小菜蛾对Bt毒素Cry1Ac和Bt制剂抗性的选育及其抗性种群的生物学适应性

用Bt制剂和Bt毒素Cry1Ac分别对源自深圳田间的小菜蛾Plutella xylostella在室内进行抗性种群选育,获得相对抗性倍数分别为24.36、38.16倍的抗性种群DBM.1Ac-R30和DBM.Bt-R46。对这2个抗性种群及其敏感种群（DBM.Bt-S）的生长发育、存活及繁殖特征进行了详细地观察与比较,并以甘蓝为饲喂材料构建了2个抗性实验种群的生命表。结果表明,DBM.1Ac-R30和DBM.Bt-R46种群较DBM.Bt-S种群的产卵量和孵化率下降,幼虫发育历期延长,雌雄比显著降低,雌成虫数量、寿命减少。DBM.1Ac-R30和DBM.Bt-R46种群相对于DBM.Bt-S种群的相对适合度分别为0.75和0.65,抗性种群在繁殖能力上存在明显的生存劣势。     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

小菜蛾对苦皮藤素抗性选育及交互抗性测定

敏感小菜蛾Plutella xylostella (L.)经苦皮藤素20代的抗性选育,其抗性增长21.57倍。选育的小菜蛾抗性品系对杀虫双、杀螟丹和叶蝉散分别有4.63、4.11和3.71倍的交互抗性;对溴氰菊酯、氯菊酯、氯氰菊酯分别有0.22、0.01和0.26倍的负交互抗性。高抗杀螟丹、杀虫双小菜蛾品系对苦皮藤素无明显交互抗性,而高抗溴氰菊酯小菜蛾品系对苦皮藤素有3.61倍的交互抗性。     

Evidence for behavioural resistance by the diamondback moth, Plutella xylostella (L.)

Abstract:  The diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), is an important pest of brassicaceous crops worldwide. It has developed resistance to almost every synthetic insecticide applied in the field and consequently is often difficult to control in crucifer-growing areas. DBM oviposition behaviour was investigated in greenhouse and growth chamber conditions with seven cruciferous and one non-cruciferous plant varieties. In free choice tests, females deposited more eggs on the stem near the soil-stem interface than on leaves. Our findings suggest that DBM is capable of developing behavioural resistance through oviposition site selection to avoid lethal doses of foliar-applied insecticides in the field.     

小菜蛾酪氨酸酶生物化学性质研究

采用时间动力学法对小菜蛾Plutella xylostella酪氨酸酶的最适反应条件、对抑制剂的敏感度以及发育期变化规律进行了研究。结果表明：小菜蛾酪氨酸酶反应的最适pH值为6.4,反应线性时间为0～2 min,在22℃和4℃下其活性分别可保持12 h 和72 h,在-20℃下至少可保持7 天。不同发育阶段小菜蛾酪氨酸酶活性的大小依次为预蛹＞4龄幼虫＞蛹＞1龄、2龄、3龄幼虫＞成虫。苯基硫脲（phenyl thiourea,PTU）对小菜蛾阿维菌素抗性品系和敏感品系酪氨酸酶活性的抑制中浓度（I₅₀）值分别为1.1796 μmol/L和1.2795 μmol/L,二者无明显差异。     

cDNA characterization and expression analysis of two arylphorin-like hexameric protein genes from the diamondback moth, Plutella xylostella (L.)

We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investigated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin-like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the developmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome.     

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.     

小菜蛾抗药性及其治理对策的研究进展

小菜蛾（Plutella xylostella L.）的抗药性研究是一项全球性的重要课题。本从其抗药性发展、抗药性机理研究进展、抗药性检测及研究手段的状况、抗药性治理对策等方面,对小菜蛾抗药性的研究进展进行综述。目前,对小菜蛾抗药性的分子水平上的探讨甚少。     

