The biochemical basis of malathion resistance in the sheep blowfly,Lucilia cuprina

The in vivo and in vitro metabolism of [¹⁴C]malathion was studied in susceptible (LS) and malathion resistant (RM) strains of the sheep blowfly, Lucilia cuprina (Wiedemann). No difference was found between strains in the penetration, excretion, storage, or inhibitory potency of the insecticide. However, RM degraded malathion to its α- and β-monocarboxylic acid metabolites more rapidly than LS, both in vivo and in vitro. This enhanced degradation of [¹⁴C]malathion occurred in vitro in both mitochondrial and microsomal fractions of resistant flies. Kinetic analysis revealed that these fractions degraded malathion by discrete mechanisms. The enzymes from the mitochondria of both strains had the same K_m, whereas the microsomal enzyme from the RM strain had a fivefold higher K_m than that from the LS strain. Studies of esterase activities and the effect of enzyme inhibitors showed that both the mitochondrial and microsomal resistance mechanisms were the result of enhanced carboxylesterase activity. It was concluded that increased carboxylesterase detoxification of malathion adequately explained the high level of malathion resistance in RM if rate-limiting factors such as cuticular penetration were taken into account.     

Glutathione S-transferase in the Australian sheep blowfly,Lucilia cuprina (Wiedemann)

Glutathione S-transferase in the Australian sheep blowfly, Lucilia cuprina, was studied using 3,4-dichloronitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The optimum pHs for enzyme activity were 7.5–8.0 and 6.7–7.4 for DCNB and CDNB conjugations, respectively. Inclusion of glutathione and bovine serum albumin in the homogenizing buffer protected the glutathione S-transferase from inhibition by endogenous compounds present in extracts of final instar larvae and of adults less than 7–8 days old. Conjugation activities for DCNB and CDNB increased throughout larval development to reach a peak early in the pupal stage. Activity then decreased through the remainder of the pupal stage and for the first 6–7 days after emergence of the adult. Almost all of the decrease in activity during the first 6 days of the adult occurred in the abdomen, which accounted for 85% of total activity in the adult female at emergence but only 47% at 6 days. Larval DCNB conjugation activity was localized almost entirely in the fat body (94%), whereas only 50% of the CDNB conjugation activity was in the fat body with the remainder in the cuticle (25%), gut (15%), and blood (10%). Adult and larval enzyme was induced ca. three- to four-fold by sodium phenobarbital. The induction was associated with changes in apparent V_max rather than apparent K_m, suggesting that phenobarbital caused increased production of forms of enzymes already present rather than inducing synthesis of altered or new forms.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

内生放线菌A-1对苹果果实轮纹病的防效及防御性酶活性的影响

为揭示内生放线菌A-1对苹果果实轮纹病的防效及防病机制,采用平板法和喷雾处理刺伤接种法,测定了其对苹果轮纹病菌的抑制作用及果实内防御性酶活性的影响。结果表明：菌株A-1能显著抑制轮纹病菌菌丝生长,10⁴ CFU/mL菌悬液的抑制率达90%以上;喷施10⁷ CFU/mL A-1菌悬液后,间隔12 h以上接种的各处理,3 d和7 d时防效分别为91.79%~95.67%和77.41%~94.00%,均与对照农药苯醚甲环唑相当。喷施10⁷ CFU/mL A-1菌悬液后接种或不接种轮纹病菌的处理,果实内过氧化物酶、过氧化氢酶、多酚氧化酶和苯丙氨酸解氨酶活性显著升高,且均高于只接种轮纹病菌的处理;喷施A-1菌悬液后接种病原菌的处理酶活性增加最显著,其峰值是对照的2.30~11.00倍。表明内生放线菌A-1可通过产生拮抗物质、提高寄主防御性酶活性等机制实现对苹果轮纹病的有效防治。     

Inhibition of the enzymatic hydration of the epoxide HEOM in insects

Enzymatic epoxide hydration, a significant mechanism in the regulation of insect development and in the detoxication of certain cyclodiene insecticides, has been investigated in vitro in the blowfly, Calliphora erythrocephala, the southern armyworm, Prodenia eridania, and the Madagascar cockroach, Gromphadorhina portentosa. Characterization of the hydrases involved in cyclodiene epoxide hydration has been achieved using as substrate a cyclodiene insecticide (HEOM) susceptible to enzymatic epoxide ring cleavage. The enzymes, which are microsomal but different from the oxidases, are inhibited in varying degrees by microsomal oxidase inhibitors as well as by certain epoxides, esterase inhibitors and compounds with reported juvenilizing ability. Group-bulk and electronegative effects are important requirements for HEOM-hydrase inhibition, the best inhibitor of the system being 1,1,1-trichloro-2,3-epoxypropane. Differences between the structural requirements for HEOM-hydrase inhibition and those for inhibition of the epoxide hydrase responsible for the degradation of juvenile hormone are discussed.     

