Target site insensitivity and mutational analysis of acetylcholinesterase from a carbofuran-resistant population of Colorado potato beetle, Leptinotarsa decemlineata (Say)

The BERTS strain of Colorado potato beetle (CPB) was found to be highly resistant to N-methyl carbofuran but relatively susceptible to azinphosmethyl. N-Methyl carbofuran resistance was found to correlate well with acetylcholinesterase (AChE) insensitivity. In becoming resistant to N-methyl carbofuran, the AChE of the BERTS strain became more sensitive to N-propyl carbofuran inhibition. This negative cross-insensitivity correlated well to the increased relative toxicity of the BERTS strain to N-propyl carbofuran compared to the susceptible SS strain. BERTS beetles were sorted into BERTS-R and BERTS-S substrains using their AChE activity profiles. Sequence comparisons of AChE cDNAs from the two substrains revealed the presence of the point mutation that results in the S291G substitution previously found in the AChE of the azinphosmethyl-resistant AZ-R strain of CPB. A novel mutation present only in BERTS-S CPB, however, resulted in an additional I392T substitution in the AChE and apparently reverses the resistance conferring properties of the S291G substitution.     

新疆北疆马铃薯甲虫成虫对新烟碱类杀虫剂的敏感性变化

采用点滴法于2009和2010年监测了新疆维吾尔自治区北疆马铃薯甲虫Leptinotarsa decemlineata 9个田间种群成虫对新烟碱类杀虫剂吡虫啉、啶虫脒、噻虫嗪和噻虫啉的敏感性变化,发现其对吡虫啉和噻虫嗪的敏感性逐年降低。2009年监测的6个种群中有3个对啶虫脒和噻虫嗪低抗(抗性倍数5.0~10.0);2010年监测的6个种群全部对噻虫嗪产生了抗性,其中中抗(抗性倍数10.1~40.0)和低抗种群各3个。噻虫嗪与高效氯氟氰菊酯可能存在交互抗性。     

Resistance and cross-resistance to neonicotinoid insecticides and spinosad in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

The Colorado potato beetle, Leptinotarsa decemlineata (Say), has developed resistance to many insecticides used for its control, recently including imidacloprid, a neonicotinoid compound. Other neonicotinoids are now being deployed to control this pest. A key point in the strategies of resistance management is the monitoring of resistance and cross-resistance. In the summer of 2003, imidacloprid-resistant adult Colorado potato beetles collected from Long Island, New York, USA were bioassayed using topical applications of imidacloprid and nine other neonicotinoids. Compared to a standard susceptible strain, the Long Island beetles showed 309-fold resistance to imidacloprid, and lower levels of cross-resistance to all other neonicotinoids, despite these never having been used in the field, i.e., 59-fold to dinotefuran, 33-fold to clothianidin, 29-fold to acetamiprid, 28-fold to N-methylimidacloprid, 25-fold to thiacloprid, 15-fold to thiamethoxam, 10-fold to nitenpyram, but less than 2-fold to nicotine. In injection bioassays, high resistance to imidacloprid was also found (116-fold). Piperonyl butoxide partially suppressed resistance to imidacloprid, but the resistance level was still over 100-fold, indicating that other mechanisms were primarily responsible for resistance. Low levels of resistance (8- to 10-fold) were found to the nicotinic activator, spinosad, in an imidacloprid-resistant strain collected from the same field in 2004. The cross-resistance seen with all the neonicotinoids tested suggests that the rotation of imidacloprid with other neonicotinoids may not be an effective long-term resistance management strategy. Rotation with spinosad also carries some risk, but it is unlikely that spinosad resistance in this case is mechanistically related to that for the neonicotinoids.     

