Inhibition of erythrocyte membrane (Ca2+ + Mg2+)-ATPase by the organophosphorus insecticides parathion and methylparathion

Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca²⁺ + Mg²⁺)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca²⁺ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca²⁺ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (K_i) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. K_i values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca²⁺ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.     

Biotechnological Production of Plant-Based Insecticides

ABSTRACT:?

The demand for natural and nonpersistent insecticides is increasing day by day. Plant cell cultures could be an alternative to conventional methods of production of insecticides from field-grown plants. In vitro cultured plant cells produce a wide array of insecticides as a part of their secondary metabolism. Their ability to synthesize key enzymes and the manipulation of these could lead to the enhanced production of many insecticides of industrial importance. The development of a high-yielding hairy root culture system for thiophenes, nicotine, and phytoecdysones is of considerable interest. In this article, the current literature on various factors that influence the growth, production, and secretion of six insecticidal compounds, namely, pyrethrins, azadirachtin, thiophenes, nicotine, rotenoids, and phytoecdysones which have been prospects for the scale-up of cell cultures, genetic engineering to obtain transgenic plants, and metabolically engineered plants for increased production of bio-molecules, has been discussed. Environmental safety clearance and the future prospects of application of bio-molecules for plant-derived insecticides are presented.     

Comparative study of insecticide susceptibility and activities of detoxification enzymes in larvae and adults of cotton bollworm,Heliothis armigera

Susceptibility of moths and larvae of cotton bollworm to ten different insecticides by topical application and their effect on enzymes involved in insecticide detoxification were determined. The moths were more susceptible than larvae to the insecticides tested, with the exception of pyrethroids and sulprofos. Combination of several insecticides with the synergists piperonyl butoxide (PB) and S,S,S-tributylphosphorotrithioate (TBPT) showed that lower level of carbamate and organophosphate toxicity in larvae, as compared to moths, was the result of higher detoxification enzyme activities. Studies of the post-treatment fate of ¹⁴C-labeled malathion and ³H-trans-permethrin indicated that both the cuticular penetration, internal accumulation, and excretion of applied toxicants and their metabolites occurred more rapidly in larvae than in moths. The activities in vitro of esterases, glutathione S-transferases, and monooxygenases were determined but there were no correlations with either toxicity of insecticides or synergistic effect for combination of insecticides with PB and TBPT in moths. © 1996 Wiley-Liss, Inc.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

6种杀虫剂对南美番茄潜叶蛾的毒力及田间防效

[目的]研究6种杀虫剂对南美番茄潜叶蛾卵、幼虫和成虫的毒力及其在温室番茄上的防治效果,为南美番茄潜叶蛾防治提供高效杀虫剂和施药技术。[方法]采用浸渍法和药膜法测定了6种杀虫剂对南美番茄潜叶蛾卵、幼虫和成虫的毒力,田间调查毒力较高杀虫剂对温室番茄上南美番茄潜叶蛾防效。[结果] 6种杀虫剂中的乙基多杀菌素、甲氨基阿维菌素苯甲酸盐和阿维菌素对南美番茄潜叶蛾卵有毒力作用,致死中浓度（LC₅₀）分别为1.415、13.588和23.194 mg·L^-1。6种杀虫剂对南美番茄潜叶蛾幼虫的LC₅₀分别为：阿维菌素0.026 mg·L^-1、四唑虫酰胺0.052 mg·L^-1、甲氨基阿维菌素苯甲酸盐0.057 mg·L^-1、乙基多杀菌素0.072 mg·L^-1、氯虫苯甲酰胺0.484 mg·L^-1和呋虫胺2.039 mg·L^-1。对于南美番茄潜叶蛾成虫,24 h时,仅甲氨基阿维菌素苯甲酸盐和四唑虫酰胺对成虫有较高毒力;72 h时,6种杀虫剂对成虫的LC₅₀分别为：甲氨基阿维菌素苯甲酸盐0.390 mg·L^-1、乙基多杀菌素1.646 mg·L^-1、四唑虫酰胺2.630 mg·L^-1、呋虫胺5.577 mg·L^-1、阿维菌素22.502 mg·L^-1和氯虫苯甲酰胺39.636 mg·L^-1。在成虫盛发期第4天施药,阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐、乙基多杀菌素在南美番茄潜叶蛾危害严重的温室番茄上防效达80%以上。[结论]阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐、乙基多杀菌素、氯虫苯甲酰胺和呋虫胺对南美番茄潜叶蛾卵、幼虫或成虫有较高毒力,其中阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐和乙基多杀菌素田间防效较好。     