酮醇酸还原异构酶抑制剂的设计、合成及除草活性

酮醇酸还原异构酶(KARI)是一个有前景的除草剂靶标酶,有关其抑制剂的设计研究鲜有报道。在文献报道的菠菜KARI酶复合物0.165 nm高分辨率晶体结构基础上,利用分子对接DOCK 4.0方法进行MDL/ACD三维数据库搜寻,得到了279个与KARI酶结合能较低的小分子结构信息,并从中选取部分小分子进行化学合成,进而测试了其除草活性。在合成的17个化合物中,发现个别化合物对油菜 (抑制率70.8 %) 和稗草 (抑制率48.8 %)具有较好的生长抑制活性。     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

Toxicity of the entomopathogenic fungus, Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) to adult females of the blowfly Lucilia sericata (Diptera: Calliphoridae)

The fungus Metarhizium anisopliae (Metchnikoff) (Deuteromycotina: Hyphomycetes) is a natural component of soil flora world-wide and is a causal agent of the green muscardine diseases of insects. The use of this pathogen as a potential biocontrol agent against adult females of the blowfly, Lucilia sericata (Meigen) (Diptera: Calliphoridae) was considered. The blowfly L sericata is an economically important agent of cutaneous myiasis of sheep, particularly in northern Europe. Exposure of flies to suspensions of fungal spores by immersion, topical application or tarsal contact with treated surfaces all resulted in high levels of fatal infection. Spores suspended in silicone oil resulted in higher levels of infection (50-70%) than those formulated in a 0.3 glitre(-1) solution of the detergent Tween 80 (10-20%). Spore concentration had a significant effect on levels of infection of flies, with the highest levels of mortality (64%) resulting from a suspension of 1 x 10(7) conidia ml(-1). The duration of tarsal contact had a significant effect on levels of infection. Mean infection levels of 30% were observed following exposure of free-flying adults to a single spore-treated, 5 x 15 cm2 cloth surface suspended from the roof of a cage (30 cm cube). The implications of the results for the potential use of M anisopliae in the biocontrol of blowflies are discussed.     

氰戊·辛硫磷对绿翅绢野螟4龄幼虫呼吸代谢、解毒酶及保护酶活性的影响

以广西大学东校园内糖胶树上的绿翅绢野螟[Diaphania angustalis(Snellen)]4龄幼虫为研究对象,研究了氰戊·辛硫磷处理后不同时间(10、30、60、90 min和120 min)对绿翅绢野螟4龄幼虫呼吸代谢、解毒酶和保护酶活性的影响。结果表明,在25℃时,氰戊·辛硫磷药液浓度128.35 mg/L处理绿翅绢野螟4龄幼虫30 min后出现明显的呼吸峰,CO_2释放率达到15.68μL/h,随后迅速降到对照水平。绿翅绢野螟4龄幼虫的乙酰胆碱酯酶和超氧化物歧化酶活性在氰戊·辛硫磷处理初期显著升高,分别达到5.57×10~(-2) n mol/s和12.3 n mol/s,60 min后迅速下降到对照水平,分别为1.87×10~(-2) nmol/s和9.42 nmol/s;过氧化物酶活性变化初期与乙酰胆碱酯酶和超氧化物歧化酶相似,但在60 min后酶活性开始升高,120 min时达到307 n mol/s,极显著高于对照。     

HPPD抑制剂的研究进展

对新型除草剂对羟苯基丙酮酸双氧化酶 (HPPD)抑制剂的发现过程、作用机制、结构特征、构效关系以及合成方法作了较为详细的综述     

寄主植物对草地螟中肠解毒酶及保护性酶活性的影响

[目的]探讨寄主植物对草地螟中肠解毒酶及保护性酶活性的影响,为研究草地螟对不同寄主植物的生理适应机制奠定基础。[方法]测定草地螟幼虫取食藜、大豆、向日葵、玉米和马铃薯等5种不同寄主植物后中肠解毒酶羧酸酯酶、谷胱甘肽S-转移酶、乙酰胆碱酯酶和保护酶超氧化物歧化酶、过氧化氢酶、过氧化物酶活力的时序变化。[结果]取食不同寄主植物会显著影响幼虫中肠解毒酶活性。取食适宜寄主植物时幼虫中肠解毒酶活力在初期明显高于取食非适宜寄主植物的,但随着取食时间的延长,取食非适宜寄主植物的酶活力快速增长。取食初期,取食不同寄主植物对于中肠保护酶活性也有一定影响,但随着取食时间的延长,保护酶活力基本趋于一致。[结论]试验结果表明,草地螟老龄幼虫中肠解毒酶和保护酶对不同寄主的适应性反应是其取食非适宜寄主能够存活的生理机制之一。     