菌株NDBJJ-BFG的鉴定及其对马铃薯甲虫不同虫态的毒力测定

为筛选出对马铃薯甲虫Leptinotarsa decemlineata(Say)具有防治潜力的菌株,本研究从马铃薯甲虫僵虫虫体上分离菌株NDBJJ-BFG,通过形态学特征和分子生物学方法明确其分类地位,采用喷雾法和覆土法测定了不同浓度下该菌株对马铃薯甲虫室内毒力的致死中浓度LC50及半致死时间LT50。结果表明,经形态特征与r DNA ITS序列分析最终确定菌株NDBJJ-BFG为球孢白僵菌Beauveria bassiana(Bals.)Vuill。该菌株对1、2、3和4龄马铃薯甲虫幼虫的LC50分别为0.91×10~6、1.51×10~6、5.09×10~6和6.84×10~6个/m L;采用喷雾法和覆土法处理蛹的LC50分别为1.43×10~7个/m L和8.15×10~6个/m L,成虫的LC50分别为5.08×10~7个/m L和2.97×10~7个/m L。在孢子悬浮液浓度相同时LT50随虫龄的增大而延长,其中成虫的LT50最长,其次是蛹;在相同龄期下LT50随着孢子悬浮液浓度的增大而缩短。表明菌株NDBJJ-BFG对马铃薯甲虫1龄和2龄幼虫具有高毒力,其在马铃薯甲虫的生物防治中将具有较大的应用潜力。     

Resistance to neonicotinoid insecticides in field populations of the Colorado potato beetle (Coleoptera: Chrysomelidae)

BACKGROUND: Neonicotinoid insecticides were first used commercially for Colorado potato beetle [Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae] control in the United States in 1995, and since then have been critical for management of this pest. Field populations from the northeastern and midwestern United States were tested from 1998 to 2010 for susceptibility to imidacloprid and thiamethoxam using standard topical dose assays with adults. RESULTS: From 1998 to 2001, imidacloprid resistance was present in only a few locations in the eastern United States. By 2003, imidacloprid resistance was common in the northeastern Unites States. In 2004, imidacloprid resistance in Colorado potato beetle was detected for the first time in the midwestern United States. In 2003, the first case of resistance to thiamethoxam was found in a population from Massachusetts. Neonicotinoid resistance in summer‐generation adults was higher than in overwintered adults from the same locations. By 2009, 95% of the populations tested from the northeastern and midwestern United States had significantly higher LD₅₀ values for imidacloprid than the susceptible population. CONCLUSIONS: The increasing resistance to neonicotinoid insecticides raises concerns for the continued effective management of Colorado potato beetles in potatoes and highlights the need for more rigorous practice of integrated pest management methods. Copyright © 2011 Society of Chemical Industry     

马铃薯甲虫热激蛋白基因Ld-hsp90的克隆及温度胁迫下的表达

马铃薯甲虫Leptinotarsa decemlineata是一种抗逆性极强的世界性检疫害虫,对温度胁迫具有很强的适应性,为进一步明确其对温度胁迫适应性的分子生态机制,采用RT-PCR及RACE技术克隆得到了1个hsp90基因的cDNA全长序列,命名为Ld-hsp90,并利用实时荧光定量PCR技术检测其在温度胁迫后相对表达量的变化。Ld-hsp90全长为2 489 bp,开放阅读框(ORF)长度为2 160 bp,编码719个氨基酸,理论等电点为4.98,分子量约为82.09 kD,5'端与3'端非编码区长度分别为113 bp和216 bp。氨基酸序列中含有HSP90家族的5个签名序列及胞质特征序列MEEVD。实时荧光定量PCR检测结果显示:38℃和44℃高温热激1 h,马铃薯甲虫成虫体内的Ld-hsp90的表达量均显著高于对照组,而0℃与-10℃低温处理1 h后的表达量与对照组均无显著差异。研究表明Ld-hsp90的上调表达在马铃薯甲虫抗高温中起着重要的作用。     

苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展

马铃薯甲虫是重要的入侵害虫,严重威胁着我国粮食作物马铃薯的生产。苏云金芽胞杆菌是重要的农业害虫生防细菌,对马铃薯甲虫有良好的防治效果。本文围绕苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展与应用进行综述。主要从马铃薯甲虫的入侵与防治手段、苏云金芽胞杆菌的晶体蛋白结构与杀虫机制、对马铃薯甲虫有活性的Bt毒蛋白研究进展、Bt毒蛋白对马铃薯甲虫的作用机制以及马铃薯甲虫对Bt毒蛋白的抗性机制等方面进行了综述。最后,从Bt新基因的挖掘和杀虫机理方面对苏云金芽胞杆菌在马铃薯甲虫防治上的研究进行了展望。     

