基于转录组测序解析草地贪夜蛾对溴氰虫酰胺的解毒代谢分子机制

为阐明草地贪夜蛾Spodoptera frugiperda对溴氰虫酰胺的解毒代谢分子机制,通过LC₅₀的溴氰虫酰胺诱导草地贪夜蛾3龄幼虫后,利用酶活测定和转录组测序鉴定解毒代谢相关基因,并采用实时荧光定量PCR技术对细胞色素P450单加氧酶(cytochrome P450 monooxygenase,P450)基因进行验证分析。结果表明,经LC₅₀的溴氰虫酰胺处理后,草地贪夜蛾3龄幼虫体内3种解毒代谢酶活性较对照均有所升高,但仅P450活性较对照显著升高,而谷胱甘肽S-转移酶和羧酸酯酶与对照无显著差异。经LC₅₀的溴氰虫酰胺处理后草地贪夜蛾3龄幼虫转录组中共筛选到1 408个差异表达基因,其中上调表达的基因有935个,下调表达的基因有473个。药物代谢-细胞色素P450通路、药物代谢-其他酶通路及细胞色素P450对异生物质的代谢通路中有超过20个基因存在差异表达。在草地贪夜蛾转录组中筛选鉴定到121个P450基因,其中,属于CYP2、CYP3、CYP4以及Mito家簇的基因分别有9、45、58和9个,而经LC_5...     

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。     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

Regulation of cytochrome P450 expression in Drosophila: Genomic insights

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

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。     

A cytochrome P450 gene involved in methidathion resistance in Amblyseius womersleyi Schicha (Acari: Phytoseiidae)

This study reports on a CYP4 gene possibly involved in methidathion resistance in Amblyseius womersleyi Schicha. This is the first report on a CYP4 gene from a phytoseiid mite. Four different CYP4 genes (CYP4-a, CYP4-b, CYP4-c, and CYP4-d) were detected in this predaceous mites. No differences in CYP4 expression levels were detected for the strains Ishigaki S, Kanaya S (methidathion susceptible strains) and Kanaya R (methidathion resistant strain), using RT-PCR method for CYP4-a, CYP4-b, and CYP4-c. In the case of CYP4-d, the expression level in Kanaya R was significantly higher than in susceptible strains. Studies using RT-PCR indicated that CYP4-d gene is expressed in all stages of A. womersleyi, with a little higher expression in protonymph, deutonymph and adult, in comparison to egg and larva. Significant correlation between CYP4-d expression and monooxygenase activity was observed for the different strains and life stages of A. womersleyi.     

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.     

Target site insensitivity mutations in the AChE and LdVssc1 confer resistance to pyrethroids and carbamates in Leptinotarsa decemlineata in northern Xinjiang Uygur autonomous region

A survey of resistance to five conventional insecticides was conducted in 2009 and 2010 for the first generation 4th-instar larvae of Leptinotarsa decemlineata from Urumqi, Changji, Qitai and Qapqal. Compared with the Tekes population, a reference susceptible population, the Changji and Qapqal populations exhibited very high to moderate levels of resistance to cyhalothrin and deltamethrin, moderate to high levels of resistance to carbosulfan and carbofuran, and low levels of resistance to azinphosmethyl. Moreover, the Urumqi and the Qitai populations reached a high and a moderate level of resistance to carbosulfan, respectively. Synergistic effects of triphenyl phosphate, diethylmeleate, and piperonyl butoxide on cyhalothrin and carbosulfan in Changji population revealed that cytochrome P450s were involved in the resistance to cyhalothrin but not carbosulfan. A modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVssc1 genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were 17.6% and 14.7%, 50.6% and 42.2%, 49.9% and 41.7%, 51.3% and 41.4%, and 44.8% and 47.4%; to pyrethroids (L1014F mutation) were 5.8% and 8.7%, 36.1% and 27.0%, 41.8% and 24.8%, 12.2% and 9.7%, and 7.9% and 10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. I392T point mutation in the AChE was detected by RT-PCR among 18 individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVscc1 are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region.     

