Genetic Structure of Three Populations of Oxya chinensis in Shanxi,China

Starch gel electrophoresis was used to analyze genetic structure of three populations of Oxya chinensis from Shanxi Province,China.Four allozyme polymorphic loci (MDH-1,MDH-2,LDH,ME) were found.The data suggest that two populations of O.chinensis about 41 kilometers apart are genetically similar,but differentiated from the third population with a geographic distance of 357 kilometers.However,the heterozygosity levels of those two genetically similar populations were much different.The Nei's genetic distance between two geographically close populations is 0.068,which is much smaller than those of geographically distant populations (0.23 in average).The observed overlap of geographical distance and genetic distance implies that for O.chinensis population divergence may correlate with geographic isolation.

Oxidative stress related enzymes in response to chromium(VI) toxicity in Oxya chinensis (Orthoptera: Acridoidae)

The toxic effects of Cr（Vl） on antioxidant enzymes of Oxya chinensis（Orthoptera： Acridoidae） were determined. Changes in the activities of the antioxidant enzymes superoxide dismutase（SOD）, catalase（CAT）, and guaiacol peroxidase（GPx） were measured in O. chinensis insects injected with Cr（VI）. Fifth-nymphs of O. chinensis insects were injected with Cr（VI） with different concentrations （0, 75, 150, 225, 300, 375, 450 mg/kg of body weight）. The results showed that Cr（VI） led to the change of SOD, CAT, and GPx activities at different concentrations, which revealed that： （1） The oxidative stress of SOD increased with the increase of Cr （VI） concentration. （2） With the increase of Cr （VI） concentrations, CAT activities for females increased at lower concentrations, but decreased at higher concentration range, which indicated that antioxidant system of O. chinensis was not influenced by the presence of Cr （VI）. A very similar response to Cr（VI） effect for males indicated that Cr（VI） concentrations were not high enough to damage O. chinensis in terms of CAT. （3） The GPx activity for females increased in all treatments, which revealed that the damage power of Cr（VI） was increased with the increase of Cr（Vi） concentrations in terms of GPx, but the effect was not so remarkable. There was not a consistent trend of GPx activities for males in all treatments of Cr（VI）. Cr（VI）-induced changes in antioxidant enzymes were different for SOD, CAT and GPx, of which the tendency was that activities generally changed with increase of concentrations of Cr（VI） suggesting SOD, CAT, and GPx could serve as indices of oxidative stress to some extent.

Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg) (Orthoptera: Acridoidea)

The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17- fold higher specific activities than the adults in females with α-NA, α-NB and β-NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α-NA, α-NB, and β-NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α-NA, α-NB, and β-NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type, and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.

Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg(Orthoptera:Acrididae)and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATCincreased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Suchinhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE fromChangzhi population. The kinetic study showed that there were no significant differences between the two populations inthe Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmax value in Changzhi populationwas 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from bothpopulations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of theirbimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon > paraoxon >demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhipopulation. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition byparaoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It isinferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxyachinensis to malathion between the two populations.

The Susceptibilities of Oxya chinensis(Orthoptera:Acridoidea) to Malathion and Comparison of the Esterase Properties from Three Collected Populations in Tianjin Area,China

The susceptibilities of Oxya chinensis to malathion were studied in three populations collected from outskirt of Tianjin, China, using bioassays and biochemical analysis. Populations were chosen to represent different exposure to insecticides： BDG （Beidagang; low exposure）, BD （Baodi; high exposure previously but low exposure now）, and JN （Jinnan; high exposure）. The results showed that the LD50 values of BD and JN populations were 3.95- and 12.02-fold and 3.64- and 10.07- fold higher than that of BDG population in females and males, respectively. The LD50 values in females were higher than those in males. The results of biochemical analysis indicated that the esterase （EST） activities in JN population were higher than those in BD and BDG populations. They showed that when α-NA, α-NB, and α-NA were used as substrates, females＇ EST activities of JN population were 1.11-, 1.30-, and 1.14-fold and 1.39-, 1.59-, and 1.54-fold higher than those of BD and BDG populations, respectively. When α-NA, α-NB, and β-NA were used as substrates, males＇ EST activities of JN population were 1.13-, 1.12-, and 1,00-fold and 1.20-, 1.14-, and 1.07-fold higher than those of BD and BDG populations, respectively. The results also showed that the specific activities of the females were higher than those of the males in the BD and JN populations, whereas the specific activities of the males were higher than those of the females in the BDG population. The results of bioassay were consistent with those of biochemical analysis. Thus, it was inferred that the elevated ESTs activities might play an important role in conferring the differences of susceptibility of O. chinensis to malathion in the three collected populations. Enzyme kinetic studies indicated that the Km and Vmax values were different among the three collected populations and between the females and the males. The observed changes in the kinetic parameters might be explained by differential expression patterns of isozymes so that the insect esterases have different affinities and maximum velocities toward the same substrate.