噻虫嗪和吡虫啉拌种对马铃薯甲虫幼虫生长发育的影响

噻虫嗪和吡虫啉是具有触杀、内吸和胃毒活性的新烟碱类杀虫剂,对植食性害虫效果显著,近年来已成为新农药创制领域研究的热点。马铃薯甲虫Leptinotarsa decemlineata (Say)是国际公认的毁灭性检疫害虫,以成、幼虫取食茄科植物茎叶、花蕾和叶芽等部位。一般田块减产30%~50%,严重地块达90%以上。目前,我国马铃薯甲虫仅在新疆马铃薯种植区发生,但对其周边省份马铃薯生产构成威胁。本试验选用噻虫嗪和吡虫啉于马铃薯播前进行一次性拌种,探讨出苗90天后的叶片对马铃薯甲虫幼虫生长发育的影响。     

马铃薯甲虫空间分布型及序贯抽样

马铃薯甲虫Leptinotarsa decemlineata Say是一种重要的害虫,为明确其在新疆的空间分布,及时采取有效的防控措施,在田间调查的基础上,采取随机取样和Iwao回归分析法对新疆乌鲁木齐地区的马铃薯甲虫成虫、卵块和幼虫的空间分布型、理论抽样数及幼虫的序贯抽样方法进行了研究。结果表明,马铃薯甲虫各虫态空间格局的聚块性指标均大于1,空间分布型为普通群聚型;对低龄幼虫进行序贯抽样,当防治阈值为50头/样方、置信水平为1.96时,防治上、下限方程分别为:d1=50n+81n和d0=50n-81n,当百株虫量达到756头以上时需要进行防治。     

Effect of Neem extract on Lymantria dispar L. (Lepidoptera: Lymantriidae) and Leptinotarsa decemlineata Say. (Coleoptera: Chrysomelidae)

High antifeedancy and low toxicity of the plant preparation Neem (azadirachtin), on the gypsy moth, Lymantria dispar L. larvae were proved under laboratory conditions. A high index of Neem antifeedancy was confirmed in all tested concentrations.Under laboratory conditions the Neem had satisfying antifeedancy on Leptinotarsa decemlineata Say third instar larvae too. Field experiments also proved that the foliage protective effect of Neem was very significant.Experiments showed that some antifeedants or repellents, such as Neem, should control olygophagous (L. decemlineata, Coleoptera) insects as well as very polyphagous insects such as L. dispar (Lepidoptera).As a biological, plant-derived preparation, Neem could be suitable for integrated pest management (IPM) programs, especially in small orchards, parks, and tree rows in urban environments and on small private gardens.     

马铃薯甲虫空间分布型及抽样技术研究

马铃薯甲虫Leptinotarsa decemlineata (Say)是我国马铃薯恶性入侵害虫。本研究利用聚集度指标检验、Taylor幂法则和Iwao回归法对马铃薯甲虫成虫、幼虫和卵在田间的空间分布型进行了研究。聚集度指标检测各虫态均为聚集分布,且种群聚集主要是由马铃薯甲虫各虫态本身的聚集行为,或由其本身的聚集行为与环境异质性共同作用所致。建立了马铃薯甲虫成虫、幼虫和卵在田间调查的理论抽样数学模型。田间抽样方法,成虫应采用大五点、对角线法;幼虫最适应采用Z字型法;卵最适应采用对角线法。     

Incidence and spread of knockdown resistance (kdr) in German Colorado potato beetle (Leptinotarsa decemlineata Say) populations

The Colorado beetle, Leptinotarsa decemlineata (Say), is one of the most important pests in many potato‐producing regions. Colorado beetle infestations are normally kept under economic damage thresholds by applying insecticides such as pyrethroids. Pyrethroids are known to act on voltage‐gated sodium channels and have been used for several decades to control L. decemlineata. Their continuous and widespread use has resulted in the development of resistance, which is often linked to a L1014F target‐site mutation in the voltage‐gated sodium channel, known as knockdown resistance (kdr). Since pyrethroids are used in many potato‐growing regions in Germany, more than 140 L. decemlineata samples were collected and analysed for the presence of the kdr allele by pyrosequencing diagnostics. Results showed that kdr is present in many German L. decemlineata populations, but even its homozygous presence does not substantially compromise the efficacy of recommended label rates of pyrethroids, as demonstrated by bioassays and crossing experiments. The implications of these findings for resistance management are briefly discussed.     