Response of adult parasitoids ofBemisia tabaci (Hom.: Aleyrodidae) to leaf residues of selected cotton insecticides

The contact toxicity of eight insecticides to adults of four parasitoids of the sweetpotato whiteflyBemisia tabaci was evaluated in the laboratory. Two common Texas species,Eretmocerus sp. andEncarsia pergandiella Howard, and two exotic species,Eretmocerus mundus Mercet from Spain andEncarsia formosa Gahan from Greece were tested. Insecticides, applied as sprays to greenhouse-grown cotton plants at recommended rates were amitraz (Ovasyn^®), azinphosmethyl (Guthion^®), bifenthrin (Capture^®), buprofezin (Applaud^®), cypermethrin (Ammo^®), methyl parathion and thiodicarb (Larvin^®), with a water control. Parasitoid adults were confined on discs cut from leaves (1) sprayed the same day and (2) sprayed two days previously. Survival in both treatments was measured two and four days following exposure. Significant differences in toxicity were detected among the insecticides. Buprofezin was not toxic to any of the four parasitoids. When caged on leaves sprayed two days previously, only amitraz of the remaining compounds allowed significant general parasitoid survival after two days.E. mundus exhibited the greatest overall tolerance to insecticides, with 40% or more surviving 48 hr after confinement on leaves sprayed with amitraz, thiodicarb and cypermethrin. Survival was generally much reduced after 96 hr. In a separate test, fresh residues of endosulfan (Thiodan^®) were highly toxic at the two rates tested, but two day old residues at the lower rate allowed 76.7% survival ofE. mundus and 35% survival ofE. pergandiella after 48 hrs.     

Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization

Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni²⁺-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k _cat/K _m of 100 s^?1 μM^?1 for α-NA, and the value is 29.78 s^?1 μM^?1 for β-naphthyl acetate. The V _max and K _m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu²⁺ and Mg²⁺ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.     

Molecular identification of glutathione S-transferase genes and their potential roles in insecticides susceptibility of Grapholita molesta

Grapholita molesta is one kind of serious fruit-boring pests in the world, which caused huge economic losses to the fruit industry including pear and apple. The heavy usage of synthetic insecticides resulted in different degrees of resistance of G. molesta to some commonly used insecticides. However, the tolerance mechanisms of G. molesta to insecticides are still unclear. Glutathione S-transferases (GSTs) belong to the superfamily of detoxifying enzymes and mainly play important roles in detoxification metabolism and insecticide susceptibility of insects. In this study, we first evaluated the toxicity of three insecticides including emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin on G. molesta at different developmental stages. Synergism study showed that the susceptibility of G. molesta larvae to these three insecticides obviously increased when the enzyme activity of GST was inhibited by diethyl maleate compared with the controls. Next, we screened out six GmGST genes which are upregulated after insecticides treatment from a total of 21 GmGST genes and analysed their sequence characteristics and expression profiles. RNAi and bioassay results further revealed that the mortality of 5th instar larvae significantly increased after exposure to LC₃₀ of emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin when silencing of GmGSTo2, GmGSTs1 and GmGSTz1 compared with the controls. In conclusion, this study indicated that GmGSTo2, GmGSTs1 and GmGSTz1 play important roles in insecticides susceptibility of G. molesta to three commonly used insecticides, which is significance for its future integrated management.     

Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.     