小菜蛾碱性磷酸酶基因cDNA片段的克隆及其序列分析

利用DNAman设计简并引物,通过反转录一多聚酶链式反应(RT-PCR)克隆小菜蛾Plutella xylostella(L.)抗性、敏感种群中可能与苏云金芽孢杆菌(Bt)抗性相关的碱性磷酸酶基因。琼脂糖电泳结果显示扩增条带与预期片段长度一致,阳性克隆经测序获得了403bp的基因片断。经blast比较得出所克隆基因属于碱性磷酸酶基因。与敏感种群相比,抗性种群中该基因片段有18个碱基发生变化,有1个氨基酸发生替换(缬氨酸转变为苏氨酸)。研究结果对于进一步研究小菜蛾全基因结构、功能有重要的意义。     

表皮穿透和GABAA受体不敏感性在小菜蛾对阿维菌素抗性中的作用

用氚标记阿维菌素点滴处理阿维菌素敏感（ABM-S）和抗性（ABM-R）种群小菜蛾幼虫,结果显示,在5～360 min内的7个不同处理时间,ABM-R种群的平均表皮穿透量比ABM-S种群少1.5倍,处理24 h后,ABM-R种群仍有45.9%的3H-阿维菌素滞留于体表,而ABM-S种群却有98.4%的药剂穿透表皮。放射配体结合分析表明,GABAA受体结合性质的改变是小菜蛾对阿维菌素的另一抗性机制,ABM-S种群（K_d=10.9368±0.4374 nmol/L）和ABM-R种群（K_d=9.8328±0.3933　nmol/L）的受体亲和力无显著差异,但抗性种群的最大结合量（B_max=71.2842±4.9910　fmol/mg 蛋白）比敏感种群（B_max=112.0255±7.8418　fmol/mg 蛋白）降低63.6%,即抗性是受体数目的减少而非结构上的改变。     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

小菜蛾溴氰菊酯敏感和抗性品系蛋白质表达谱的比较分析

杀虫剂抗性是害虫防治中的一个主要挑战。为有效治理抗性, 我们必须了解杀虫剂诱导的害虫生理生化变化。目前害虫抗药性的一些机理已经清楚, 但更多的相关机制还有待探究。本研究通过蛋白质组学方法检测了小菜蛾Plutella xylostella溴氰菊酯敏感和抗性品系间蛋白质组的表达差异。结果显示： SDS-PAGE胶上有大约300个蛋白差异点, 其中23个蛋白点具2.5倍以上的表达差异, 通过MALDI-TOF-MS, 我们成功鉴定出8个蛋白, 其中包括化感蛋白CSP2、 铜锌超氧化物歧化酶和peroxiredoxin样蛋白。通过实时定量PCR（real-time quantitative PCR, qPCR）分析了其中5个蛋白的mRNA 表达水平, 结果表明mRNA 表达水平不能真实反映蛋白的表达水平。免疫印迹验证了双向电泳中SOD1的表达差异。本研究有力地证明溴氰菊酯诱导小菜蛾成虫蛋白质组表达变化, 这为进一步筛选抗性靶标提供很大的帮助。     

Seasonal population fluctuations of the diamondback moth,Plutella xylostella (L.) (Lep.: Plutellidae) on different cauliflower cultivars

The diamondback moth, Plutella xylostella (L.) (Lep.: Plutellidae) is one of the most important pests of cruciferous plants throughout the world. In recent years, it has been identified as a serious pest of the cauliflower fields in Tehran province. Resistance of P. xylostella to all main groups of insecticides has been recorded and it is ranked in the 20 most resistant pest species reported until now. According to many researchers, to solve the problem of pest resistance to chemical pesticides, an integrated pest management programme should be used. Despite this condition, it seems that the use of resistant cauliflower cultivars is an appropriate policy for integrated control of the pest in the field. In order to identify the most resistant cultivar in the field, eight cauliflower cultivars in a completely randomised design with five replicates were planted at the Shahed University research field (south of Tehran). Density of eggs, larvae and pupae of P. xylostella were measured every 10 days in these cultivars. The results showed that there is no significant difference between numbers of eggs per plant on different cultivars. But number of larvae and pupae per plant were significantly different among different cultivars. Smilla and Snow mystique cultivars had the highest number of larvae and pupae. On the other hand, Buris and Snow crown cultivars had the lowest number of pupae and Snow crown and SG cultivars had the lowest number of larvae per plant. According to the results, the Buris and Snow crown cultivars had the lowest infestation and had a kind of resistance to pest.     

