absence of a specific chromosome translocation and with production of E4 or FE4.
Project description:BackgroundAphid attack induces defense responses in plants activating several signaling cascades that led to the production of toxic, repellent or antinutritive compounds and the consequent reorganization of the plant primary metabolism. Pepper (Capsicum annuum L.) leaf proteomic response against Myzus persicae (Sulzer) has been investigated and analyzed by LC-MS/MS coupled with bioinformatics tools.ResultsInfestation with an initially low density (20 aphids/plant) of aphids restricted to a single leaf taking advantage of clip cages resulted in 6 differentially expressed proteins relative to control leaves (3 proteins at 2 days post-infestation and 3 proteins at 4 days post-infestation). Conversely, when plants were infested with a high density of infestation (200 aphids/plant) 140 proteins resulted differentially expressed relative to control leaves (97 proteins at 2 days post-infestation, 112 proteins at 4 days post-infestation and 105 proteins at 7 days post-infestation). The majority of proteins altered by aphid attack were involved in photosynthesis and photorespiration, oxidative stress, translation, protein folding and degradation and amino acid metabolism. Other proteins identified were involved in lipid, carbohydrate and hormone metabolism, transcription, transport, energy production and cell organization. However proteins directly involved in defense were scarce and were mostly downregulated in response to aphids.ConclusionsThe unexpectedly very low number of regulated proteins found in the experiment with a low aphid density suggests an active mitigation of plant defensive response by aphids or alternatively an aphid strategy to remain undetected by the plant. Under a high density of aphids, pepper leaf proteome however changed significantly revealing nearly all routes of plant primary metabolism being altered. Photosynthesis was so far the process with the highest number of proteins being regulated by the presence of aphids. In general, at short times of infestation (2 days) most of the altered proteins were upregulated. However, at longer times of infestation (7 days) the protein downregulation prevailed. Proteins involved in plant defense and in hormone signaling were scarce and mostly downregulated.