两种金小蜂体内Wolbachia的wsp基因分子检测及序列分析

采用Wolbachia的通用引物及A和B大组特异性引物对蝶蛹金小蜂Pteromalus puparum和丽蝇蛹集金小蜂Nasonia vitripennis体内Wolbachia的wsp基因进行PCR扩增及序列测定，并对测定的序列进行了同源性比较和基因特征分析。结果表明：蝶蛹金小蜂和丽蝇蛹集金小蜂均被A和B两个大组的Wolbachia复合感染；同种寄生蜂的雌蜂和雄蜂的wsp基因片段序列完全一致。采用通用引物从蝶蛹金小蜂中扩增出Wolbachia的wsp基因片段序列的长度为540bp，属于B大组中Pip组，而从丽蝇蛹集金小蜂中扩增出Wolbachia的wsp基因片段序列的长度为558bp，属于A大组中Uni组。用A-Wolbachia引物从蝶蛹金小蜂和丽蝇蛹集金小蜂中扩增出的wsp基因片段序列长度为548bp，同源性达99．8％；而用B-Wolbachia引物从两者中扩增的两条wsp基因片段序列长度分别为424bp和439bp同源性达87．5％．     

寄生蜂细胞色素P450基因家族进化分析

寄生蜂是重要的天敌昆虫,被广泛应用于生物防治,但在实际生产应用中面临诸多问题,如环境适应性和耐药性问题。细胞色素P450是真核生物中广泛存在的超基因家族酶系,参与农药、植物次生代谢物及环境有害物质等的代谢,同时也参与昆虫体内多种内源性化合物的合成与代谢。本文通过对植食性榕小蜂和9种寄生蜂基因组数据的生物信息学分析,共获得615个P450基因,分析表明不同寄生蜂P450基因家族的差异主要发生在CYP3和CYP4簇,而CYP2和Mito簇相对保守;相较于外寄生蜂,内寄生蜂的P450超基因家族有明显的收缩现象;此外,发现了2个在寄生蜂中1：1：1高度保守的P450基因,CYP18和CYP314,以及茧蜂特有的CYP304基因和内寄生蜂特有的CYP28基因。本研究从基因组水平系统地分析了寄生蜂P450超基因家族的进化,为寄生蜂的遗传改良和规模化饲养等提供理论基础。     

宿主蝇蛹对丽蝇蛹集金小蜂寄生与产子蜂量的影响

本文报道宿主蝇种、蜂蛹比例和蝇蛹日龄对丽蝇蛹集金小蜂寄生率及产子蜂量的影响。该蜂在家蝇、丝光绿蝇、巨尾阿丽蝇和红尾粪麻蝇蛹中的寄生率分别为:85％、92％、76％和14％。蜂蛹最适比例为:1:2.5～5。该蜂可产子蜂于1～7日龄的巨尾阿丽蝇蛹,以1～2日龄蝇蛹产子量最高。     

两种金小蜂类icarapin蛋白潜在致敏性的初步评价

西方蜜蜂icarapin蛋白是一种常见的人畜过敏原,在寄生蜂类群中也有其同源物存在。为探究寄生蜂中类icarapin蛋白的潜在致敏性,本文克隆并表达了蝶蛹金小蜂和丽蝇蛹集金小蜂中类icarapin蛋白编码基因。序列分析表明,两种金小蜂类icarapin蛋白编码基因开放阅读框分别为645 bp和714 bp,生物信息学预测其均具有潜在的致敏性。经Ni离子亲和层析柱纯化得到了高纯度的重组类icarapin蛋白,在此基础上建立了小鼠致敏模型,初步探究了其潜在致敏性。结果表明,注射2种寄生蜂和西方蜜蜂重组icarapin蛋白的小鼠血清中组胺和免疫球蛋白IgE浓度均高于注射PBS的阴性对照组,表明这2种金小蜂类icarapin蛋白有潜在的致敏风险。本研究为寄生蜂源活性蛋白在应用前的安全性评估提供了一定的参考。     

丽蝇蛹集金小蜂寄生行为的观察

丽蝇蛹集金小蜂Nasonia uitripennis是外栖性有害蝇类的重要天敌,可寄生于13种蝇蛹,对丽蝇科种类,尤其是绿蝇的虫口具有一定的调节作用。为了更好地了解和掌握该蜂的生物学特性与饲养技术,以发挥其防制蝇类的作用,作者于1984年对其某些行为学做了初步的研究,现报告如下。     