Neural actions of imidacloprid and their involvement in resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say)

BACKGROUND: The development of resistance to imidacloprid in eastern US populations of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), threatens this critical use for neonicotinoid insecticides. Previous pharmacokinetic studies with resistant adult CPBs provided no explanation for the high resistance level (over 200-fold) to topically applied imidacloprid. The authors assessed the neural activity of imidacloprid by recording spontaneous activity from a motor nerve leaving the isolated central nervous system to compare the sensitivity of the latter to imidacloprid between susceptible and resistant CPBs. RESULTS: On the isolated central nervous system, imidacloprid was initially neuroexcitatory, and neuroinhibitory at higher concentrations. The neuroexcitatory action of imidacloprid was blocked by coapplication of a specific nAChR antagonist, methyllycaconitine, indicating that it is a result of action on nAChRs. The sensitivity to the neuroexcitatory and inhibitory activities of imidacloprid varied independently among individuals in each population. The sensitivity of the central nervous system of resistant CPBs to excitation by imidacloprid did not differ from that of susceptible insects, but the sensitivity to inhibition by imidacloprid was reduced 52- to 58-fold, indicating a possible change in the sensitivity of at least one subgroup of nAChRs. CONCLUSION: This study provides evidence that reduced nerve sensitivity to the blocking action of imidacloprid is associated with imidacloprid resistance in the CPB.     

The effect of host plant on colorado potato beetle (coleoptera: Chrysomelidae) susceptibility to pyrethroid insecticides

In laboratory studies, Colorado potato beetle (CPB) (Leptinotarsa decemlineata Say) reared on eggplant were significantly more susceptible to permethrin than CPB reared on tomato. Field studies conducted in 1984 and 1985 showed that the percentage reduction of CPB larvae reared on eggplant due to foliar applications of fenvalerate was significantly greater than the percentage reduction of CPB larvae reared on either potato or tomato. Plant defoliation was greater on eggplant and potato than on tomato. Eggplant and potato yields increased as the dose of fenvalerate increased and the potato beetle population decreased, but tomato yields were unaffected by fenvalerate treatments. The effects and biological activity of individual plant chemical compounds (i.e., glvcoalkaloids) in various Solanum species need to be examined in detail before the full significance of the role of these compounds in host-plant resistance and their interaction with CPB insecticide resistance are understood.     

Insecticide resistance patterns among colorado potato beetle,leptinotarsa decemlineata (Say) (Coleoptera: chrysomelidae), populations in north carolina

A residual bioassay was used to determine insecticide susceptibility in populations of Colarado potato beetle, Leptinotarsa decemlineata (Say), in eastern North Carolina. Mean bioassay mortalities were subjected to correlation analysis to assess patterns of resistance among tested insecticides. Within insecticide classes, cross-resistance patterns were observed between carbaryl and carbofuran (carbamates), fenvalerate and permethrin (pyrethroids), and among azinphosmethyl, methamidophos, and phosmet (organophosphates). Significant associations between insecticides from different insecticide classes were concluded to be due to multiple resistance. Knowledge of multiple and cross-resistance patterns are discussed in relation to the bioassay as a tool for monitoring insecticide resistance.     

Control of the Colorado potato beetle (Leptinotarsa decemlineata [Say]) on potato under field conditions: a comparison of the efficacy of foliar application of two strains of Steinernema feltiae (Filipjev) and spraying with thiametoxam

Journal of Plant Diseases and Protection - The efficacy of entomopathogenic nematodes in controlling Leptinotarsa decemlineata on potato was tested in a field experiment. A Slovenian strain of...     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.