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.     

Cloning,mutagenesis, and expression of the acetylcholinesterase gene from a strain of Musca domestica; the change from a drug-resistant to a sensitive enzyme

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.     

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.     

Impact of introduction of Bactrocera dorsalis (Diptera: Tephritidae) and classical biological control releases of Fopius arisanus (Hymenoptera: Braconidae) on economically important fruit flies in French Polynesia

Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), was discovered on Tahiti Island in July 1996. Eradication programs were conducted from 1997 to 2001, but failed. From 1998 to 2006, B. dorsalis was recovered from 29 different host fruit from the five Society Islands: Tahiti, Moorea, Raiatea, Tahaa, and Huahine. Analysis of coinfestation patterns by B. dorsalis, Bactrocera tryoni (Froggatt), and Bactrocera kirki (Froggatt) suggested B. dorsalis had displaced these two species and become the most abundant fruit fly in coastal areas. To suppress B. dorsalis populations, a classical biological control program was initiated to introduce the natural enemy Fopius arisanus (Sonan) (Hymenoptera: Braconidae) into French Polynesia from Hawaii. Wasps were released and established on Tahiti, Moorea, Raiatea, Tahaa, and Huahine Islands. In guava, Psidium guajava L., collections for Tahiti, F. arisanus parasitism of fruit flies was 2.1, 31.8, 37.5, and 51.9% for fruit collected for 2003, 2004, 2005 and 2006, respectively. Based on guava collections in 2002 (before releases) and 2006 (after releases), there was a subsequent decrease in numbers of B. dorsalis, B. tryoni, and B. kirki fruit flies emerging (per kilogram of fruit) by 75.6, 79.3, and 97.9%, respectively. These increases in F. arisanus parasitism and decreases in infestation were similar for other host fruit. Establishment of F. arisanus is the most successful example of classical biological control of fruit flies in the Pacific area outside of Hawaii and serves as a model for introduction into South America, Africa, and China where species of the B. dorsalis complex are established.     

基于mtDNA COI基因序列的中国柑橘果实蝇的分子鉴定及柑橘大实蝇种群的遗传多样性分析

【目的】探明危害我国柑橘的实蝇种类以及柑橘大实蝇Bactrocera minax不同地理种群和不同寄主种群的遗传多样性。【方法】利用mtDNA COI基因对危害柑橘的果实蝇进行种类鉴定,采用MEGA软件对其中28个地理种群的535头果实蝇COI基因片段(约505 bp)序列进行比对,分析种间及种内遗传距离,构建系统发育树。使用DnaSP软件分析柑橘大实蝇不同地理种群和不同寄主种群的遗传多样性。【结果】从柑橘虫果内共鉴定出4种实蝇,分别为柑橘大实蝇B.minax、桔小实蝇B.dorsalis、蜜柑大实蝇B.tsuneonis和瑞丽果实蝇B.ruiliensis。这4种实蝇的种间遗传距离为0.0264～0.2410,种内遗传距离为0.0000～0.0140,种间与种内遗传距离没有重叠区域。单个柑橘虫果内一般仅有1种实蝇,极个别柑橘果实可同时被两种实蝇危害(4/43);在这些为害柑橘的实蝇种类中,以柑橘大实蝇的个体数量比例最大,占90.70%。柑橘大实蝇地理种群遗传多样性高,28个种群共有17个单倍型。【结论】柑橘大实蝇是所调查地区柑橘实蝇的绝对优势种,其种群遗传分化程度较高,扩散危害风险大。本研究结果对柑橘果实蝇类的监测和防控具有重要意义。     

Fenitroxon insensitive acetylcholinesterases of the housefly, Musca domestica associated with point mutations

The cDNA of AChE in the housefly, Musca domestica, was sequenced and individual flies were genotyped by this gene in an inhibition assay of AChE activity with an organophaspate, fenitroxon. Mutations at Gly(342) and Tyr(407), which are reportedly conserved in resistant strains of Drosophila, were associated with the insensitivity to fenitroxon. Two other mutations, Ile(162) and Val(260), did not have an apparent effect on insensitivity. However, the four mutations are located in the active site of the enzyme, and therefore the non-neutral mutations in this gene are considered to cause the insensitivity of AChE in the development of insecticide resistance of the housefly.     

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.     

