Toxicological and biochemical characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in the resistant and susceptible strains (SS) of Liposcelis bostrychophila were investigated. The two resistant strains were the dichlorvos-resistant strain (DDVP-R) and the phosphine-resistant strain (PH₃-R) with resistance ratios of 22.36 and 4.51, respectively. Compared to their susceptible counterpart, the AChE activity per insect and the specific activity of AChE in DDVP-R and PH₃-R were significantly higher. There were also significant kinetic differences between DDVP-R and PH₃-R. The apparent Michaelis-Menten constant (K_m) for acetylthiocholine iodide (ATChI) was obviously lower in SS than that in PH₃-R, indicating a higher affinity to the substrate ATChI in the susceptible strains. The affinity for the substrate ATChI in DDVP-R and SS were not significantly different. The V_max value of the PH₃-R was significantly greater when compared to the V_max for the SS suggesting a possible over expression of AChE in this resistant strain. The inhibition of AChE to insecticide exposure in vitro revealed that all six insecticides were inhibitory for the extracted AChE’s. Based on the I₅₀ values, AChE of the SS were more sensitive to dichlorvos, paraoxon-ethyl, malaoxon and demeton-S-methyl than those of the two resistant strains. As for carbaryl and eserine, the PH₃-R suggested a significantly higher I₅₀s compared to the susceptible strain, while, no significant differences were found between SS and DDVP-R.     

几种代谢酶基因与柑橘全爪螨对双甲脒抗性的关系

为了明确羧酸酯酶(carboxylesterase,CarE)基因、谷胱甘肽S-转移酶(glutathione S-transferases,GST)基因和过氧化氢酶(catalase,CAT)基因与柑橘全爪螨Panonychus citri对双甲脒抗性的关系,通过BLAST检索,从柑橘全爪螨转录组数据库中对这3种代谢酶抗性相关基因进行鉴定,并采用RPKM法对双甲脒抗性品系和敏感品系代谢抗性相关基因进行表达差异分析,对差异较大的基因作定量PCR检测.基因差异性分析发现,抗性品系中有9条CarE基因、12条GST基因及6条CAT基因表达量发生上调,13条CarE基因、12条GST基因和3条CAT基因表达量发生下调;Pc29773nrt、Pcl7807nlg和Unigene31477为上调倍数最高的3个基因,其log2 ratio (RS/SS)分别为12.95、10.81、10.01.定量分析显示,Pc29773 nrt、Pcl7807nlg和Unigene31477的上调倍数分别为3.72、2.03和3.09,Pc29773 nrt和Unigene31477上调显著.研究表明柑橘全爪螨Pc29773nrt和Unigene31477上调与其对双甲脒的抗性相关.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Molecular and biochemical mechanisms of organophosphate resistance in laboratory-selected lines of the oriental fruit fly (Bactrocera dorsalis)

Genetic and biochemical factors leading to resistance to various organophosphate (OP) based insecticides were studied in lines selected for OP-resistance in the oriental fruit fly Bactrocera dorsalis. Lines were separately selected for resistance to naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion. Overall, these lines showed increased resistance ratios ranging from 13.7- to 814-fold relative to a susceptible (S) line. Also, in these newly selected lines the same three point mutations in the ace gene, previously identified in resistance studies and designated as I214V, G488S and Q643R, were found. As expected, the enzyme from the resistant lines showed lower overall activity and reduced sensitivity to inhibition by fenitrothion, methyl-paraoxon and paraoxon compared to the wild type acetylcholinesterase (AChE) enzyme. The apparent V_max values for esterase from the resistant lines were 1.2-3.69 times higher than that of the S line. Although only the naled-, trichlorfon- and fenthion-r lines showed lower esterase affinities (based on apparent K_m values) compared with the S line, all of the V_max/K_m ratios were higher in the resistant lines compared to that of the S line. The OP-resistant lines also displayed an overall similar pattern of isozyme expression, except for one additional band found only in the naled-r line and one band that was absent in the trichlorfon-, malathion-, and fenthion-r lines. Our results also show that overall, multiple examples of high OP resistance in selected lines of B. dorsalis exhibiting the same genetic alterations in the ace gene seen previously resulted in different effects on esterase enzyme activity in relation to various OP compounds.     

