Mitochondrial impacts of insecticidal formate esters in insecticide‐resistant and insecticide‐susceptible Drosophila melanogaster

BACKGROUND: Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide‐susceptible strain (Canton‐S) and a strain resistant by cytochrome P450 overexpression (Hikone‐R). RESULTS: In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant‐induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton‐S strain (r² = 0.70). The resistant Hikone‐R strain showed no correlation (r² < 0.0001) between formate ester hydrolysis and cytochrome c release. CONCLUSION: The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate‐ester‐induced neurotoxicity. Copyright © 2009 Society of Chemical Industry     

11种植物源杀虫活性成分对草地贪夜蛾的毒力测定

草地贪夜蛾是2018年入侵我国的重大迁飞性害虫, 对玉米等农作物生产构成重大威胁。为开发新型生物农药产品,提供防效更高的生物农药品种, 本研究采用点滴法评价了蛇床子素、大蒜素、补骨脂素、闹羊花毒素Ⅲ、印楝素、对叶百部碱、莨菪碱、血根碱、辣椒碱、苦参碱和丁香酚等共11种植物源杀虫活性成分对草地贪夜蛾2龄幼虫的毒力水平。结果显示:这11种杀虫活性成分对草地贪夜蛾2龄幼虫均有毒杀作用, 但起效时间都有不同程度的滞后。用药后48、72 h和168 h, 不同杀虫活性成分对草地贪夜蛾2龄幼虫的LD50范围分别为0.212～14.382 μg/头、0.191～11.675 μg/头和0.164～9.463 μg/头。药后72 h 毒力大小顺序为:丁香酚＞印楝素＞闹羊花毒素Ⅲ＞补骨脂素＞血根碱＞大蒜素＞苦参碱＞莨菪碱＞蛇床子素＞辣椒碱＞对叶百部碱。研究结果为利用丁香酚等活性成分开发防治草地贪夜蛾的生物农药新产品提供了技术支撑。     

Synthesis and insecticidal activity of lipophilic amides. Part 1: Introductory survey,and discovery of an active synthetic compound

A group of naturally occurring isobutylamides and related compounds exhibit a range of biological properties including activity against insects. Their potential as a starting point for developing new insecticides, needed to control pests resistant to other classes of insecticides, is discussed, following an extensive survey of known structures and activities. Few of the known compounds were sufficiently active and well characterised to point to sound structure-activity relationships, so synthetic analogues were examined. One of these [(2E,4E)-N-(2-methylpropyl)-6-phenylhexa-2,4-dienamide] was sufficiently active, and prompted the investigations reported here and in subsequent papers in the series. Analogues of this compound designed to establish areas where structural variation is possible are also described.     

Ethyl formate,formic acid and ethanol in air,wheat, barley and sultanas: analysis of natural levels and fumigant residues

Ethyl formate and ethanol in air, eg in fumigant studies, were readily detected by gas chromatography (GC) (flame ionisation). Residues in wheat, barley and sultanas were analysed by GC, after extraction in polar solvents (eg methanol, aqueous propanol). Both natural levels and levels resulting from fumigation with ethyl formate were measured. Formic acid was extracted from commodities with polar solvents (eg methanol, water) and analysed by GC after esterification. Solvent extracts of commodities were concentrated after addition of disodium hydrogen orthophosphate, and an aliquot added to acidified alcohols (several combinations of acids and alcohols were tested) in a sealed container. Formic acid esters were determined by GC, from headspace sampling over the esterification solvent. Esterification was faster with strong acids than with boric acid. However, esterification with boric acid/butanol gave the least interference of all tested methods. Product identity was confirmed by GC/mass spectrometry. High natural levels of formic acid, and low natural levels of ethyl formate and ethanol, presented problems in identifying residues arising from fumigation. These natural levels are relevant to food regulations for ethyl formate, especially those based on ‘total formic acid, free and combined’. Polar columns (eg FFAP, carbowax) were useful for measurement of formic acid esters, which eluted before the alcohols used for esterification or extraction, whereas elution followed the molecular mass on non-polar columns, such as GS-Q or DB-624. © 1999 Society of Chemical Industry     

Design, synthesis, insecticidal activity and structure-activity relationship of 3,3-dichloro-2-propenyloxy-containing phthalic acid diamide structures