Detection of insecticide resistance in Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations in South Australian crucifer crops

In response to reports of failures of sprays to control Plutella xylostella infestations in vegetable-growing areas around Adelaide in 1991, the extent of resistance to six insecticides in several South Australian populations of P. xylostella was determined. Populations sampled from vegetable crops in the Adelaide region were five- to 200-fold resistant to organophosphates (chlorpyrifos, methamidophos and mevinphos), eight- to 400-fold resistant to pyrethroids (esfenvalerate and permethrin) and six-fold resistant to the carbamate methomyl compared with a susceptible strain. Mevinphos and esfenvalerate were the most effective of the tested insecticides. These, together with Bacillus thuringiensis products, have continued until 1998 to be used almost exclusively for the control of P. xylostella in South Australian vegetable crops in the absence of any resistance management strategy.     

Microsatellites reveal a lack of structure in Australian populations of the diamondback moth, Plutella xylostella (L.)

The diamondback moth, Plutella xylostella, is renowned for developing resistance to insecticides and causing significant economic damage to Brassica vegetable crops throughout the world. Yet despite its economic importance, little is known about the population structure and movement patterns of this pest both at local and regional scales. In Australia, the movement patterns and insecticide resistance status of P. xylostella infesting canola, vegetables, forage brassicas and weeds have fundamental implications for the management of this pest. Here we use six polymorphic microsatellite loci to investigate population structure and gene flow in Australian populations of P. xylostella. Samples of P. xylostella from New Zealand, Malaysia, Indonesia and Kenya were also scored at these loci. We found no evidence of population structure within Australia, with most populations having low inbreeding coefficients and in Hardy-Weinberg equilibrium. In addition, a sample from the North Island of New Zealand was indistinguishable from the Australian samples. However, large genetic differences were found between the Australia/New Zealand samples and samples from Kenya, Malaysia and Indonesia. There was no relationship between genetic distance and geographic distance among Australian and New Zealand samples. Two of the loci were found to have null alleles, the frequency of which was increased in the populations outside the Australia/New Zealand region. We discuss these results with reference to insecticide resistance management strategies for P. xylostella in Australia.     

氰氟虫腙对小菜蛾阿维菌素抗性和敏感种群的亚致死效应(英文)

在实验室条件下采用生命表技术研究了氰氟虫腙亚致死剂量（LC₂₅）对小菜蛾Plutella xylostella阿维菌素抗性（AV-R）和敏感（AV-S）种群的亚致死效应, 旨在为小菜蛾对阿维菌素的抗性治理提供理论基础。结果表明： 氰氟虫腙对小菜蛾3龄幼虫抗性种群的LC₅₀和LC₂₅分别为0.24 mg/L和0.09 mg/L; 对敏感种群的LC₅₀和LC₂₅分别为0.20 mg/L和0.07 mg/L。氰氟虫腙亚致死剂量0.09 mg/L 处理小菜蛾后, 对处理代的影响表现为显著降低处理种群的化蛹率、 蛹重、 羽化率、 繁殖力; 明显延长蛹期, 缩短成虫产卵期和寿命; 对子代种群的影响表现为显著降低卵的孵化率、 幼虫各龄期的存活率, 延长发育历期。处理种群的内禀增长率（r_m）、 周限增长率（λ）和净增值率（R₀）显著低于对照种群（P<0.0001）。亚致死剂量的氰氟虫腙对小菜蛾抗性种群的影响大于敏感种群, 对处理代种群的影响大于子代种群。氰氟虫腙亚致死剂量可以极大地影响小菜蛾尤其是阿维菌素抗性种群的种群动态, 因此氰氟虫腙对于小菜蛾的抗性治理具有积极的作用。     

Conserving the efficacy of insecticides against Plutella xylostella (L.) (Lep., Plutellidae)

