Mutation Report for: A201S_pluxy-ACHE1

BACKGROUND: The fall armyworm (FAW), Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), is now an economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. Variations in sub-population genetics and the processes of rapid adaptation underpinning the invasion remain unclear. For this, the genetic identity and diversity of FAW populations in Uganda were revealed by sequencing 87 samples (collected across the country). Based on the partial mitochondrial cytochrome oxidase I (COI) gene polymorphisms, we further examined the mitochondrial haplotype configuration and compared the FAW in Uganda with sequences from other parts of the world. The molecular target for organophosphate and carbamate resistance, acetylcholinesterase, was also investigated. RESULTS: Analysis of the partial COI gene sequences showed the presence of both rice (predominant) and corn strain haplotypes, with a haplotype diversity of 0.382. Based on the COI marker, pairwise difference distribution analyses, and neutrality tests, showed that the FAW populations in Uganda and the rest of Africa are evolving neutrally, but those in America and Asia are undergoing expansion. Our findings support observations that invasive FAW populations throughout the rest of Africa and Asia share a common origin. Sequencing of the S. frugiperda ace-1 gene revealed four amino acid substitutions, two of which (A201S and F290V) were previously shown to confer organophosphate resistance in both S. frugiperda and several other insect species. The other two previously reported new variations in positions g-396 and g-768, are presumed to be related to the development of insecticide resistance. CONCLUSIONS: This research has increased our knowledge of the genetics of FAW in Uganda, which is critical for pest surveillance and the detection of resistance. However, due to the low gene polymorphism of COI, more evolutionary studies incorporating the Spodoptera frugiperda whole-genome sequence are required to precisely understand the FAW population dynamics, introduction paths, origin, and subsequent spread.

Acetylcholinesterase-1 (AChE1) is a vital enzyme involved in neurotransmission and represents an attractive insecticide-target for organophosphates and carbamates in Plutella xylostella (Linneaus), an important pest of cruciferous crops worldwide. However, insecticide-resistance often occurs due to mutations, making many organophosphates and carbamates ineffective. In particular, A298S and G324A mutations in AChE1 significantly lower the binding affinity of insecticides. In the present study, the wild-type and mutant AChE1 structures were constructed and their structural stabilities, residual flexibilities were investigated through molecular dynamics simulations. Subsequently, the structural and energetic changes responsible for the insecticide-resistance in AChE1 were analyzed using molecular docking. The results of molecular dynamics simulation showed that the mutant AChE1 shows little structural deviation than the wild-type, indicate the structural instability. Furthermore, the docking results demonstrated that these mutations break the intermolecular hydrogen bonding interactions and thereby affect the prothiofos as well as all insecticide binding. Hence, the results could provide some insights into the resistance mechanism of AChE1 in insecticides binding and helpful in the development of novel insecticides that are less susceptible to insecticide-resistance.

Diamondback moth (Plutella xylostella L.) causes enormous damage on cruciferous vegetables and can rapidly develop resistance to all kinds of insecticides. To effectively manage the insecticide resistance of P. xylostella, an understanding of its inheritance and stability is essential. Here we investigated the phenotypic and genotypic basis of mevinphos resistance by crossing two genetically pure lines of P. xylostella, an SHggt wild-type strain and an SHMTCN resistant strain carrying 892T/T, 971C/C, and 1156T/G (TCN) mutations of the acetylcholinesterase 1 gene (Pxace1). Similar median lethal concentrations and degrees of dominance in the reciprocal cross progeny, and no plateau on the log concentration-probit line of F1 backcross and self-cross progeny, suggest that the mevinphos-resistance in P. xylostella is inherited as an autosomal and incomplete dominant trait governed by more than one gene. In the absence of mevinphos exposure, the resistance ratio and Pxace1 mutation frequency declined concomitantly in the SHMTCN strain. After 20-generation relaxation, the mevinphos resistance decreased from 52- to 6-fold and the Pxace1 mutation frequency of the TCN haplotype pair decreased from 100% to 0%. A good correlation was found between the resistance ratio and TCN frequency within the range of 12.5- to 25-fold resistance. Since there was no TCN haplotype pair detected below a resistance level of 12.5-fold, we speculate that resistance mechanisms other than target site insensitivity may exist. These observations are important for the prediction and management of mevinphos and related organophosphate resistance in field populations of P. xylostella.