短管赤眼蜂Argonaute蛋白基因家族鉴定及表达分析

动物和植物Argonaute蛋白在RNA诱导的基因沉默中发挥重要作用。本研究采用生物信息学方法在全基因组水平鉴定短管赤眼蜂Argonaute蛋白基因家族,鉴定并命名6个TpAGO蛋白。蛋白结构及系统进化分析将TpAGO家族蛋白分为PIWI和Ago两个亚家族。TpAGO蛋白二级结构的数量比例差异不大,分布却存在一定差异,其中α-螺旋分布差异尤为显著。TpAGO蛋白基因结构表现明显的多样性,基因外显子数量差异明显,从1到20个数量不等;Ka/Ks分析表明TpAGO2和TpAGO5,TpAGO3和TpAGO4两对基因间进化动力为正选择作用,其他组合间表现为纯化选择。EST表达分析发现TpAGO基因在昆虫的卵期、幼虫期、蛹期和成虫期均有不同程度的表达,而且靶向EST在同一物种中具有单一性别(雌性或雄性)表达特性,表明短管赤眼蜂TpAGO蛋白基因可参与赤眼蜂性别及发育调控。该研究不仅有助于揭示赤眼蜂产雌孤雌生殖的表观遗传机理,也有助于推动赤眼蜂等天敌的生防应用。     

丽蝇蛹集金小蜂在山西的寄主范围及其发生季节

丽蝇蛹集金小蜂 Nasonia vitripennis(Walker)是寄生于常见蝇类的一种金小蜂,世界性分布,对重要的医学蝇类,如家蝇、绿蝇及麻蝇等虫口具有一定的控制作用(Legner 1967,Rutz 1980a)。了解该蜂的寄主范围及其季节活动有助于对该蜂的培养、释放、保护和利用。作者自1980年发现该蜂寄生于丝光绿蝇幼虫(薛瑞德1982)以来,     

寄生于丝光绿蝇的一种金小蜂

丝光绿蝇Lucilia sericata是常见的丽蝇科昆虫,可传播数种肠道传染病及引起人体蝇蛆病,与人类关系较为密切。1980年在研究蝇类中,发现一种金小蜂Nasonia sp.寄生于丝光绿蝇的蛹内,使之不能羽化。 1981年6月,在山西医学院动物室腐败兔尸体周围的泥土中挖取蝇蛹15头,在室内     

外源哌啶酸对丽蝇蛹集金小蜂生长发育及耐寒性的影响

本研究以丽蝇蛹集金小蜂及其寄主红尾肉蝇为研究对象,评价了寄主饲料中添加哌啶酸对金小蜂生长发育和耐寒性的影响。结果表明,寄主饲料中添加哌啶酸的最适浓度为2%,无论金小蜂幼虫滞育与否,添加哌啶酸均能一定程度上降低金小蜂的鲜重、干重、含水量、水合量及过冷却点,其中对鲜重、含水量及水合量的影响显著。添加哌啶酸也能显著缩短非滞育金小蜂成蜂的寿命,降低其繁殖力,但对其发育历期和性比无显著影响。添加哌啶酸还能显著提高金小蜂幼虫的耐寒性,其中对滞育幼虫的耐寒性提高最显著。本研究结果证明哌啶酸对金小蜂的生长发育具有一定抑制作用,但可显著提高其耐寒性,这为深入研究哌啶酸的功能机制及促进金小蜂的利用提供了理论参考。     

小豆AP2/ERF基因家族鉴定及其应答锈菌侵染的表达分析

 为明确小豆VaAP2/ERF家族基因在小豆应答豇豆单胞锈菌(Uromyces vignae)侵染中的作用,本研究采用生物信息学方法从小豆基因组中鉴定VaAP2/ERFs家族基因186个,包括AP2(24个)、RAV(2个)、Soloist(1个)及ERF(159个)四个亚家族,其中ERF亚家族又可分为DREB和ERF两个亚类。基因结构分析发现,68.3%的VaAP2/ERFs基因不具内含子。保守基序分析发现,各亚族成员保守域组成类似,可作为基因家族分类的重要依据。前期转录组测序发现VaAP2/ERF中的6个成员接种后24 h显著上调,进一步应用qPCR技术对其在‘1-D-3’(免疫)及‘宝清红’(感病)中应答锈菌侵染的表达分析发现,免疫品种中6个成员均于接种后6 h及120 h显著上调,另有2个于接种后12 h也显著上调,表明上述基因均在小豆抗锈病中发挥正调控作用。     

Spinosad resistance in female Musca domestica L. from a field-derived population

BACKGROUND: Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. RESULTS: In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC₅₀, whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. CONCLUSION: This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry     

Biochemical and molecular mechanisms of diafenthiuron resistance in the whitefly,Bemisia tabaci