基于性信息诱捕监测广东桔小实蝇和瓜实蝇发生动态及其对2种常用农药的抗药性评价

利用性信息素诱捕的方法对广东省茂名、佛山、梅州和韶关的桔小实蝇Bactrocera dorsalis (Hendel)和瓜实蝇Bactrocera cucurbitae (Coquillett)种群动态进行监测;并用药膜法测定了两种实蝇不同地理种群成虫对阿维菌素和甲维盐的抗药性情况。结果表明,桔小实蝇和瓜实蝇在所监测的4个地市全年均有发生,桔小实蝇和瓜实蝇种群数量发生高峰期集中在5月中旬至8月中旬,从4月中旬开始田间种群数量开始增加,10月中旬以后种群数量急剧下降,高峰期与果实成熟期基本吻合。抗药性监测结果表明,桔小实蝇和瓜实蝇对阿维菌素和甲维盐的抗药性有逐年上升趋势,广东4个地市桔小实蝇和瓜实蝇已对阿维菌素产生中等抗性水平;2019年11月监测梅州地区桔小实蝇种群和韶关瓜实蝇种群对甲维盐抗性倍数分别为4.32和3.42倍,仍处于敏感水平,其余地区种群对甲维盐均达到了低水平或中等水平抗性。     

福州实蝇监测初报

自2005年5月29日至2006年5月28日在福州金山福建农林大学校园内进行了实蝇监测。采用甲基丁香酚(M e)、诱蝇酮(Cue)和地中海实蝇诱芯(T),诱集到橘小实蝇[Bactrocera(Bactrocera)dorsalis]、瓜实蝇[B.(Zeugodacus)cucurbi-tae]、南瓜实蝇[B.(Zeugodacus)tau]和具条实蝇(B.scutellata)4种实蝇,但未诱到地中海实蝇(Ceratitis capitata)。其中,橘小实蝇诱集量最大,8月中下旬达到高峰。本研究为掌握福州地区实蝇发生动态提供了基础资料。     

Fitness costs of an insecticide resistance and their population dynamical consequences in the oriental fruit fly

Naled is a commonly used insecticide for controlling populations of the oriental fruit fly, Bactrocera dorsalis (Hendel), in Taiwan and other countries. B. dorsalis has developed resistance to the insecticide, and the resistance management is an important issue. Ecological effects (e.g., fitness costs) of the resistance, when fully understood, can be used for the resistance management. This study examined the effects of the insecticide resistance on important life history traits (i.e., survival rates, stage durations, and fecundity) of the oriental fruit fly by comparing the traits of insecticide resistant individuals and susceptible individuals. Population dynamical properties were also examined using a stage-structured matrix model that was parameterized with the empirical data. The results revealed that susceptible individuals had shorter stage durations (e.g., grew faster) and reproduced more than resistant individuals. The average longevity of sexually mature susceptible adults was longer than that of sexually mature resistant adults. The matrix population model predicted that a population of the susceptible individuals would grow faster than a population of the resistant individuals in the absence of the insecticide. The sensitivity analysis of the model suggests that the sexually immature adult stage is a good candidate for controlling B. dorsalis populations.     

橘小实蝇成虫肠道可培养细菌群落结构分析

【目的】研究橘小实蝇(Bactrocera dorsalis) 3个种群(实验室正常喂养种群、实验室无菌糖水喂养种群和野生种群)成虫肠道可培养细菌的群落结构组成。【方法】利用16S rRNA基因的聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)分析技术,结合菌落形态观察和生理生化特征鉴定细菌种类。【结果】从橘小实蝇3个种群成虫肠道600株可培养细菌得到53种不同细菌遗传型,分属于肠杆菌科(Enterobacteriaceae)、肠球菌科(Enterococcaceae)和芽孢杆菌科(Bacillaceae)等3个科。其中肠杆菌科是肠道可培养细菌最优势的细菌种类。同样以序列相似性大于97%的菌株归为相同的细菌种类为标准,找到了橘小实蝇3个种群可培养细菌的共有菌种,结合菌落形态观察和生理生化特征鉴定,确定共有菌种为肠杆菌属5株,克雷伯氏菌属2株,柠檬酸杆菌属1株,泛菌属1株,肠球菌属2株,以及芽孢杆菌属4株。【结论】通过研究橘小实蝇成虫肠道可培养细菌群落结构组成,可为探讨肠道菌群对寄主的生理功能和生态学意义奠定基础,最终为利用微生物防治此类害虫提供新思路。     

