Frequencies of organophosphate resistance-associated mutations in the acetylcholinesterase gene of field collected olive fly (Bactrocera oleae) populations under different insecticide regimes

In the present study, the frequencies of three organophosphate (OP) resistance-associated mutations in acetylcholinesterase gene of Bactrocera oleae (BoAce) populations collected from 8 different important olivegrowing areas in the west part of Turkey were determined. Populations were sampled from the areas that have been treated with only the pyrethroid α-cypermethrin; pyrethroids plus OPs; deltamethrin with pheromone eco-traps, and no insecticide treatment applied areas for many years. For Ile214Val and Gly488Ser point mutations PCR-RFLP and for Δ3Q deletion mutation PCR diagnostic tests were carried out. Seventy-two percent of the total individuals analyzed in the study were exhibited heterozygous genotype (RS) for both Ile214Val and Gly488Ser point and homozygous susceptible genotype (SS) for Δ3Q deletion mutations. This RS/RS/SS combination together with RS/RR/SS with the frequency of 13% were the most common two combinations observed in all of the populations under different insecticide regimes, even in the populations under no insecticide pressure for many years. Independent evaluation of the three mutations resulted in 0.450, 0.534 and 0.037 frequency values for the resistant alleles of 214Val, 488Ser and Δ3Q mutations, respectively. Among the studied populations, the frequencies of resistant alleles for the positions of 214 and 488 were not differed from each other. However, in 3 of the populations the frequency of the R allele of Δ3Q was zero and it changed between 0.025 and 0.100 in the remaining five populations. Results of this study contributed to the distribution pattern of the two point mutations in Europe and a pattern for Δ3Q mutation was determined for the first time in the field collected olive fly samples.     

Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion

Alterations of the structure and activity of the enzyme acetylcholinesterase (AChE) leading to resistance to organophosphate insecticides have been examined in the oriental fruit fly, Bactrocera dorsalis (Hendel), an economic pest of great economic importance in the Asia-Pacific region. We used affinity chromatography to purify AChE isoenzymes from heads of insects from lines showing the phenotypes of resistance and sensitivity to insecticide treatments. The AChE enzyme from a strain selected for resistance to the insecticide fenitrothion shows substantially lower catalytic efficiency for various substrates and 124-, 373- and 5810-fold less sensitivity to inhibition by paraoxon, eserine and fenitroxon, respectively, compared to that of the fenitrothion susceptible line. Using peptide mass fingerprinting, we also show how specific changes in the structure of the AChE enzymes in these lines relate to the resistant and sensitive alleles of the AChE (ace) gene characterized previously in this species (described in Hsu, J.-C., Haymer, D.S., Wu, W.-J., Feng, H.-T., 2006. Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem. Mol. Biol. 36, 396-402). Polyclonal antibodies specific to the purified isoenzymes and real-time PCR were also used to show that both the amount of the isoenzyme present and the expression levels of the ace genes were not significantly different between the R and S lines, indicating that quantitative changes in gene expression were not significantly contributing to the resistance phenotype. Overall, our results support a direct causal relationship between the mutations previously identified in the ace gene of this species and qualitative alterations of the structure and function of the AChE enzyme as the basis for the resistance phenotype. Our results also provide a basis for further comparisons of insecticide resistance phenomena seen in closely related species, such as Bactrocera oleae, as well as in a wide range of more distantly related insect species.     

A molecular marker associated with the H21 Hessian fly resistance gene in wheat

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (Coker 797 * 4 / Hamlet) and differed by the presence or absence of H21 (on 2RL) derived from Chaupon rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.     

Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides

Mutations in the gene encoding the enzyme acetylcholinesterase (AChE) of the oriental fruit fly, Bactrocera dorsalis, associated with resistance to an organophosphorus insecticide have been characterized. Three point mutations producing nonsynonymous changes in the predicted amino acid sequence of the product of the B. dorsalis ace gene in resistant vs. susceptible flies have been identified. One of these changes is unique to B. dorsalis while the other two occur at sites that are identical to mutations previously described for another Bactrocera species. Although the precise role of the third mutation is not clearly established, the independent origin of two identical alterations in these two species strongly supports the idea proposed previously that molecular changes associated with insecticide resistance in key genes and enzymes such as AChE are largely constrained to a limited number of sites. The results obtained here also suggest that the widespread use of organophosphorus insecticides will likely lead to a predictable acquisition of resistance in wild populations of B. dorsalis as well as other pest species. For surveys of B. dorsalis populations that may develop resistance, diagnostic tests using PCR-RFLP based methods for detecting the presence of all three mutations in individual flies are described.     