Effects of lindane and deltamethrin on zooplankton communities of experimental ponds

Two insecticides, lindane (321 µg l^–1) and deltamethrin (13 µg l^–1) were employed in a four mesocosm experiment (two ponds of 10 m³ and two of 16 m³) to asses the impact of water pollution by pesticides. Resistance of the different zooplankton species was variable and depended upon both the group and the insecticide concentration. No effect of lindane was observed on macrozooplancton such as Cladocera and Copepoda. In the deltamethrin-treated pond, all species of zooplankton were found dead a day after the treatment. The microzooplankton (Rotifera and copepod nauplii) were highly susceptible to both insecticides. Although the larvae of Chaoborus were present in the ponds after the treatments, their density decreased (less than 1 individual l^–1). The elimination of filter-feeding zooplankton by deltamethrin was followed by an increase of the concentration of chlorophyll a in the post-treatment period. Two months later the original zooplankton population recovered, with the addition of a new and dominant species: Ceriodaphnia reticulata.     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Effect of Fungicides and Insecticides on Growth and Enzyme Activity of Four Cyanobacteria

Cyanobacterial populations introduced into crop fields as biofertilizer become non-target organisms for the pesticides and fungicides applied in the field. Effect of four commonly used pesticides viz. Bagalol, Mancozeb (fungicides), Thiodan and Phorate (insecticides) was studied on growth and different enzymes of four cyanobacterial species viz. Nostoc ellipsosporum, Scytonema simplex, Tolypothrix tenuis, and Westiellopsis prolifica. EC 50 concentration of each pesticide was determined for all cyanobacteria. Bagalol and Thiodan were found to be the most toxic. Both the fungicides and insecticides inhibited the activity of nitrogenase and glutamine synthetase (GS) at EC 50 concentration in all the four species studied. Bagalol incurred maximum inhibition of nitrogenase and GS activity on N. ellipsosporum and S. simplex while Thiodan and Phorate had maximum effect on T. tenuis, and W. prolifica. Mancozeb had lesser effect on all the above enzymes. One catabolic enzyme of carbohydrate metabolism, isocitrate dehydrogenase (ICDH) and one anabolic enzyme isocitrate lyase (ICL), which is related to glyoxylate pathway as well as gluconeogenesis, were also assayed. Cell free extracts of cyanobacteria treated with pesticides for 7 days show a drastic reduction of ICDH activity. ICL activity was induced in the organisms when treated with pesticides.     

Some properties of a mitochondrial malic enzyme from the flight muscle of the tsetse fly (Glossina)

An active malic enzyme [l-malate-NAD⁺-oxidoreductose (decarboxylating)] has been isolated from flight muscle mitochondria of the tsetse fly (Glossina Morsitans). The enzyme utilizes NAD⁺ preferentially as a hydrogen acceptor and is activated by low concentrations of Mn²⁺ or higher concentrations of Mg²⁺. The enzyme exhibits oxaloacetate decarboxylase activity and is inhibited by pyruvate, oxalate, malonate, oxaloacetate, ATP, and ADP. In some the nature of the inhibition depends on the concentration of Mn²⁺. The inportance of this enzyme in the pathway of proline metabolism is discussed.     

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot-forming (SF) and non-shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [¹⁴C]-l,2-acetate into various metabolites. Early in culture, the lipid fraction contained the most ¹⁴C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg^-1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21-day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited later in culture.     

Pyruvate Kinase Activity of Wheat Plants Grown under Potassium Deficient Conditions

The activity of pyruvate kinase was determined in the first leaves of wheat plants grown under K⁺-deficient conditions. An enhancement of the enzyme activity compared with the normal plants was found to start from eighth day of growth, and about 4-fold increase in the enzyme activity was observed in 14-day wheat leaves. The addition of K₂SO₄ to the nutrient solution given to the K⁺-deficient plants at tenth day resulted in the restoration of the enzyme activity to the normal level after 3 days. The levels of K⁺ as well as carbohydrates and chlorophyll were found to return normal over the same period. These findings are discussed in relation to the metabolic pattern of plants at the early stages of K⁺-deficiency.     