Activation kinetics of cetylpyridinium chloride on the prophenol oxidase from pupae of blowfly (Sarcophaga bullata)

Prophenol oxidase (PPO) (EC 1.14.18.1) is isolated from pupae of blowfly (Sarcophaga bullata) and purified by employing ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100 column chromatography. The enzyme exists in a latent or inactive state. Cetylpyridinium chloride (CPC), a cationic detergent, is found to activate the PPO activity. The activation of the enzyme by CPC has first been studied by using the kinetic method of the substrate reaction described by Tsou. The results show that the enzyme is activated by a complexing scheme that has not been previously identified. The enzyme first reversibly and quickly binds CPC and then undergoes a slow reversible active course. The activation reaction is a single molecule reaction and the apparent activation rate constant is dependent on the CPC concentration with the function relationship fit with a hyperbola. The micro rate constants of activation and the association constant are determined from the measurements. Substrate binding does not affect the micro rate constants of activation by CPC.     

Insect juvenile hormones: Induction of detoxifying enzymes in the housefly and detoxication by housefly enzymes

The cecropia juvenile hormone and three of its analogs were compared as inducers of microsomal epoxidase, O-demethylase, and DDT dehydrochlorinase in the housefly, Musca domestica L. The compounds were the cecropia juvenile hormone, methoprene, hydroprene, 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene, and piperonyl butoxide, a well known insecticide synergist. The compounds were administered by feeding at levels up to 1% in the diet for 3 days to 1-day-old female adults. Enzymes were then prepared and assayed for their activity using heptachlor, p-nitroanisole, and DDT as substrates.There was approximately a twofold increase in the microsomal oxidases and a 50% increase in DDT dehydrochlorinase after the treatment with the cecropia juvenile hormone, while methoprene had some activity as an inducer of the epoxidase (30% increase) but no activity in the case of the O-demethylase or the dehydrochlorinase. Hydroprene had no effect on any of the enzyme systems, while 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene was an inhibitor of the two microsomal oxidases. The latter compound and piperonyl butoxide were strong inducers of DDT dehydrochlorinase, causing approximately twofold increases in the activity of this enzyme.There was evidence that the microsomal preparations were able to metabolize and inactivate methoprene and hydroprene, the action being oxidative in the case of methoprene and both oxidative and hydrolytic in the case of hydroprene. The oxidative metabolism of the two juvenile hormone analogs by the microsomal preparations was inducible by the cecropia juvenile hormone and by phenobarbital and dieldrin.     

Induction of host defence enzymes by the endophytic bacterium Serratia marcescens,in banana plantlets

Pre-inoculation with the endobacterium Serratia marcescens (strain UPM39B3) induced the production of host defence enzymes such as peroxidase, polyphenoloxidase, phenylalanine ammonia lyase, total soluble phenols and lignothioglycolic acid in banana plantlets. The levels of these enzymes were evidently higher in plantlets pre-treated with the endobacterium compared to the control. The production of host-induced enzymes benefitted the crop plants as they may have a role in suppressing Fusarium wilt incidence in the plantlets. This was evident when plantlets pre-treated with the endobacterium showed a lower disease severity (50%) compared to diseased plantlets lacking the endobacterium (74%). The results of this study thus highlight the potential of the isolate Serratia marcescens (strain UPM 39B3) as a biological control agent for Fusarium wilt management in bananas, reducing disease severity via stimulation of host defences.     

Arginine kinase of the sheep blowfly Lucilia cuprina: Gene identification and characterization of the native and recombinant enzyme

Arginine kinase (ATP:l-arginine ω-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme with crucial functions in the energy metabolism of insects and other invertebrates as well as some protozoa and has been proposed as a parasiticidal and pesticidal drug target. In this study we report the identification, cDNA cloning, genomic gene structure and functional expression of an AK gene from the parasitic sheep blowfly Lucilia (L.) cuprina. The blowfly AK-encoding gene is devoid of introns and present in a single copy in the L. cuprina genome. AK is present in L. cuprina flies as a highly expressed soluble enzyme and is more abundant in the thorax, where it represents up to 2% of the total soluble protein, compared to head or abdomen. Guanidino substrate specificity studies show that L. cuprina AK is stereospecific for the l-form over the d-form of its specific substrate arginine. Furthermore, the presence of a free or esterified carboxylic group as well as an unsubstituted α-amine group are essential for acceptance of the l-arginine substrate while modifications in the aliphatic side chain are better tolerated. The apparent Michaelis-Menten constants K_M and the molecular size of the recombinant enzyme are virtually identical to that of the native enzyme suggesting that the gene cloned in this study encodes the highly expressed AK enzyme of the sheep blowfly.     