BACKGROUND: Phthalic acid diamide derivatives are among the most important classes of synthetic insecticides. In this study, a 3,3‐dichloro‐2‐propenyloxy group, the essential active group of pyridalyl derivatives, was incorporated into phthalic acid diamide derivatives with the aim of combining the active groups to generate more potent insecticides. RESULTS: Thirty‐one new phthalic acid diamides were obtained, and these were characterised by ¹H and ¹³C NMR. The structure of N²‐[1,1‐dimethyl‐2‐(methoxy)ethyl]‐3‐iodo‐N¹‐[4‐(3,3‐dichloro‐2‐propenyloxy)‐3‐(trifluoromethyl)phenyl]‐1,2‐benzenedicarboxamide was determined by X‐ray diffraction crystallography. The insecticidal activities of the compounds against Plutella xylostella were evaluated. The title compounds exhibited excellent larvicidal activities against P. xylostella. Structure‐activity relationships revealed that varying the combination of aliphatic amide and aromatic amide moieties, or the nature and position of substituent Y on the aniline ring, could aid the design of structures with superior performance. CONCLUSION: A series of novel phthalic acid diamides containing a 3,3‐dichloro‐2‐propenyloxy group at the 4‐position of the aniline ring were designed and synthesised. Structure‐activity relationships with the parent structure provided information that could direct further investigation on structure modification. Copyright © 2012 Society of Chemical Industry     

气相色谱法同时检测水果中氟吡菌酰胺、肟菌酯及其代谢物肟菌酸的残留量

采用气相色谱法建立了水果中氟吡菌酰胺、肟菌酯及其代谢物肟菌酸残留的分析方法。样品用乙腈匀浆提取,氯化钠和无水硫酸镁盐析后,经N-丙基乙二胺（PSA）分散固相萃取净化,气相色谱-电子捕获检测器（GC-ECD）检测。结果表明:在0.05~1 mg/kg添加水平下,氟吡菌酰胺、肟菌酯及其代谢物肟菌酸的平均回收率为79%~120%,相对标准偏差（RSD）为0.7%~16%。氟吡菌酰胺、肟菌酯及其代谢物肟菌酸在不同水果样品中的检出限（LOD）分别为0.01、0.015和0.01 mg/kg,定量限（LOQ）均为0.05 mg/kg。该方法快速、简单和稳定,可以满足水果中氟吡菌酰胺、肟菌酯及其代谢物肟菌酸残留量的检测要求。     

超高效液相色谱-串联质谱法快速检测核桃中戊唑醇､肟菌酯及肟菌酸残留

建立了超高效液相色谱-串联质谱法(UHPLC-MS/MS)同时测定戊唑醇?肟菌酯及其代谢物肟菌酸在核桃中的残留检测方法?样品中待测农药组分采用2%乙酸乙腈溶剂振荡提取, 弗罗里硅土(florisil)净化, 利用乙腈和0.2%甲酸水作为流动相梯度洗脱, C18色谱柱分离, 在多反应监测模式下定量分析, 基质外标法定量?结果表明:戊唑醇添加水平为5~500 μg/kg时, 戊唑醇在核桃中添加回收率为80.3%~100.8%, 相对标准偏差(RSD)为2.5%~11.7%; 肟菌酯和肟菌酸添加水平为2~200 μg/kg时, 肟菌酯在核桃中添加回收率为91.1%~102.9%, 相对标准偏差(RSD)为1.7%~12.5%; 肟菌酸在核桃中添加回收率为91.7%~101.9%, 相对标准偏差(RSD)为3.7%~9.2%?戊唑醇?肟菌酯和肟菌酸溶剂标准曲线和基质标准曲线在1~1 000 μg/L范围内线性关系良好, 相关系数均大于0.99?戊唑醇定量限为5 μg/kg, 肟菌酯和肟菌酸定量限均为2 μg/kg?实际样品检测中, 戊唑醇残留结果为6~99 μg/kg, 肟菌酯残留结果为<2~103 μg/kg, 肟菌酸均小于检测限?该方法操作简便?快捷和准确, 满足在核桃实际样品中戊唑醇?肟菌酯及其代谢物肟菌酸的残留检测要求?     