Effectiveness of protein baits on melon fly and oriental fruit fly (Diptera: Tephritidae): attraction and feeding

Attraction and feeding responses of oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), were determined for different protein baits. In separate choice attraction assays for each species, significantly more flies arrived at stations with bait than water, but no differences existed among baits of GF-120 Fruit Fly Bait, GF-120 NF Naturalyte Fruit Fly Bait, Provesta 621 autolyzed yeast extract, and Mazoferm E802. In comparison with B. dorsalis, B. cucurbitae had 2.8 times more responders and a 4.8 times better discrimination between baits and water. In a second attraction assay with only B. dorsalis, volume of bait was negatively correlated to numbers of flies alighting on the bait. Feeding assays for both species demonstrated that time spent feeding and duration on a leaf were both significantly affected by bait type. B. dorsalis fed the longest on Provesta 621, with significantly less feeding on the other baits, and with all baits resulting in more feeding than water. The longest feeding times for B. cucurbitae resulted with Mazoferm E802 and Provesta 621, and all baits except GF-120 NF resulted in eliciting a significantly longer feeding duration than water. In separate toxicology assays for each species, significantly higher mortality resulted from bait formulations containing spinosad compared with blank baits, but no differences existed between GF-120 and GF-120 NF formulations. The differences are discussed between the two Bactrocera species primarily in regard to bait preference, extent of response, and previous work on laboratory flies.     

基于mtDNA Cytb 的六种果实蝇的分子鉴定

本研究首次对果实蝇属的桔小实蝇Bactrocera dorsalis、瓜实蝇B. cucurbitae、南瓜实蝇B. tau、番石榴实蝇B. correcta、具条实蝇B. scutellata、黑漆实蝇B. scutellaris等6种实蝇mtDNA Cytb基因进行了测序。对这6种实蝇72个个体mtDNA Cytb基因中段420 bp的碱基序列进行分析,得到38种单倍型,发现了116个变异位点,其中30个位点较为稳定。对这6种实蝇与其各自鉴别位点的对应关系研究表明,mtDNA Cytb基因可以作为这6种实蝇种类鉴别的分子标记。     

无脊椎动物乙酰胆碱酯酶研究进展

乙酰胆碱酯酶(AChE)是生物体中一种十分重要的神经递质水解酶,也是有机磷和氨基甲酸酯类杀虫剂的作用靶标。AChE在不同生物中的性质显著不同,如编码基因个数、序列保守性、表达分布及生理功能等。作为杀虫剂的主要作用靶标之一,AChE不但可以通过单个点突变引起昆虫抗药性,还能够通过多个点突变联合作用、靶标表达量变化及基因复制等方式引起抗药性并且改变昆虫的适合度代价。本文主要从AChE的基因类型、分子进化、蛋白结构、生理功能、与昆虫的抗药性关系、同一物种中不同AChE的性质等6个方面对昆虫纲、蛛形纲和线虫等无脊椎动物AChE的研究进展作一综述。     

橘小实蝇复合体分类学研究进展

橘小实蝇复合体Bactroceradorsaliscomplex是以橘小实蝇命名的实蝇类群,已知75种,广泛分布于亚洲、澳洲及环太平洋周边地区。文章概述基于形态学和遗传学特征基础上的橘小实蝇复合体的分类鉴定及系统发育研究进展,介绍近年来分子生物学技术在橘小实蝇复合体分类及系统发育研究中的应用情况,展望系统发育和行为学研究是橘小实蝇复合体未来研究的重点领域。     

Trap capture of three economically important fruit fly species (Diptera: Tephritidae): evaluation of a solid formulation containing multiple male lures in a Hawaiian coffee field