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

A novel wheat gene encoding a putative chitin-binding lectin is associated with resistance against Hessian fly

The gene-for-gene interaction triggering resistance of wheat against first-instar Hessian fly larvae utilizes specialized defence response genes not previously identified in other interactions with pests or pathogens. We characterized the expression of Hfr-3 , a novel gene encoding a lectin-like protein with 68–70% identity to the wheat germ agglutinins. Within each of the four predicted chitin-binding hevein domains, the HFR-3 translated protein sequence contained five conserved saccharide-binding amino acids. Quantification of Hfr-3 mRNA levels confirmed a rapid response and gradual increase, up to 3000-fold above the uninfested control in the incompatible interaction 3 days after egg hatch. Hfr-3 mRNA abundance was influenced by the number of larvae per plant, suggesting that resistance is localized rather than systemic. In addition, Hfr-3 was responsive to another sucking insect, the bird cherry-oat aphid, but not to fall armyworm attack, wounding or exogenous application of methyl jasmonate, salicylic acid or abscisic acid. Western blot analysis demonstrated that HFR-3 protein increased in parallel to mRNA levels in crown tissues during incompatible interactions. HFR-3 protein was detected in both virulent and avirulent larvae, indicating ingestion. Anti-nutritional proteins, such as lectins, may be responsible for the apparent starvation of avirulent first-instar Hessian fly larvae during the initial few days of incompatible interactions with resistant wheat plants.     

Mutations in the Bemisia tabaci para sodium channel gene associated with resistance to a pyrethroid plus organophosphate mixture

The voltage-gated sodium channel is the primary target site of pyrethroid insecticides. In some insects, super knockdown resistance (super-kdr) to pyrethroids is caused by point mutations in the linker fragment between transmembrane segments 4 and 5 of the para-type sodium channel protein domain II (IIS4-5). Here, we identify two mutations in the IIS4-5 linker of the para-type sodium channel of the whitefly, Bemisia tabaci: methionine to valine at position 918 (M918V) and leucine to isoleucine at position 925 (L925I). Although each mutation was isolated independently from strains >100-fold resistant to a pyrethroid (fenpropathrin) plus organophosphate (acephate) mixture, only L925I was associated with resistance in strains derived from the field in 2000 and 2001. The L925I mutation occurred in all individuals from nine different field collections that survived exposure to a discriminating concentration of fenpropathrin plus acephate. Linkage analysis of hemizygous male progeny of unmated heterozygous F1 females (L925I×wild-type) shows that the observed resistance is tightly linked to the voltage-gated sodium channel locus. The results provide a molecular tool for better understanding, monitoring and managing pyrethroid resistance in B. tabaci.     

A high level of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits

Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in "hot spots" in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.     

Molecular analysis of the fungal microbiome associated with the olive fruit fly Bactrocera oleae

A molecular approach was used to investigate the fungal microbiome associated with Bactrocera oleae a major key pest of Olea europea, using the ITS2 region of the ribosomal DNA (rDNA) as barcode gene. Amplicons were cloned and a representative number of sequenced fragments were used as barcode genes for the identification of fungi. The analysis of the detected sequence types (STs) enabled the identification of a total of 34 phylotypes which were associated with 10 fungal species, 3 species complexes and 8 genera. Three phylotypes remained unresolved within the order Saccharomycetales and the phylum Ascomycota because of the lack of closely related sequences in GenBank. Cladosporium was the most abundantly detected genus, followed by Alternaria and Aureobasidium, well-known components of olive sooty moulds. Interestingly, Colletotrichum sp. and other fungal plant pathogens were also detected, leading to potential new insights into heir epidemiology.     

A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis

Peripherin is a neuronal intermediate filament associated with inclusion bodies in motor neurons of patients with amyotrophic lateral sclerosis (ALS). A possible peripherin involvement in ALS pathogenesis has been suggested based on studies with transgenic mouse overexpressors and with a toxic splicing variant of the mouse peripherin gene. However, the existence of peripherin gene mutations in human ALS has not yet been documented. Therefore, we screened for sequence variants of the peripherin gene (PRPH) in a cohort of ALS patients including familial and sporadic cases. We identified 18 polymorphic variants of PRPH detected in both ALS and age-matched control populations. Two additional PRPH variants were discovered in ALS cases but not in 380 control individuals. One variant consisted of a nucleotide insertion in intron 8 (PRPH(IVS8)(-36insA)), whereas the other one consisted of a 1-bp deletion within exon 1 (PRPH(228delC)), predicting a truncated peripherin species of 85 amino acids. Remarkably, expression of this frameshift peripherin mutant in SW13 cells resulted in disruption of neurofilament network assembly. These results suggest that PRPH mutations may be responsible for a small percentage of ALS, cases and they provide further support of the view that neurofilament disorganization may contribute to pathogenesis.     