Influence of pesticides on the activity and kinetics of invertase,urease and acid phosphatase enzymes

The effects of four pesticides (three herbicides, atrazine, paraquat, glyphosate, and an insecticide, carbaryl) on the activity and kinetics of an invertase (from yeast), a urease (from jack bean) and an acid phosphatase (from potato) were investigated. Glyphosate and paraquat showed a marked activation effect on invertase activity. From the dependence of V_max and K_m parameters on pesticide concentrations, a mixed-type activation mechanism was suggested. The kinetic behaviour of urease and acid phosphatase appeared to be uninfluenced by both pesticides. Methanol, used as solvent for both carbaryl and atrazine, inhibited invertase, urease and acid phosphatase with, respectively, competitive, non-competitive and uncompetitive mechanisms. The extent of the inhibition was different, as estimated by the values of the inhibition constant K₁. An additional inhibition effect of urease and invertase activities was achieved in the presence of carbaryl, whereas no influence was detected on the activity and kinetics of acid phosphatase. Finally, atrazine was shown to alter the kinetics of the enzymes only at higher concentrations.     

Effect of Mono-oxygenase Inhibitors on Uptake,Metabolism and Phytotoxicity of Propanil in Resistant Biotypes of Jungle-Rice,Echinochloa Colona

The effect of the mono-oxygenase inhibitors tridiphane, piperonyl butoxide and prochloraz on propanil uptake, metabolism and phytotoxicity was measured in a resistant (R) biotype of Echinochloa colona. The uptake of propanil was not significantly affected by any of the three mono-oxygenase inhibitors. The first metabolite of propanil metabolism, 3,4-dichloroaniline, was found to accumulate to higher levels in E. colona treated with each of the mono-oxygenase inhibitors mixed with formulated propanil, compared to propanil applied alone. Accumulation of further metabolites of propanil (glucosyl-3,4-dichloroaniline and bound products) was reduced in the presence of mono-oxygenase inhibitors, compared with propanil application alone. Leaf damage caused by a single drop of propanil compared to propanil+mono-oxygenase inhibitor was used to assess the degree of propanil tolerance in E. colona biotypes. Leaf damage was significantly greater in propanil+mono-oxygenase inhibitor treatments. No leaf damage was observed in mono-oxygenase inhibitor treatments alone at the concentrations used. Peroxidase activity was measured in crude extracts of the R-biotype of E. colona using 3,4-dichloroaniline as substrate, in the presence and absence of mono-oxygenase inhibitors and the specific peroxidase inhibitor salicylhydroxamic acid. Peroxidase activity was inhibited by all three mono-oxygenase inhibitors at 10 μM and by salicylhydroxamic acid at 1 μM . Glucosyl-3,4-dichloroaniline was found not to be a substrate for peroxidase activity. These results suggest that the incorporation of 3,4-dichloroaniline into bound residues involves peroxidase activity which can be inhibited by mono-oxygenase inhibitors. When peroxidase activity is inhibited, the precursor metabolite 3,4-dichloroaniline accumulates, and propanil resistance in E. colona is reduced, possibly as a consequence of phytotoxicity of this metabolite and/or product inhibition of the first step in propanil metabolism, responsible for the formation of 3,4-dichloroaniline. Glasshouse trials have demonstrated that the application of mono-oxygenase inhibitors, (particularly tridiphane which is also known to inhibit glutathione transferase activity) with propanil offers a promising approach to the control of propanil resistant biotypes of Jungle-Rice. © 1997 SCI.     

NaCI胁迫对塔里木河下游罗布麻抗性酶的影响

研究了不同盐浓度胁迫下罗布麻种子的抗性酶活性及含量.罗布麻种子萌发采用盐溶液水培方法,设0、0.05、0.1、0.2、0.3、0.4、0.5、0.6mol/L8个盐梯度.分析罗布麻丙二醛(MDA)、超氧化物歧化酶(SOD)和游离脯氨酸(Pro)三种酶的含量和活性随盐浓度的变化趋势.得出结论:MDA含量和SOD活性变化趋...     

增强UV-B辐射对立枯丝核菌抗氧化酶及细胞壁降解酶活性影响研究

作为太阳光谱的一部分,到达地球表面的UV-B辐射有逐渐增加的趋势,影响地球生态系统.为揭示增强UV-B辐射对立枯丝核菌的影响,本文采用UV-B灯辐照处理病菌菌丝,并对菌丝生长、菌核形成,活性氧稳态和细胞壁降解酶进行分析.结果表明,增强UV-B辐射可导致供试菌株菌丝生长抑制、刺激菌核提早形成.处理菌株O2和H2O2的含量...