Peach-potato Aphid Myzus Persicae Research Articles

High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR

The peach–potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman ® MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.

Transgenic Tobacco Expressing Zephyranthes candida Agglutinin Showing Enhanced Resistance to Aphids

AbstractAphid is one of the most serious, sap‐sucking insect pests which cause significant losses of crop yields. The aim of this study is to investigate whether transgenic plants expressing Zephyranthes candida agglutinin (ZCA) could confer enhanced resistance to aphids. Tobacco was transformed with a plasmid, pCAMBIAZCA, containing the marker genes nptII and gusA and the Zephyranthes candida agglutinin gene (zca) via Agrobacterium tumefaciens‐mediated transformation. Twenty‐six independent transgenic plants were regenerated. Western blot analysis revealed ZCA expression at various levels in transgenic plants. Insect bioassay tests showed that transgenic plants expressing a high level of ZCA significantly inhibited the growth of the population of peach potato aphids (Myzus persicae Sulzer). This is the first report in which transgenic plants expressing ZCA conferred enhanced resistance to aphids. Our study suggests the zca gene could be a useful candidate for genetic engineering strategies in plants to provide aphid resistance.

DNA methylation in insects.

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri.

Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids.

Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.

Incidence of insecticide resistance alleles in sexually-reproducing populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae) from southern France.

Intensive chemical treatments have led to the development of a number of insecticide resistance mechanisms in the peach-potato aphid Myzus persicae (Sulzer). Some of these mechanisms are known to be associated with negative pleiotropic effects (resistance costs). Molecular and biochemical methods were used to determine the genotypes or phenotypes associated with four insecticide resistance mechanisms in single aphids from sexually-reproducing populations in southern France. The mechanisms considered were E4 and FE4 carboxylesterase overproduction, modified acetycholinesterase, and kdr and rdl resistance-associated mutations. A new method for determining individual kdr genotypes is presented. Almost all resistant individuals overproduced FE4 carboxylesterase, whereas modified acetylcholinesterase was rare. Both the kdr and rdl resistance mutations were present at high frequencies in French sexually-reproducing populations. The frequencies of insecticide resistance genes were compared before and after sexual reproduction in one peach orchard at Avignon to evaluate the potential impact of selection on the persistence of resistance alleles in the over-wintering phase. The frequencies of the kdr and rdl mutations varied significantly between autumn and spring sampling periods. The frequency of the kdr mutation increased, probably due to pyrethroid treatments at the end of the winter. Conversely, the frequency of the rdl mutation decreased significantly during winter, probably because of a fitness cost associated with this mutation.

Variation in response to neonicotinoid insecticides in peach-potato aphids, Myzus persicae (Hemiptera: Aphididae).

Laboratory bioassays applying the neonicotinoid insecticides imidacloprid, acetamiprid and nitenpyram against clones of the peach-potato aphid Myzus persicae (Sulzer) demonstrated that these compounds effectively circumvent the known carboxylesterase, modified acetylcholinesterase (MACE) and knock-down (kdr) insecticide resistance mechanisms in this species. However, some clones showed cross-tolerance (up to 18-fold) of these compounds relative to susceptible standards. A survey assessing the frequency of neonicotinoid tolerance in M persicae in the UK, based on samples collected from the field and glasshouses between 1997 and 2000, showed that such tolerance is still rare. Experiments on neonicotinoid-susceptible and -tolerant populations of M persicae under simulated field conditions in the laboratory showed that, although the latter were well controlled by imidacloprid applied at recommended application rates, they were more likely to survive and reproduce when this compound was applied at lower concentrations. Such conditions are probably periodically present in imidacloprid-treated field and glasshouse crops. Selection favouring tolerant forms of M persicae could lead to increases in their frequency and the evolution of more potent resistance to neonicotinoids.

Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae).

The effects of the hta gene encoding Helianthus tuberosus agglutinin (HTA) on an insect in the order Homoptera were investigated. Homologous cDNAs of hta-a, hta-b, hta-c and hta-d with CaMV35S as promoter were introduced into tobacco via Agrobacterium tumefaciens. Southern blot results showed that the exogenous hta gene was inserted into the genome of host plants, and northern blot analysis confirmed that hta was expressed in transgenic plants. A bioassay with peach-potato aphid (Myzus persicae) demonstrated that transgenic plants had deleterious effects on the insect. The average population of aphids fed on transgenic T0 plants during an 11-day assay decreased by 70%, compared controls. In transgenic plants of T1 generation, aphid fecundity inhibitions were 53.0% (hta-b) and 64.6% (hta-c), respectively. The development of aphids was notably retarded. We conclude that hta could be a novel and promising candidate for plant transgenic engineering against homopteran insect pests.

Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape

When administered in artificial diets, the cysteine protease inhibitor (PI) oryzacystatin I (OC-I) induced moderate but significant growth inhibition on the pea aphid ( Acyrthosiphon pisum Harris), the cotton/melon aphid ( Aphis Gossypii Glover) and the peach potato aphid ( Myzus persicae Sulzer). In transgenic oilseed rape plants ( Brassica napus L. cv. Drakkar) expressing OC-I under the control of the CaMV-35S RNA promoter, oryzacystatin was detected in both leaves and phloem sap. Transgenic plants from three independent homozygous lines were used to test the effect of in planta-expression of OC-I on M. persicae. Mean adult weight and fecundity were significantly reduced, and aphid biomass produced in 2 weeks was decreased by 25–40% for aphids fed transgenic plants, when compared to those fed control plants. The effects of OC-I on M. persicae were correlated with the decrease of a major cathepsin L/H-type cysteine protease activity, detected in whole insect extracts. Immuno-histological analysis showed OC-I labeling along the gut epithelium, but also its association with aphid bacteriocytes and oenocytes. These results suggest that OC-I affects M. persicae through digestive tract targets, but also by reaching the haemolymph, thereby inhibiting extra-digestive proteolytic activities and interacting with functions related to aphid reproduction. Overall, it appears that PIs can display deleterious effects against phloem-feeding insects in addition to their activity on leaf-feeding insects.

Interactions of aphid herbivory and nitrogen availability on the total foliar glycoalkaloid content of potato plants.

In plant growth room (PGR) and open-air pot (OAP) experiments, potato cvs King Edward and Maris Piper were grown under two nitrogen levels or two different nitrogen release patterns. Plants were subjected to infestation by peach potato aphids Myzus persicae (Homoptera: Aphididae). Total glycoalkaloid (GA) levels were measured in the foliage of both infested and non-infested plants, before, during and after aphid infestation. In the PGR experiment, aphid infestation reduced the amounts of total GAs in both cultivars. This reduction is attributed to the sugar deficiency induced in the plants owing to the dense aphid colonization. Results from the OAP experiment showed a temporal increase of GAs produced by potato cv. King Edward plants subjected to aphid infestation. Elevated amounts of nitrogen in the nutrient solutions (PGR experiment) reduced total GAs, while no differences were observed between manure and fertilizer treated plants (OAP experiment). It is concluded that the source of available nitrogen does not affect foliar GA synthesis in potatoes, and as a consequence, does not affect its endogenous chemical defense against insect herbivory. The case for insect-induced chemical defense mechanisms as triggered by low rates of aphid infestation is discussed.

The potential of some adjuvants in promoting infection with Verticillium lecanii: laboratory bioassays with Myzus persicae

Summary The potential of 14 adjuvants, including mineral oils, vegetable oils, stickers, spreaders, surfactants and nutrients, in promoting the virulence of the entomopathogenic fungus Verticillium lecanii were compared relative to a commercial formulation of the pathogen. The rate of infection over time with each adjuvant relative to the commercial formulation was tested using the peach potato aphid Myzus persicae. The proportion of infected aphids was modelled binomially with a logistic rate function assumed for the proportion infected over the time period of the experiment. Four of the adjuvant treatments, Codacide, skimmed milk, glycerol and Cutinol together with the commercial control had similar logistic slope parameters and were modelled using parallel regressions. The parallel regressions model showed significant differences between the intercepts with both glycerol and Cutinol significantly better than V. lecanii alone. Glycerol had a more positive intercept than Cutinol but the difference was not statistically significant. Although highly variable, the numbers of progeny produced by the treated aphids were significantly reduced by all the treatments relative to untreated control aphids. Glycerol and Cutinol were the only treatments that significantly reduced the number of progeny relative to the V. lecanii control.