Toxicological and metabolic parameters of the teleost fish (Leporinus obtusidens) in response to commercial herbicides containing clomazone and propanil

Pesticides, such as herbicides can affect the metabolic and toxicological parameters on fish. For this reason, an experiment was carried out with the objective of to evaluate the effects of commercial formulations of clomazone and propanil herbicides on acetylcholinesterase (AChE), thiobarbituric acid-reactive substances (TBARS), catalase (CAT) and metabolic parameters in teleost fish (Leporinus obtusidens). Fish were exposed during 90 days to field measured concentration of the herbicides clomazone and propanil (376 and 1644 μg/L, respectively) on rice paddy water. Specific AChE activity in the brain and muscle decreased and TBARS levels decreased in brain, muscle and liver tissues. Liver catalase decreased after exposure to both herbicides. Metabolic parameters in the liver and white muscle showed different changes after exposure to both herbicides. In summary, the results showed that clomazone and propanil affects toxicological and metabolic parameters of piavas. These results suggest that environmentally relevant herbicides concentrations are toxic to Leporinus obtusidens.     

Characterization of imidacloprid resistance in the housefly Musca domestica (Diptera: Muscidae)

In recent years, imidacloprid was introduced to control the housefly in China and it was documented that the housefly indeed showed signs of resistance to imidacloprid somewhere but not in China. Therefore, a housefly population collected from filed (IFS) was selected continuously with imidacloprid to establish the resistant strain (IRS) and the basic characteristics were investigated in this study. After continuous selection over 21 generations, the resistance ratio increased from 9.01 to 140, and different levels of cross-resistance were developed to beta-cypermethrin, chlorpyrifos, chlorfenapyr, acetamiprid and azamethiphos in the IRS strain. The realized heritability of resistance was 0.10. The synergistic ratios for IRS pretreated with DEF, DEM and PBO were 1.68, 1.52 and 2.53, and the corresponding ones for IFS were 3.17, 1.87 and 2.67, respectively. Synergistic and biochemical assays suggested that the cytochrome P450 may play an important role in the imidacloprid resistance comparing with GSTs- and carboxylesterases-mediated detoxification in the IRS strain, and there might be additional mechanisms (e.g. reduced target-site sensitivity) contributed to imidacloprid resistance in the IRS strain.     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

Cholinesterase (ChE) inhibition in pumpkinseed (Lepomis gibbosus) as environmental biomarker: ChE characterization and potential neurotoxic effects of xenobiotics

Inhibition of cholinesterases (ChEs) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. More recently, this biomarker has been suggested as a putative biomarker for exposure to detergents. The use of cholinesterase inhibition as effect criterion in Ecotoxicology requires the previous characterization of the specific enzymatic forms that may be present in different tissues or organs. Different ChEs isoforms may be present in the same tissue and may exhibit distinct sensitivities towards environmental contaminants. This work intended to characterize the soluble ChEs present in pumpkinseed sunfish (Lepomis gibbosus) total head and dorsal muscle homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Also, the in vitro effects of sodium dodecylsulphate (SDS - anionic detergent) and chlorfenvinphos (organophosphate pesticide) on the enzymatic activity of the mentioned species were investigated. In general terms, the predominant cholinesterasic form present in both tissues was acetylcholinesterase. Chlorfenvinphos was responsible for inhibitory effects on AChE activity, while SDS did not cause any significant effect. These results suggest that in environmental monitoring programs, L. gibbosus head and dorsal muscle AChE can be an adequate diagnostic tool for exposure to OP pesticides; this conclusion however is not applicable to detergent residues. We also discuss the usefulness of L. gibbosus as an alternative model system and valuable option for freshwater ecotoxicological monitoring programs.     