Invasive fruit flies (Diptera: Tephritidae) pose a global threat to agriculture through direct damage to food crops and the accompanying trade restrictions that often result. Early detection is vital to controlling fruit flies, because it increases the probability of limiting the growth and spread of the invasive population and thus may greatly reduce the monetary costs required for eradication or suppression. Male-specific lures are an important component of fruit fly detection, and three such lures are used widely: trimedlure (TML), cue lure (CL), and methyl eugenol (ME), attractive to Mediterranean fruit fly, Ceratitis capitata (Wiedemann); melon fly, Bactrocera cucurbitae (Coquillett); and oriental fruit fly, Bactrocera dorsalis (Hendel), respectively. In California, Florida, and Texas, the two Bactrocera lures are applied to separate species-specific traps as liquids (with a small amount of the insecticide naled added), whereas TML is delivered as a solid plug in another set of traps. Thus, the detection protocol involves considerable handling time as well as potential contact with a pesticide. The purpose of this study was to compare trap capture between liquid male lures and "trilure" wafers that contain TML, ME, raspberry ketone (RK, the hydroxy equivalent of CL), and the toxicant DDVP embedded within a solid matrix. Field studies were conducted in a Hawaiian coffee (Coffea arabica L.) field where the three aforementioned species co-occur, showed that the wafer captured at least as many flies as the liquid baits for all three species. This same result was obtained in comparisons using both fresh and aged (6-wk) baits. Moreover, the wafers performed as well as the single-lure traps in an ancillary experiment in which TML plugs were substituted for liquid TML. Additional experiments demonstrated explicitly that the presence of ME and RK had no effect on captures of C. capitata males and similarly that the presence of TML had no effect on the capture of B. cucurbitae or B. dorsalis males.     

从东南亚进口水果截获桔小实蝇复合种五近缘种的鉴别(双翅目:实蝇科)

介绍了近年从进口东南亚水果中经常截获的、最具经济重要性的桔小实蝇复合种5个近缘种:杨桃实蝇B.carambolae、桔小实蝇B.dorsalis、芒果实蝇B.occipitalis、木瓜实蝇B.papayae及菲律宾实蝇B.philippinensis,分别记述了翅、胸、足、腹及雌虫产卵器主要鉴别特征,并列出桔小实蝇复合种5近缘种鉴别特征检索表。     

Toxicity of spinosad in protein bait to three economically important tephritid fruit fly species (Diptera: Tephritidae) and their parasitoids (Hymenoptera: Braconidae)

The feeding toxicity of the natural insecticide spinosad in Provesta protein bait was evaluated for three economically important fruit fly species, the Mediterranean fruit fly, Ceratitis capitata (Wiedemann); the melon fly, Bactrocera cucurbitae Coquillett; and the oriental fruit fly, Bactrocera dorsalis Hendel. Both females and males were evaluated. Spinosad was remarkably similar in toxicity to all three fruit fly species. Male C. capitata (24 h LC50 values and 95% fiducial limits = 2.8 [2.60-3.0] mg/liter spinosad) were significantly, although only slightly more susceptible to spinosadthan females (4.2 [3.8-4.6] mg/liter). Male (5.5 [4.7-6.6] mg/liter) andfemale (4.3 [3.7-4.9] mg/liter) B. cucurbitae were equally susceptible to spinosad. Female (3.3 [3.1-3.6] mg/liter) and male (3.1 [2.9-3.3] mg/liter) B. dorsalis also were equally susceptible to spinosad. Provesta bait containing spinosad also was evaluated against two parasitoids of tephritid fruit flies, Fopius arisanus (Sonan) and Pysttalia fletcheri (Silvestri). These parasitoids did not feed on the bait, so a contact toxicity test was conducted. Significant amounts of mortality were found only after exposure of parasitoids to spinosad-coated glass vials with concentrations > or =500 mg/liter spinosad. Parasitoids were less susceptible than fruit flies to such a degree that use of spinosad in bait spray should be compatible with these parasitoid species. Because the fruit flies tested in this study were so susceptible to spinosad, this product seems to be promising as a bait spray additive and a replacement for malathion for control of these species.     

Characteristics of protein variants in trichlorphon-resistant Bactrocera dorsalis (Diptera; Tephritidae) larvae

Functional proteins in larvae of Bactrocera dorsalis, a major fruit pest, play a central role in their resistance to organophosphorus insecticides. Changes in proteins in B. dorsalis larvae after trichlorphon treatment may have a role in the resistance response to trichlorphon. We analyzed 14 protein spots of crude proteins from B. dorsalis larvae post-treatment with trichlorphon in two-dimensional gel electrophoresis through mass spectrometry and protein sequencing. We found functional proteins that are responsible for signal transduction (pkaap and dual specificity tyrosine-phosphorylation-regulated kinase), immunity (hemolectin), synthesis and decomposition (twinstar, cathepsin B, RE66325p), oxidative stress metabolism (glutathione S transferase and CG7320), energy metabolism (Act57B), and cytoskeleton formation (actin). These proteins appear to be involved in the resistance response to trichlorphon.