Abstract:  The diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), is one of the most destructive insect pests of crucifers worldwide. It was the first crop insect reported to be resistant to DDT and now in many crucifer-producing regions it has shown significant resistance to almost every insecticide applied in field including biopesticides such as crystal toxins from Bacillus thuringiensis and spinosyns from Saccharopolyspora spinosa . In certain parts of the world, economical production of crucifers has become almost impossible because of its resistance to insecticides and resulting control failure. A coordinated resistance management program needs to be implemented with the involvement of pesticide industry, local pesticide regulatory authorities, scientists and farmers. The judicious use of chemicals in conjunction with other control measures (e.g. biological control agents, resistant varieties, proper fertilization rates) is the best way to manage DBM and other pests of cruciferous crops. Introduction of glucosinolate-sulphatase inhibitors as plant-incorporated-products or sprayable material may also lead to a novel pest management strategy.     

小菜蛾有机磷护性季节性变化及毒理机制研究

1998～ 2 0 0 3年期间的田间监测结果显示 ,福州地区小菜蛾 Plutella xylostella对甲胺磷和敌敌畏的抗性水平呈季节性变化 ,每年春秋季的抗性水平显著高于夏季 7月期间的抗性水平。与 2 5℃ 2 d的饲养条件相比 ,35℃ 2 d和 5℃ 30 d可使小菜蛾羧酸酯酶 (Car E)和谷胱甘肽 - S-转移酶 (GST)活力显著抑制 ,35℃ 2 d还可使小菜蛾乙酰胆碱酯酶 (ACh E)活力显著抑制 ,但 5℃4 d对小菜蛾 ACh E、Car E和 GST活力影响差异不显著。当反应温度均为 2 5℃时 ,在 35℃ 2 d、2 5℃ 2 d和 5℃ 30 d下饲养的小菜蛾 ACh E对甲胺磷和克百威的敏感性 (K i值 )没有明显变化 ,但酶与杀虫剂在 37℃的反应温度下 ,甲胺磷、敌敌畏和克百威对在 2 5℃下饲养的小菜蛾 ACh E的抑制作用显著增高。上述结果表明环境温度会影响小菜蛾的解毒代谢及杀虫剂对 ACh E的抑制作用。探讨了环境温度的季节性变化对田间小菜蛾有机磷抗性水平影响的毒理学机制。     

氯虫苯甲酰胺抗性小菜蛾的产卵行为和交配节律观察

【目的】为了明确氯虫苯甲酰胺抗性小菜蛾Plutella xylostella(L.)的交配节律、产卵节律和产卵部位偏好是否发生变化,以及这些行为变化是否有利于小菜蛾抗药性的形成。【方法】采用室内观察的方法,比较了小菜蛾氯虫苯甲酰胺抗性品系与室内品系在产卵行为和交配节律上的差异。【结果】(1)抗性品系在甘蓝植株上产卵时偏好选择中下部叶片,L1-L5叶上卵量占总卵量的比例,抗性品系显著高于室内品系;抗性品系在叶背面产卵的比例也高于室内品系,但差异不显著;而在心叶、叶柄和茎干部位3个品系间的产卵比例基本相当;(2)3个品系交配比例最高的时间段均在0:00—6:00,但交配比率第二高的时间段3个品系间存在差异,室内品系(29.40%)和中抗品系(25.91%)出现在18:00—24:00,而高抗品系(28.67%)出现在12:00—18:00;(3)室内品系、中抗品系和高抗品系白天产卵量占总产卵量的比例分别为39.49%、37.57%和42.67%,高抗品系白天产卵比例明显增加。但3个品系在4个时间段产卵从多到少规律一致,依次是0:00—6:00,12:00—18:00,18:00—24:00,6:00—12:00。【结论】抗性品系在甘蓝植株上产卵时,选择的部位更靠近植株的中下部,趋向于叶片背部。抗性品系和室内品系的交配节律也存在一定的差异,交配高峰期有一定程度的错开。氯虫苯甲酰胺抗性品系的这些行为变化有利于抗性基因的纵向传递,但是否影响抗性形成的速度需要进一步研究。