西花蓟马抗甲氨基阿维菌素苯甲酸盐种群的交互抗性与生化抗性机制

为了解西花蓟马Frankliniella occidentalis(Pergande)对甲氨基阿维菌素苯甲酸盐的抗性风险,采用生物和生化测定方法研究了西花蓟马甲维盐抗性种群与其它杀虫剂的交互抗性和生化抗性机制。西花蓟马甲维盐抗性种群对阿维菌素有高水平交互抗性,抗性倍数为31.656,对啶虫脒有中等水平交互抗性,为12.182,对吡虫啉、溴虫腈、氯氟氰菊酯、毒死蜱和灭多威有低水平交互抗性,为5.517~8.568,而对多杀菌素无明显交互抗性。增效剂胡椒基丁醚(PBO)、马来酸二乙酯(DEM)、三丁基三硫磷酸酯(DEF)和磷酸三苯酯(TPP)对甲维盐抗性种群和田间种群均有显著增效作用。甲维盐抗性种群多功能氧化酶细胞色素P₄₅₀和b₅含量、O-脱甲基酶、谷胱甘肽S-转移酶和羧酸酯酶活性均显著提高,分别为敏感种群的3.89、3.61、5.32、4.42和1.30倍,表明多功能氧化酶、谷胱甘肽S-转移酶和羧酸酯酶等解毒代谢酶活性的提高是西花蓟马对甲维盐产生抗性的重要机制。     

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH₃-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I₅₀ value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.     

Increased activity and reduced sensitivity of acetylcholinesterase associated with malathion resistance in a field population of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibility to malathion, and the activity and sensitivity of acetylcholinesterase (AChE, EC 1.1.1.7) were compared between two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen) collected from Wudi County of Shandong Province in East China and Huangliu County of Hainan Province in South China. Huangliu population showed 8.5-fold resistance to malathion compared with Wudi population. AChE from Huangliu population showed 4.8-fold higher activity than that from Wudi population toward the model substrate acetylthiocholine (ATC). Kinetic studies indicated that AChE from Huangliu population had 2.6-fold lower affinity, but 5.0-fold higher catalytic activity toward ATC than AChE from Wudi population. Significantly increased activity of AChE in Huangliu population was also confirmed by non-denaturing polyacrylamide gel electrophoresis. Inhibition kinetics revealed that AChE from Huangliu population was 9.8-, 2.4-, 8.0- and 7.7-fold less sensitive to inhibition by paraoxon, malaoxon, chlopyrifos oxon, demeton-S-methyl, respectively, than that from Wudi population. Our studies revealed that a mild resistance to malathion in Huangliu population was associated with reduced sensitivity and increased catalytic activity of AChE. Our results suggest that alterations of AChE may play an important role conferring or contribute to malathion resistance in Huangliu population of the locust.     

Insecticide resistance in the tropical bedbug Cimex hemipterus

Insecticide resistance in the bedbug Cimex hemipterus was investigated using 4211 bedbugs collected from three districts of Sri Lanka. Insecticide bioassays were carried out with discriminating dosages of deltamethrin, permethrin, DDT, malathion, and propoxur. Activity levels of insecticide metabolizing enzymes and the insecticide target site acetylcholinesterase were monitored using biochemical assays. Percentage survivals after DDT, malathion, and propoxur exposure were 41-88%, 18-64%, and 11-41%, respectively. For deltamethrin and permethrin, KT₅₀/KT₉₀ (time to knock-down 50%/90% of the population) values were 0.5-24/1.0-58 and 1.3-10/2.5-47 h, respectively. Both elevated esterase and malathion carboxylesterase mechanisms were present in bedbug populations. Monooxygenase levels were heterogeneous. Organophosphate and carbamate target site acetylcholinesterase, was insensitive in 29-44% of the populations. High DDT resistance was probably due to glutathione S-transferases. Malathion carboxylesterases are mainly responsible for high malathion resistance. High tolerance to both DDT and pyrethroids suggests the presence of ‘kdr’ type resistance mechanism in one population.     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