Pyrethroid and organophosphate pesticide resistance in field populations of horn fly in Brazil

Pesticides are used worldwide to control arthropod parasites in cattle herds. The indiscriminate and/or inappropriate use of pesticides without veterinary guidance is a reality in several countries of South America. Improper pesticide use increases the chances of contamination of food and the environment with chemical pesticides and their metabolites. Reduction of these contamination events is an increasing challenge for those involved in livestock production. The horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), is one of the most economically important parasites affecting cattle herds around the world. As such, horn fly control efforts are often required to promote the best productive performance of herds. Pesticide susceptibility bioassays revealed that pyrethroid resistance was widespread and reached high levels in horn fly populations in the Brazilian state of Rondônia. The knockdown resistance (kdr) sodium channel gene mutation was detected in all horn fly populations studied (n = 48), and the super kdr sodium channel gene mutation was found in all homozygous resistant kdr individuals (n = 204). Organophosphate resistance was not identified in any of the fly populations evaluated.     

Enantioselective interaction with acetylcholinesterase of an organophosphate insecticide fenamiphos

Enantioselectivity in the environmental behavior and ecotoxicity of chiral pesticide is widely observed. However, the investigation of the enantioselective mechanisms remains limited. In this study, we used fenamiphos (FAP), an organophosphorus insecticide, to study enantioselectivity in toxicity to arthropods and the inhibition potential towards acetylcholinesterase (AChE) in the rat pheochromocytoma 12 (PC 12) cell line. Furthermore, we carried out molecular docking to help explain the mechanisms of enantioselective toxicity of FAP. The two enantiomers of FAP were successfully separated and identified as R‐(+)‐FAP and S‐(?)‐FAP. Toxicological assays revealed that R‐(+)‐FAP was 2.4‐fold more toxic than S‐(?)‐FAP to Daphnia magna and approximately threefold more to PC12 cells. Based on molecular docking results, dynamic simulation shows that strong hydrophobic interactions and a key hydrogen bond can only exist between R‐(+)‐FAP and AChE, which helps explain the preference of R‐(+) binding to AChE over that of the S‐(?)‐enantiomer, and supports our biological results. Our present study considers the impact of stereochemistry on ecotoxicological effects and, ultimately, on development of environmentally safe, insecticidally efficient pesticides. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

昆虫乙酰胆碱酯酶基因变异抗药性机制研究

有机磷和氨基甲酸酯类杀虫剂的大量使用导致昆虫对其产生抗药性。乙酰胆碱酯酶是昆虫对这类杀虫剂产生抗性的重要的靶标酶,昆虫产生抗药性的重要原因之一,就是因为乙酰胆碱酯酶的基因表达量上升,或基因突变而导致其敏感性下降。文章简要论述昆虫乙酰胆碱酯酶基因发生变异而导致的抗药性,分析了变异对其结构和功能的影响。     

Potential for insecticide resistance in populations of Bactrocera dorsalis in Hawaii: spinosad susceptibility and molecular characterization of a gene associated with organophosphate resistance

The potential for populations to become resistant to a particular insecticide treatment regimen is a major issue for all insect pest species. In Hawaii, for example, organophosphate (OP)‐based cover sprays have been the chemical treatment most commonly applied against oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), populations since the 1950s. Moreover, bait spray treatments using spinosad were adopted as a major control tactic in the Hawaii area‐wide fruit fly pest management program beginning in the year 2000. To determine the current level of spinosad and OP tolerance of wild B. dorsalis populations, bioassays were conducted on flies collected from a range of geographic localities within the Hawaiian islands. Adult B. dorsalis flies were tested (1) for the level of susceptibility to spinosad using LC₅₀ diagnostic criteria, and (2) for the presence of alleles of the ace gene previously shown to be associated with OP resistance. Regarding spinosad tolerance, only flies from Puna, the one area lacking prior exposure to spinosad, showed any significant difference compared to controls, and here the difference was only in terms of non‐overlap of 95% fiducial limit values. With respect to OP tolerance, specific mutations in the ace gene associated with resistance to these insecticides were found in only two populations, and in both cases, these alleles occurred at relatively low frequencies. These results suggest that at the present time, populations of B. dorsalis in Hawaii show no evidence for having acquired resistance to the insecticides widely used in control programs.     

A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors

Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae)

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.     

Interaction of an organophosphate with a peripheral site on acetylcholinesterase

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.     

Maternal duplication associated with gene deletion in sporadic hemophilia.

Sporadic occurrences of X-linked disorders can give insights into mutagenesis in man. In a case of sporadic hemophilia, associated with a partial deletion of the factor VIII gene, an unexpected inheritance pattern of gene rearrangements was observed. The factor VIII gene was found to be partially duplicated in the hemophiliac's mother. A pedigree analysis indicates that the mother has contributed both aberrant genes as well as the normal gene to her offspring. One simple model for the evolution of the deletion in this family is that the duplication is the precursor to the deletion.