Relationship among expression, amplification, and methylation of FE4 esterase genes in Italian populations of Myzus persicae (Sulzer) (Homoptera: Aphididae)

The wide use of insecticides containing an esteric group selected resistant Myzus persicae populations characterised by the overproduction of one of two closely related carboxylesterases (E4 and FE4). In this paper, we present data collected from Italian population indicating that all the 22 populations analysed possess amplified FE4 gene only. The estimation of FE4 copy number, carried out by densitometric scanning of dot and Southern blots, puts in evidence that the different populations possess a gene copy number ranging from 6 to 104. Statistical analysis shows the existence of a high positive correlation between gene copy number and total esterase activity. In aphid strain with low FE4 copy number, these genes are almost totally methylated. On the contrary, aphid strains with high FE4 gene number evidenced highly variable methylation levels and absence of correlation between the number of genes and their methylation state. The same result has been observed when comparing FE4 methylation levels and esterase activity.     

A new derivative of fipronil: Effect of adding a glycinyl group to the 5-amine of pyrazole on phloem mobility and insecticidal activity

Glycinyl fipronil, a new fipronil derivative with 5-amine acylated by glycine, was synthesized. The phloem mobility of the synthesized derivative was higher that of the parent compound in intact soybean seedling and its insecticidal activity against the 3rd instar larvae of Plutella xylostella was comparable with that of fipronil.     

Selectivity, acetylcholinesterase inhibition kinetics, and quantitative structure-activity relationships of a series of N-(2-oxido-1,3,2-benzodioxa-phosphol-2-yl) amino acid ethyl or diethyl esters

The inhibitory effects of a recently introduced series of the titled compounds on insect and mammalian acetylcholinesterase (AChE) activity were examined, where the median inhibition concentration (I₅₀) and the inhibition kinetic parameters, bimolecular inhibition rate constant (k_i), affinity constant (K_a), and phosphorylation rate constant (k_p), were determined for each compound. Results indicated that all examined dioxaphospholenes had less inhibitory effects on mammalian AChE than fenitrothion, a commercial pesticide with moderate mammalian toxicity. The highest selectivity was obtained with compounds containing glutamic and leucine moieties (2.70 and 2.18, respectively) while selectivity of fenitrothion was 0.93. The low inhibitory effects of the examined dioxaphospholenes on mammalian AChE were attributed to their low phosphorylation rates (k_p < 2.2 min⁻¹) compared to that of fenitrothion (k_p = 4.84 min⁻¹). QSAR equations indicated that the inhibition process is controlled mainly by both the phosphorylation rate (direct effect) and the affinity of compounds toward the enzyme (inverse effect). Although the compounds’ hydrophobicity had no effects on the inhibition process, it affects the compounds’ toxicity since it affects the ability of compounds to penetrate insects to reach the enzyme active site.     

Efficacy of vaporised ethyl formate/carbon dioxide formulation against stored‐grain insects: effect of fumigant concentration,exposure time and two grain temperatures

BACKGROUND: The ethyl formate/carbon dioxide (CO₂) formulation Vapormate? is a rapid‐acting fumigant being developed for the control of stored‐grain insects. The effects have been investigated of concentration, exposure times of 1, 3, 24 and 72 h and two grain temperatures, 15 and 25 °C, on its efficacy against mixed‐stage cultures of Sitophilus oryzae (L.) Tribolium castaneum (Herbst) and strongly phosphine‐resistant Rhyzopertha dominica (F.) strain QRD569. RESULTS: High mortalities (≥92%) of mixed‐stage cultures of all three species were obtained when grain was fumigated with the formulation (193 g m^?3 ethyl formate) for 1 h. Complete control of R. dominica QRD569 and T. castaneum was achieved with 63 and 76 g m^?3 ethyl formate respectively, with exposure for 24 h, whereas mean mortality of S. oryzae was 86% under the same conditions. Mortalities of S. oryzae juvenile stages were significantly lower than adults under the conditions tested, which was due to pronounced tolerance of mid‐stage pupae to the fumigant. Reducing grain temperature from 25 to 15 °C had no effect on insect mortality. CONCLUSION: Ethyl formate/CO₂ formulation is highly effective against stored‐grain insects over a range of concentrations and exposure times. Efficacious fumigations were conducted in as little as 1 h, and a strongly phosphine‐resistant R. dominica strain was readily controlled with the fumigant. Copyright © 2009 CSIRO, Australia. Published by John Wiley & Sons, Ltd     