Comparative studies of acetylcholinesterases purified from two field populations of the oriental migratory locust (Locusta migratoria manilensis): implications of insecticide resistance

Acetylcholinesterase (AChE) was purified by affinity chromatography from two populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), collected from Huanghua and Pingshan Counties, Hebei Province of China. The purification factors and yields were 1661-fold and 19.3%, respectively, for the Huanghua population, and 3897-fold and 39.6% for the Pingshan population. Both the purification factor and yield were significantly lower in the Huanghua population than in the Pingshan population. AChE activity was almost completely inhibited by 10⁻⁶ M eserine and BW284C51, but ?5.8% of AChE activity was inhibited by ethopropazine at the same concentration, suggesting that purified AChE from either population was a typical insect AChE. However, AChE purified from the Huanghua population was 62-, 2.0-, and 1.6-fold less sensitive to inhibition by the three organophosphate compounds, chlorpyrifos oxon, demeton-S-methyl, and paraoxon, respectively, than that from the Pingshan population. Significantly lower purification factor and low yield associated with reduced sensitivity of AChE to inhibition by the organophosphates indicated that AChE purified from the Huanghua population was biochemically and pharmacologically different from that of the Pingshan population. Reduced sensitivity of AChE appeared to contribute to organophosphate resistance in the locust from Huanghua County, where insecticides have commonly been used to manage outbreaks of the locust.     

Fitness cost, cross resistance and realized heritability of resistance to imidacloprid in Spodoptera litura (Lepidoptera: Noctuidae)

Imidacloprid has been used as a key insecticide for controlling sucking insect pests of cotton, whereas Spodoptera litura also has been indirectly exposed to this insecticide in Pakistan. To evaluate the risk of resistance evolution and to develop a better resistance management strategy, a field collected population was selected with imidacloprid in the laboratory. Thereafter, fitness cost, realized heritability and cross resistance of imidacloprid resistance in S. litura were investigated. After 14 generations of selection with imidacloprid, S. litura developed a 137.48-fold resistance to the insecticide. Bioassay revealed that this strain showed cross-resistance to acetamiprid (RR 8.52) and a little to lamdacyhalothrin (1.92) but negative cross-resistance was found to methomyl (−0.19). The resistant strain had a relative fitness of 0.38, with substantially lower rates of larval survival, larval duration, male pupal duration, development time, emergence rate of healthy adults, fecundity, hatchability, and prolonged larval and pupal duration. Mean relative growth rate of the larvae, intrinsic rate of population increase, and biotic potential was lower for the selected populations. The estimated realized heritability (h²) of imidacloprid resistance was 0.15 in the resistant strain of S. litura. Development of the resistance may cost significant fitness for the resistant population. This study provided valuable information for further understanding the impact of imidacloprid resistance on physiological parameters of S. litura and for facilitating the development of resistance management strategies.     

辣椒品种营养组成和绒毛特征与抗侧多食跗线螨的关系

利用螨量比值法,研究11个辣椒品种叶片茸毛密度和长度、叶绿素含量、可溶性糖含量、可溶性蛋白质含量和游离氨基酸含量与抗侧多食跗线螨Polyphagotarsonemus latus (Bank)的关系,以期阐明辣椒品种对侧多食跗线螨的抗性机制.结果显示,螨量比值与辣椒品种叶片绒毛密度、单位面积叶绿素含量和可溶性糖含量呈显著负相关,相关系数分别为-0.8455、-0.8851和-0.6712;与游离丝氨酸含量呈极显著负相关,相关系数为-0.829;与游离氨基酸总量和可溶性蛋白质含量无显著相关性.研究表明,辣椒品种对侧多食跗线螨的抗性与叶片叶绿素、可溶性糖、游离丝氨酸含量及叶片绒毛密度有关.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.