B-biotype Bemisia tabaci has developed high levels of resistance to many insecticides. To investigate the risks and explore possible mechanisms of resistance to diafenthiuron in B. tabaci, a 32.8-fold diafenthiuron-resistant strain (R-DfWf) was established after selection for 36 generations compared with the susceptible strain (S-Lab). Biochemical assays showed that the activity of cytochrome P450 towards p-NA was significantly higher (4.37-fold higher) in the R-DfWf strain than in the S-Lab strain. Similarly, the carboxylesterase (COE) activity and glutathione S-transferase (GST) activity were also significantly higher (3.12- and 1.83-fold higher, respectively) in the R-DfWf strain than in the S-Lab strain. The expression of five of seven P450 genes was significantly higher (>3-fold) in the R-DfWf strain than in the S-Lab strain. The expression of COE2 was significantly higher (>2.5-fold) in the R-DfWf than in the S-Lab strain. The expression of GST and GST2 was significantly higher (>2.3-fold) in the R-DfWf than in the S-Lab. Thus, cytochrome P450, COE and GST may appear to be responsible for the resistance to diafenthiuron in B. tabaci. It is also valuable for usage of insecticides for resistance management and control of this species.     

白背飞虱两个P450基因的克隆、序列分析及杀虫剂胁迫下的表达分析

细胞色素P450属于超基因家族,与昆虫对杀虫剂抗药性产生有重要关系。本文在白背飞虱转录组数据的基础上,克隆到了两个P450基因,分别命名为SfCYP4DE1和SfCYP353D1v2,其分别编码534和471个氨基酸,分子量约为61.567和53.762 kDa,理论等电点为9.18和8.74。结构域分析发现,SfCYP4DE1和SfCYP353D1v2氨基酸序列中均包含5个P450基因共有序列。噻虫嗪、噻嗪酮和阿维菌素的LC₁₀、LC₂₅、LC₅₀和LC₉₀4个不同浓度处理白背飞虱3龄若虫48 h后,采用RT-qPCR技术测定SfCYP4DE1和SfCYP353D1v2两个基因的相对表达量,结果表明：噻虫嗪、噻嗪酮LC₁₀和LC₂₅浓度胁迫后SfCYP4DE1基因相对表达量均显著低于对照组,但随着浓度继续的加大,SfCYP4DE1基因的表达量出现上升的趋势。不同的是,经阿维菌素LC₁₀和LC₂₅浓度胁迫后,SfCYP4DE1基因的表达量均显著高于对照组,且随着浓度增加其表达量升高。与对照组相比,经噻虫嗪不同浓度胁迫后,SfCYP353D1v2基因表达量均呈现出诱导增加的趋势,且LC₁₀、LC₅₀和LC₉₀处理组显著高于对照组。噻嗪酮LC₁₀浓度处理能够显著诱导SfCYP353D1v2基因的表达,但阿维菌素LC₁₀浓度处理下其表达量被显著抑制。另外,以阿维菌素LC₅₀和LC₉₀浓度处理后,SfCYP353D1v2基因的表达量被显著诱导。以上研究结果可为进一步研究白背飞虱P450基因对杀虫剂解毒代谢途径提供基础资料。     

四种P450基因调控小菜蛾对氯虫苯甲酰胺的抗性

为研究CYP6家族P450基因在小菜蛾Plutella xylostella代谢氯虫苯甲酰胺中的作用,利用浸叶法测定不同小菜蛾种群3龄幼虫对氯虫苯甲酰的抗性水平,采用实时荧光定量PCR(quantitative realtime PCR,q RT-PCR)方法分析CYP1v3、CYP1v4、CYP6B6和CYP6f这4种P450基因在小菜蛾不同抗性种群体内的表达差异及杀虫剂的短期诱导效应,并通过RNA干扰技术沉默4种P450基因后分析小菜蛾对氯虫苯甲酰胺的敏感性。结果显示,4种P450基因在中等抗性水平小菜蛾种群体内高表达;氯虫苯甲酰胺处理可诱导4种基因显著上调表达。分别沉默4种P450基因后,处理组小菜蛾的P450酶活力显著下降37.60%～54.82%,且处理组小菜蛾的死亡率显著高于对照组;同时沉默4种基因处理组的小菜蛾P450酶活力显著低于其他各处理组。表明4种P450基因可能同时参于小菜蛾对氯虫苯甲酰胺的代谢。     

解毒酶和转运蛋白介导的害虫抗药性分子机制研究进展

害虫抗药性是导致杀虫剂防效降低的一个重要因素,而抗性机制的阐明是害虫抗药性综合治理的基础。研究表明,代谢能力增强是害虫抗药性产生的重要原因,害虫对杀虫剂等外源物质的代谢需要细胞色素P450酶系(P450s)、羧酸酯酶(CarEs)、谷胱甘肽S-转移酶(GSTs)、UDP-葡萄糖醛酸转移酶(UGTs)和ATP结合盒转运蛋白(简称ABC转运蛋白)等解毒酶和转运蛋白的参与。结合近年来对害虫抗药性分子机制的研究进展,本文综述了上述解毒酶和转运蛋白参与杀虫剂抗药性的分子机制,并对害虫抗药性治理的新方法进行了展望。     

Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster

Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, cypermethrin, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No obvious abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.