Purification and characterisation of proteins secreted by the entomopathogenic fungus Metarhizium anisopliae with insecticidal activity against adults of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae)

BACKGROUND: The control of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wied) is usually performed with protein bait sprays incorporating chemical insecticides that may have adverse effects on humans, non‐target organisms and the environment. In recent years, scientists have sought more environmentally friendly insecticides for medfly control, such as plant‐ and microorganism‐derived compounds. Among these compounds, entomopathogenic fungi are an unexplored source of natural insecticides. RESULTS: The crude soluble protein extract (CSPE) of the entomopathogenic fungus Metarhizium anisopliae (Mestch.) (strain EAMa 01/58‐Su) shows chronic insecticidal activity when administered per os. Mortality in flies exhibits a dose response. The CSPE produces an antifeedant effect in adult flies, a result probably due to a progressive deterioration of the fly midgut after ingestion of the extract. Protease and temperature treatments show that insecticidal activity against C. capitata is due to proteinaceous compounds that are highly thermostable. Four monomeric proteins from this crude extract have been purified by liquid chromatography and gel electroelution. Although all four monomers seem to be involved in the insecticidal activity of the CSPE, the 15 kDa and the 11 kDa proteins appear to be mainly responsible for the observed insecticidal effect. CONCLUSIONS: Four new fungal proteins with insecticidal activity have been purified and identified. These proteins might be combined with insect baits for C. capitata biocontrol. Copyright © 2009 Society of Chemical Industry     

茉莉酸诱导小麦抗病虫性初步研究

初步研究了茉莉酸诱导对小麦苗抗病虫能力的影响,结果显示,小麦在喷施茉莉酸后能够提高植株对麦长管蚜和小麦白粉病菌、小麦叶锈病菌的抵抗能力,可显著降低小麦白粉病、叶锈病的发病级别和病斑数量,对麦长管蚜则在体重和产仔数量上有显著的抑制作用。     

A novel probe for harpin receptor in nonhost plants: Monoclonal anti-idiotypic antibodies as internal images of HarpinXoo active sites

Harpins constitute one group of effector proteins which elicit a hypersensitive response in nonhost plants, but the subcellular localization and tissue distribution of harpin receptors are still controversial. Antigen mimicry by anti-idiotypic antibodies is employed as a reliable strategy to probe receptors that are present in very low concentrations in the organism. In this study, a monoclonal anti-idiotypic antibody (Ab2), named 6B2, was elicited by F(ab′)₂ fragments digested from the purified polyclonal antibody specific for Harpin_Xoo (Ab1). 6B2 competed with Harpin_Xoo for binding to Ab1 and the total protein extracted from tobacco leaves indicated its anti-idiotypic character and internal image property. The relevance of antigen mimicry was further confirmed by eliciting a third generation antibody (Ab3), which was shown not only to bind to Ab2 competing with Ab1 but also to react with the original antigen, Harpin_Xoo. Taken together, these results demonstrate functional and biochemical mimicry of Harpin_Xoo by Ab2 and suggest that 6B2 can be a useful tool in probing its receptor in nonhost plants and other downstream studies.     

Effects of the molluscicidal agent GA-C13:0, a natural occurring ginkgolic acid, on snail mitochondria

Ginkgolic acids (GAs) from the leaves and sarcotesta of Ginkgo biloba L. represent a new kind of molluscicide agent. To date, the mechanism(s) for the observed molluscicidal activity remains largely unknown. Since GA-C_13:0 has effectively inhibited snail mobility, we examined the effects of the compound on mitochondrial function and gene expression as compared to niclosamide. Snail mitochondrial damage induced by GAs was tested using transmission electron microscopy (TEM) and gene-expression profiling of five mitochondrial enzymes using real-time PCR. GA-C_13:0 was found to have a pronounced effect on snail mitochondria with gross ultrastructural changes. In addition, GA-C_13:0 was also found to inhibit the gene expression of four mitochondrial enzymes including cytochrome c oxidase, adenosine triphosphate (ATP) synthase, cytochrome b and dihydronicotinamide adenine dinucleotide (NADH) dehydrogenase. In contrast, niclosamide did not show such effects on mitochondrial function and gene expression, suggesting that the molluscicidal activity of GA-C_13:0 and niclosamide differed. Our results imply that snail mitochondria are a potential target for the molluscicidal activity of ginkgolic acids.