Fitness costs associated with resistance to insecticides have been well documented, usually at normal temperature conditions, in many insect species. In this study, using chlorpyrifos-resistant homozygote (RR) and chlorpyrifos-susceptible homozygote (SS) of resistance ace1 allele of Plutella xylostella (DBM), we confirmed firstly that high temperature experience in pupal stage influenced phenotype of wing venation in insecticide-resistant and insecticide-susceptible Plutella xylostella, and SS DBM showed significantly higher thermal tolerance and lower damages of wing veins under heat stress than RR DBM. As compared to SS DBM, RR DBM displayed significantly lower AChE sensitivity to chlorpyrifos, higher basal GSTs activity and P450 production at 25 degrees C, but higher inhibitions on the enzyme activities and P450 production as well as reduced resistance to chlorpyrifos under heat stress. Furthermore, RR DBM displayed significantly higher basal expressions of hsp69s, hsp72s, hsp20,hsp90,Apaf-1, and caspase-7 at 25 degrees C, but lower induced expressions of hsps and higher induced expressions of Apaf-1,caspase-9, and caspase-7 under heat stress. These results suggest that fitness costs of chlorpyrifos resistance in DBM may partly attribute to excess consumption of energy caused by over production of detoxification enzymes and hsps when the proteins are less demanded at conducive environments but reduced expressions when they are highly demanded by the insects to combat environmental stresses, or to excess expressions of apoptotic genes under heat stress, which results in higher apoptosis. The evolutionary and ecological implications of these findings at global warming are discussed.

This study examined the acetylcholinesterase 1 gene (AChE1) in Plutella xylostella strains with different sensitivities to acephate. Multiple haplotypes of the gene were found in the field-collected strains including distinct haplotypes carrying one or both previously reported mutations (A298S and G324A). Moreover, sequencing results indicated the presence of duplicated copies of the gene in the field-collected strains. No correlation was found between copy numbers of AChE1 and levels of resistance to acephate suggesting that extensive AChE1 duplication is not a major resistance factor at least in some P. xylostella strains. Proportions of the A298S and G324A mutations showed no correlation with levels of resistance to acephate. This suggests that acephate resistance of P. xylostella is complex and cannot be evaluated based on the AChE1 copy number or proportions of the resistance mutations alone.

The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field.

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.

Insensitive acetylcholinesterase (AChE) was determined to be primarily involved in a prothiofos-resistant (PR) strain of diamondback moth (DBM, Plutella xylostella L.), as judged by the AChE inhibition assay using paraoxon, where the PR strain exhibited ca. 26-fold increased I50 value. Extensive sequence analysis of the previously reported ace2-type DBM AChE gene revealed no difference between the susceptible and PR strains. To elucidate the molecular basis of the prothiofos resistance mechanism mediated by insensitive AChE, we cloned and characterized a second AChE gene from DBM. The deduced amino acid sequence of the novel AChE showed the highest homology to ace1, the second copy of insect ace, and was determined in fact as the predominant AChE in DBM compared to the ace2-type with 13- to 250-fold higher transcription levels depending on different tissues. Sequence comparison of the ace1-type cDNA between the susceptible and PR strains of DBM revealed that a total of three amino acid substitutions are closely associated with the PR strain. Among these, the Gly227Ala mutation, exclusively present in the PR strain, was located at the same position of the organophosphate resistance-conferring Gly-to-Ala mutation on the ace2 of the fruit fly and house fly. This finding suggests that the Gly227Ala mutation along with two other ones on the ace1 are likely responsible for the AChE insensitivity in DBM.