Joint action of the entomopathogenic fungus Isaria fumosorosea and four chemical insecticides against the whitefly Bemisia tabaci

The strain IfB01 of Isaria fumosorosea (Paecilomyces fumosoroseus) is a promising myco-insecticide candidate for control of Bemisia tabaci. Joint action of strain IfB01 and four chemical insecticides [spirotetramat (Spi), acetamiprid (Ace), imidacloprid (Imi) and thiamethoxam (Thi)] against the whitefly B. tabaci was evaluated using the cooperative virulence index (c.f.). Substantial synergistic actions were found in the mixtures of IfB01 conidia with Spi, Imi and Thi 2–4 days after treatment. The greatest synergism was recorded in the mixture of IfB01 (2.5 × 10⁶ conidia/L) and Imi (12.5 mg/L), which had a c.f. value of 184 at day 3 after treatment. Furthermore, the shorter LT₅₀ values recorded in mixtures indicated that effectiveness improved when the conidia were applied with the chemical insecticides. However, no substantial synergisms were recorded with Ace and from day 5 onwards.     

Metabolic flux analysis of<Emphasis Type="Italic"> pykF</Emphasis> gene knockout<Emphasis Type="Italic"> Escherichia coli</Emphasis> based on <Superscript>13</Superscript>C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

Metabolic flux analysis based on ¹³C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF^– mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF^– mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.     

不同杀虫剂对西花蓟马的室内毒力及田间药效

【目的】西花蓟马是一种世界性的危险性入侵害虫,筛选防治西花蓟马的有效药剂可为生产中科学用药提供依据。【方法】通过室内毒力测定和田间药效试验,测定了2种化学药剂和8种生物药剂对西花蓟马成虫的致死中浓度(LC_(50))和药后不同时期的防治效果。【结果】室内毒力测定依据LC_(50)值将各药剂对西花蓟马的敏感性由高到低依次排序为乙基多杀菌素(0.1958 mg·L~(-1))、印楝素(0.9399 mg·L~(-1))、苦参碱(1.2483 mg·L~(-1))、阿维菌素(1.8096 mg·L~(-1))、高效氯氰菊酯(4.4458 mg·L~(-1))、藜芦碱(10.7628 mg·L~(-1))、鱼藤酮(18.1898 mg·L~(-1))、吡虫啉(46.3964 mg·L~(-1))、松脂酸钠(131.5214 mg·L~(-1))、苏云金杆菌(446.2318 mg·L~(-1));田间药效试验发现,乙基多杀菌素和吡虫啉防治西花蓟马均表现出较强的速效性和持效性,药后1～14 d的防效分别可达84%和73%以上;其次是藜芦碱,药后1～14 d的防效可达48.15%～61.37%;高效氯氰菊酯的防效较低,药后14 d的防效为46.22%;生物药剂阿维菌素、苏云金杆菌、苦参碱、印楝素的速效性均较低,但防效随施药后时间的延长而逐渐上升;鱼藤酮持效性最低。【结论】乙基多杀菌素和吡虫啉可推荐为生产中防治西花蓟马的首选药剂,植物源药剂藜芦碱和生物药剂阿维菌素可作为交替使用药剂。     

Field efficacy of biorational insecticides against Melanaphis sacchari (Zehntner) in sorghum

Abstract

The sugarcane aphid, Melanaphis sacchari (Zehntner), is a major pest in diverse sorghum-growing regions, affecting yields if no effective control measures are implemented. The objective of this study was to evaluate the field efficacy of commercial formulations of biorational insecticides against this pest. All the evaluated biorational insecticides exerted acceptable biological efficacy for at least 7 days after application. The insecticides based on fatty acid potassium salts (Ultralux^® S and Impide^®) maintained aphid density below the established threshold of 50 aphids per leaf up to 14 days after application. The results obtained suggest that biorational insecticides can be included in the integrated management of M. sacchari.