The ups and downs of insecticide resistance in peach-potato aphids ( Myzus persicae) in the UK

The peach-potato aphid, Myzus persicae, provides an excellent demonstration of how genetic and ecological factors can interact to determine the dynamics of resistance and influence success of resistance management. The diversity of resistance mechanisms, based on both enhanced detoxification of insecticides and modifications to their target sites, that this species shows is now well understood at the genotypic, biochemical and molecular levels. These developments have yielded rapid and precise methods for detecting individual mechanisms, and for monitoring frequency changes in the presence and absence of insecticide selection. One factor influencing the prediction of these changes in the UK is the occurrence of prolonged periods of parthenogenesis in M. persicae, which results in non-random associations between particular resistance genes through the absence of genetic recombination. This has important implications for the rate at which resistance genes can accumulate in populations under selection by insecticides, and also for the speed at which they will decline when selection is relaxed. Although it is becoming apparent that resistance can be associated with various fitness costs, the relative contribution of individual resistance genes to these effects is still under investigation. However, the overall effect of the `ups’ and `downs’ of resistance in M. persicae, in the UK at least, has been to help maintain resistance phenotypes at manageable levels in most seasons. Unfortunately, the increasing genetic diversity of resistance in this species is progressively eroding the supply of effective insecticides. This in turn highlights the importance of ensuring sufficient chemical diversity to combat existing resistance mechanisms and to restrict the use of novel insecticides in order to sustain their effectiveness.

Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae.

Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%-94%). A similarity plot showed that sequences were particularly variable in the 50-100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important.

Characterisation of multiple α-bungarotoxin binding sites in the aphid Myzus persicae (Hemiptera: Aphididae)

The nicotinic antagonist α-bungarotoxin (α-BgTx) has been extensively used in studies of the vertebrate nicotinic acetylcholine receptor (nAChR) and is used here to investigate putative nicotinic acetylcholine receptors of the peach potato aphid Myzus persicae. Saturable binding is consistent with the presence of both high and low affinity binding sites for [ 125I]-α-BgTx, with dissociation constants of 1.2 and 34 nM, and maximal binding capacities of 167 and 640 fmol mg −1, respectively, with a Hill value of 0.71. Displacement studies with various nicotinic ligands, including neonicotinoid insecticides, indicate that the pharmacology of the high affinity [ 125I]-α-BgTx binding site of M. persicae is similar to that of α-BgTx sites in Apis mellifera and Manduca sexta. However, low Hill values in the displacement studies suggest the presence of either multiple receptor sub-types or cooperativity between nicotinic binding sites on the same nAChR. Isotopic dissociation of [ 3H]-α-BgTx initiated by 1μM methyllycaconitine was accelerated and biphasic in character in contrast to monophasic dissociation initiated by imidacloprid, epibatidine or α-BgTx itself. This is consistent with a model in which there is allosteric interaction between at least two nicotinic binding sites per aphid nAChR.

Tri-trophic interactions involving pest aphids, predatory 2-spot ladybirds and transgenic potatoes expressing snowdrop lectin for aphid resistance

Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin- expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata[L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2–3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.

Concanavalin A inhibits development of tomato moth (Lacanobia oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants

The effects of concanavalin A (ConA), a glucose/mannose-specific lectin from jackbean (Canavalia ensiformis), on insect crop pests from two different orders, Lepidoptera and Homoptera, were investigated. When fed to larvae of tomato moth (Lacanobia oleracea) at a range of concentrations (0.02–2.0% of total protein) in artificial diet, ConA decreased survival, with up to 90% mortality observed at the highest dose level, and retarded development, but had only a small effect on larval weight. When fed to peach-potato aphids (Myzus persicae) at a range of concentrations (1–9μM) in liquid artificial diet, ConA reduced aphid size by up to 30%, retarded development to maturity, and reduced fecundity (production of offspring) by >35%, but had little effect on survival. With both insects, there was a poor correlation between lectin dose and the quantitative effect. Constitutive expression of ConA in transgenic potatoes driven by the CaMV 35S promoter resulted in the protein accumulating to levels lower than predicted, possibly due to potato not being able to adequately reproduce the post-translational processing of this lectin which occurs in jackbean. However, the expressed lectin was functionally active as a haemagglutinin. Bioassay of L. oleracea larvae on ConA-expressing potato plants showed that the lectin retarded larval development, and decreased larval weights by >45%, but had no significant effect on survival. It also decreased consumption of plant tissue by the larvae. In agreement with the diet bioassay results, ConA-expressing potatoes decreased the fecundity of M. persicae by up to 45%. ConA thus has potential as a protective agent against insect pests in transgenic crops.