亚致死剂量氟啶虫胺腈对灰飞虱细胞色素P450的影响

为明确氟啶虫胺腈对灰飞虱Laodelphax striatellus细胞色素P450的影响,评估其抗药性风险,采用点滴法、酶活力分析法和实时荧光定量PCR法分别测定了氟啶虫胺腈对灰飞虱的毒性及对其细胞色素P450酶活力和P450基因表达量的影响。结果表明,氟啶虫胺腈对灰飞虱的致死中量LD_(50)随着虫龄的增加而升高,1~5龄若虫的LD_(50)为0.10~0.94 ng/头,雌、雄成虫的LD_(50)分别为1.09 ng/头和1.07 ng/头。氟啶虫胺腈对4龄若虫的亚致死剂量LD_(10)、LD_(30)和LD_(50)分别为0.17、0.41、0.76 ng/头,处理灰飞虱4龄若虫后可将其体内P450酶活力分别显著提高1.60、1.97和1.22倍;而各处理响应的P450基因的种类和数量不同,但相对表达量均受到诱导;CYP4DE1、CYP426A1、CYP303A1、CYP4C、CYP6FK1和CYP4C71v2的相对表达量表现出时间效应,表达量高峰在处理后24 h或48 h。表明不同亚致死剂量的氟啶虫胺腈在特定时间点可提高相应的细胞色素P450基因表达量,从而使酶蛋白量增加,可能导致灰飞虱体内细胞色素P450酶活力上升。     

In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations

Biotransformations of profenofos were studied in vitro. Two metabolites, desthiopropylprofenofos and hydroxyprofenofos, were detected by LC-MS after incubation of profenofos with human liver homogenates and different mammalian liver microsomes. The rank order of desthiopropylprofenofos formation in liver microsomes based on intrinsic clearance (V_max/K_m) was mouse > human > rat, while for profenofos hydroxylation it was mouse > rat > human. In view of the ratio between desthiopropylation and hydroxylation intrinsic clearance rates, human liver microsomes were most active in profenofos bioactivation. The interspecies differences and interindividual variation were within range of the default uncertainty/safety factors for chemical risk assessment. CYP3A4, CYP2B6 and CYP2C19 were identified as profenofos-oxidizing enzymes in human liver on the basis of recombinant expressed enzymes and correlation with CYP model activities. The rank order of CYPs in profenofos activation was CYP3A4 > CYP2B6 > CYP2C19, whereas it was the contrary for profenofos hydroxylation. Profenofos inhibited relatively potently several human liver microsomal activities: the lowest IC₅₀ values were about 3 μM for CYP1A1/2 and CYP2B-associated activities. Profenofos is extensively metabolized by liver microsomal CYP enzymes and its interaction potential with several CYP activities is considerable.     

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.     

Feeding behavior of Plutella xylostella and Leptinotarsa decemlineata on leaves treated with Bacillus thuringiensis and esfenvalerate

Studies were conducted to examine interactions between esfenvalerate and Bacillus thuringiensis Berl. in their effects on feeding behavior of larvae of Plutella xylostella L. and Leptinotarsa decemlineata Say. Droplets containing esfenvalerate, B. thuringiensis, the two in combination, and water controls were individually applied to leaf disks. Third-instar P. xylostella larvae were allowed to feed for 24 h on cabbage disks and third-instar L. decemlineata larvae were allowed to feed for approximately 18 h on potato leaf disks. For each insect species and each treatment, the proportion of leaf area removed during the feeding period was regressed on the proportion of droplets removed during that period to test for avoidance of droplet residues. No significant differences were found between treatments in these regressions for L. decemlineata. In contrast, B. thuringiensis, either alone or in combination with esfenvalerate, significantly affected the relationship of leaf and droplet consumption for P. xylostella. No evidence of individual droplet avoidance, i.e. a consistently greater proportion of leaf than droplets consumed, was found for any of the treatments. Proportions of leaf consumed and proportions of droplets consumed were compared among treatments to test for effects on feeding rate. B. thuringiensis significantly reduced feeding rate, both alone and in combination with esfenvalerate for P. xylostella and alone for L. decemlineata. We found no statistically significant differences in feeding rate due to esfenvalerate. The species studied can be expected to consume the same amount of B. thuringiensis whether it is mixed with esfenvalerate or not, whereas esfenvalerate dose by ingestion would be reduced if it were mixed with B. thuringiensis.     