The evolution of insecticide resistance in the peach–potato aphid, Myzus persicae

The peach–potato aphid Myzus persicae (Sulzer) can resist a wide range of insecticides, but until recently (1990) the only mechanism identified was the increased production of carboxylesterases (E4 or FE4), which cause enhanced degradation and sequestration of insecticidal esters. We have now identified two forms of target–site resistance involving changes in the acetylcholinesterase ( AChE ) and sodium channel ( kdr ) genes. Biochemical and DNA diagnostic methods can be used to identify all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. Amplified genes underlie the increased production of esterases but their expression is modulated by DNA methylation. Amplification of the E4 gene is in strong linkage disequilibrium with the kdr mechanism. This may reflect strong insecticidal selection favouring aphids with multiple mechanisms, tight chromosomal linkage and/or the prominence of parthenogenesis in many M. persicae populations. The decreased fitness of resistant aphids under winter conditions may be a consequence of the altered sodium–channel gene affecting behaviour and/or the perception of external stimuli.

Correction: Pestic. Sci. 53, 201–208 (1998)

In the paper ‘Proinsecticides effective against insecticide-resistant peach-potato aphid (Myzus persicae (Sulzer)),’ by D. Hedley et al., in Table 3 the units of both columns headed LC50 should be (mg litre−1)

3H]Imidacloprid Labels High- and Low-Affinity Nicotinic Acetylcholine Receptor-like Binding Sites in the AphidMyzus persicae(Hemiptera: Aphididae)

The nitroguanidine insecticide imidacloprid (IMI) is known to act as an agonist at the insect nicotinic acetylcholine receptor (nAChR). Saturable binding of [3H]IMI to membranes of the peach potato aphidMyzus persicae(R1 strain) revealed the presence of both high- and low-affinity [3H]IMI binding sites having dissociation constants and maximal binding capacities of 0.14 nM and 284 fmol/mg and 12.6 nM and 485 fmol/mg, respectively, with a Hill value of 0.61. Displacement of 500 pM [3H]IMI binding to aphid membranes by the nicotinic ligands acetylcholine, (−)-nicotine, αbungarotoxin (α-BGTX), (±)-epibatidine, methyllycaconitine (MLA) and IMI gave IC50values (nM) of 2390, 643, 62.4, 21.9, 15.1, and 1.1, respectively. The pharmacology of the aphid high-affinity [3H]IMI binding site thus resembles that of nicotinic acetylcholine receptors. Low Hill values for ACh, α-BGTX, and MLA displacement suggest either that high-affinity [3H]IMI binding occurs at multiple sites which have differing affinities for ACh, α-BGTX, or MLA or that binding to one high-affinity site is allosterically influenced by binding to another site on the same putative nAChR. Saturable [3H]IMI binding was analyzed in a panel of insects, namely,Nephotettix cincticeps(Hemiptera: Cicadellidae),Periplaneta americana(Dictyoptera: Blattidae),Lucilia sericata(Diptera: Calliphoridae),Drosophila melanogaster(Canton S) (Diptera: Drosophiloidea),Manduca sexta(Lepidoptera: Sphingidae),Heliothis virescens(Lepidoptera: Noctuidae), andCtenocephalides felis(Siphonaptera: Pulicidae). The results demonstrate that of these, only aphids and green leaf hoppers (both Hemiptera) have very high-affinity binding sites for [3H]IMI in addition to the lower affinity sites that are also seen in insects from other orders, which in these insects haveKdvalues of about 1 nM and Hill values of approximately 1. The presence of very high-affinity [3H]IMI binding sites only in the hemipteran insects studied may explain why IMI is particularly useful in controlling sucking pests.

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae.

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.

Long-term effects of elevated CO2 and temperature on populations of the peach potato aphid Myzus persicae and its parasitoid Aphidius matricariae.

Model terrestrial ecosystems were set-up in the Ecotron controlled environment facility. The effects of elevated CO2 (ambient + 200 [Formula: see text]mol/mol) and temperature (ambient + 2.0°C) on plant chemistry, the abundance of the peach potato aphid Myzus persicae, and on the performance of one of its parasitoids Aphidius matricariae, were studied. Total above-ground plant biomass at the end of the experiment was not affected by elevated atmospheric CO2, nor were foliar nitrogen and carbon concentrations. Elevated temperature decreased final plant biomass while leaf nitrogen concentrations increased. Aphid abundance was enhanced by both the␣CO2 and temperature treatment. Parasitism rates remained unchanged in elevated CO2, but showed an increasing trend in conditions of elevated temperature. Our results suggest that M. persicae, an important pest of many crops, might increase its abundance under conditions of climate change.