大螟两个mGST1基因的时空分布及经氯虫苯甲酰胺诱导后的表达情况

为明确微粒体谷胱甘肽S-转移酶（microsomal glutathione S-transferase,mGST）是否参与大螟Sesamia inferens对氯虫苯甲酰胺的解毒代谢，采用Illumina深度测序技术分析大螟转录组数据获得mGST1基因序列，利用实时荧光定量PCR技术测定其在大螟不同龄期幼虫和4龄幼虫不同组织中的表达模式，及经不同浓度氯虫苯甲酰胺处理不同时间后的相对表达量。结果表明，共获得2个mGST1基因序列，分别命名为SimGST1-1和SimGST1-2(GenBank登录号分别为OL770267和OL770268)。SimGST1-1和SimGST1-2的开放阅读框分别为456 bp和447 bp，分别编码152个和149个氨基酸，均具有昆虫mGST的特征序列。SimGST1-1和SimGST1-2在大螟不同龄期幼虫体内均有表达，且在4～6龄幼虫体内的相对表达量显著高于1～3龄幼虫。SimGST1-1在4龄幼虫中肠和脂肪体中的相对表达量显著高于在头部和表皮中的相对表达量，SimGST1-2在头部和中肠中的相对表达量显著高于在表皮和脂肪体中的相对表达量。与对照组...     

印楝素对马铃薯甲虫的控制作用

为探讨植物源杀虫剂印楝素对马铃薯甲虫的防治作用,采用叶碟法测定印楝素乳油对其成虫的拒食作用,并通过药效试验测定印楝素乳油对马铃薯甲虫的控制效果。结果显示,印楝素对马铃薯甲虫成虫有较强的非选择性拒食作用,且拒食率随取食时间的增加而升高。在取食36 h后,浓度为6、7.5、10、15和30 mg/L印楝素的拒食率分别为55.5%、59.1%、53.4%、76.1%和63.3%,拒食率与浓度无显著相关性;但印楝素对马铃薯甲虫的选择性拒食率较低,各浓度处理间均无显著差异,取食36 h时,选择拒食率约20%;在田间10 mg/L印楝素溶液施药1 d和3 d后,虫口减退率分别为26.52%和50.76%,显著高于对照组,施药9 d后,马铃薯甲虫的校正防治效果为48.86%。表明印楝素对马铃薯甲虫有一定的控制效果。     

A novel series of O,O-diethyl O-(1,3-disubstituted-1,2,4-triazol-5-yl) phosphorothioates. Part II. Quantitative structure–activity relationships

The in-vivo toxicity data of 33 O,O-diethyl O-(1-alkyl-3-R″-1,2,4-triazol-5-yl) phosphorothioates, reported in Part I of this series, were subjected to multiregression analyses for the following organisms: Leptinotarsa decemlineata (larvae), Macrosiphum euphorbiae (aphids), Culex pipiens (adults), Spodoptera littoralis (larvae) and the rat. The activities were found to depend mainly on the steric parameters of R″, these being best expressed by the values L, B₁ and B₄ of the computer program STERIMOL. It was shown that there were different steric requirements for each species and these are discussed. The predictive values of the best equations were also estimated.     

Diflubenzuron: Some aspects of its ovicidal and larvicidal mode of action and an evaluation of its practical possibilities

Diflubenzuron, 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea, possesses larvicidal and ovicidal activities. On larvae it acts mainly as a stomach poison, yet it sometimes exhibits important contact activity. Though all instars can be controlled, older instars are generally less susceptible than younger ones. Histological inspections of Leptinotarsa decemlineata larvae revealed that after ending exposure to the compound, distortions in newly deposited cuticular layers decreased gradually. Ovicidal effects resulted from direct contact of diflubenzuron with eggs or from contamination of females by contact or feeding. Electron microscopic observations of embryos of Leptinotarsa decemlineata, contaminated via the female, also showed disturbed cuticule formation, suggesting a similar activity of the compound in larvae and in eggs. Spraying the eggs of Leucoptera scitella shows the compound to be mainly ovidical at a rate of 100 mg litre^?1, whilst with lower concentrations (10 and 1 mg litre^?1) the young larval instar will be killed. The levels of cross-resistance to diflubenzuron as a larvicide are low and the compound can be used effectively in the field against populations that are highly resistant to conventional insecticides. Laboratory and field results, based on larvicidal and ovicidal activities of diflubenzuron, are discussed in respect of species belonging to the Diptera, Lepidoptera, Coleoptera, Acarina (Phyllocoptruta oleivora) and Hemiptera (Eurydema oleraceum and Psylla piri).