Study on Insecticidal Activities and Effect on Three Kinds of Enzymes by 5-Aminolevulinic Acid on Oxya chinensis

Insecticidal activities and effects on three enzymic activities caused by 5-aminolevulinic acid （ALA） on Oxya chinensis were studied. Fourth-instar nymphs of O. chinensis were treated with different doses ofALA （A1,250 mM; A2, 450 mM; A3,750 mM; A4, 1 000 mM）. Mortality and the activities of acetylcholinesterase （ACHE）, glutathione S-transferase （GSTs）, and glutathione peroxidase （GPx） were determinated. The mortality of O. chinensis rose with an increasing dose of ALA. The mortality of high-dose treatments A3 and A4 reached 66.19 and 80.21%, respectively. The value of LD50 was 3.61 （3.29-3.93） mg·g^-1 body weight （95% confidence interval）. Biochemical studies showed that the activities of AChE and GPx in the A4 treatment declined by 51.53 and 42.82% in the female, and 42.65 and 43.85% in the male compared to the control, respectively, and the degree of decline reached a significant level at P 〈 0.05. Meanwhile, the GSTs activities of O. chinensis enhanced with increasing dose of ALA. The GSTs activities of female and male O. chinensis in the A4 treatment remarkably increased by 171.05 and 97.42% compared to the control （P〈 0.05）. ALA had an obviously toxic effect on O. chinensis. Moreover, ALA caused the photoinactivation of AChE and GPx, which induced nerve transmission blocking and the capability to defend oxidation damage declining. Meanwhile, a high dose of ALA could activate GSTs, which caused a feedback inhibition of the insect to the phototoxic substance.

Analysis on Genetic Relationship of Oxya chinensis and Oxya japonica from Xuzhou and Pingshan, China

Genetic relationship among Oxya chinensis populations and Oxya japonica populations collected from Xuzhou City, Jiangsu Province and Pingshan County, Hebei Province, China were analyzed by random amplified polymorphic DNA （RAPD） markers. A total of 125 DNA bands ranging from 200 to 2 200 bp were amplified by 10 random primers in DNA samples from 43 grasshopper individuals. One hundred and twenty-three （99%） of these bands were polymorphic. Shannon＇s index showed that the genetic, diversity within O. chinensis （0.3432） was higher than that of O. japonica （0.2781）. Nei＇s genetic distance between O. chinensis population and O. japonica population from the same area was less than that between populations from two different areas. The dendrogram based on Nei＇s genetic distance of RAPD markers was constructed using the unweighted pair group method with the arithmetic average （UPGMA） and Neighbor-Joining （NJ） methods. Cluster analysis indicated that all the individuals were grouped into two main clusters. O. chinensis populations from Xuzhou and Pingshan formed one cluster, and O. japonica populations from the two regions belonged to another cluster. The results demonstrated that RAPD can detect within species, and among closely related species. polymorphisms to distinguish minor difference among individuals

A Photosensitivity Insecticide, 5-Aminolevulinic Acid, Exerts EffectiveToxicity to Oxya chinensis (Orthoptera: Acridoidea)

5-Aminolevulinic acid （ALA）, a major photosensitivity insecticide, has attracted increasing attention as a new type of highly efficient, environmental friendly pesticide to be used to control the pest. To examine whether or not ALA acts effectively to grasshopper, Oxya chinensis and elucidate the detoxification mechanism of ALA, the susceptibility to ALA was assessed in O. chinensis and two major metabolic detoxification enzymes including glutathione S-transferases （GSTs） and general esterases （ESTs）-specific activities were compared in different development stages and different body sections of O. chinensis treated by ALA and the control. The results showed that the ALA exhibited obvious toxicity to the grasshopper in different development stages. In the low-dose treatment （0.0597 mmol L-1）, the mortalities of O. chinensis reached a significant level （55.5% in the 1st instar nymphs, 61.4% in the 2nd instar nymphs, 71.4% in the 3rd instar nymphs, and 64.4% in the 4th instar nymphs. But, there was no dose-dependent toxic effect. Thereby, we proposed that ALA has the potential for acting as photosensitivity insecticide for controlling O. chinensis. GSTs activity assays using CDNB and DCNB as substrates indicated that the thorax and abdomen of the different instar nymphs treated by ALA showed 1.52-5.56 fold significantly increased GSTs activities compared with the control. However, for the ESTs-specific activity assay, there was no significant difference between O. chinensis treated by ALA and the control within different instar nymphs, when a-NA, a-NB and b-NA were used as substrates. Therefore, GSTs-mediated metabolic detoxification as evidenced by significantly increased GSTs activities might contribute to protect against oxidative damage and oxidative stress by ALA in O. chinensis.

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis(Thunberg)(Orthoptera:Acrididae)

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.

Different survival of chromium-exposed Oxya chinensis among allozyme genotypes

The goal of the present study was to compare the different survival of Oxya chinensis exposed to chromium（VI） among allozyme genotypes to gain a better understanding of the relationship between the genetic variations and environmental disturbance. This study analyzed the occurrence of genotypes in O. chinensis population exposed to Cr（VI）. O. chinensis samples were collected at Yuanping, Shanxi Province, China and used in acute toxicity tests. Specimens were assigned to Cr（VI） exposure （LDs0：291.0 mg/kg） for 24 h. The genetic composition of both dead and survived specimens was analyzed with horizontal starch gel electrophoresis in four enzymes （GPI, PGM, LDH, and ME）. The results indicated that under Cr（VI） exposure, specimens with different genotypes had various mortalities at the four loci in laboratory conditions, and there was a genetic basis for tolerance in O. chinensis during acute exposure to Cr（VI）.

Toxicological and biochemical characterizations of malathion sensitivity in two field populations of Oxya chinensis （Orthoptera： Acridoidea）

We evaluate comparative toxicity of malathion in the two populations of the grasshopper Oxya chinensis, collected from Daixian and Fanshi of Shanxi province, China. General esterases and acetylcholinesterase （ACHE） from the two populations were characterized and compared. LD50 of the Daixian population （7.58 μg/g body weight） was 2.02-fold higher than that of the Fanshi population （3.75μg/g body weight）. General esterase-specific activities in the Daixian population were 1.91,130 and 1.85-fold higher than those in the Fanshi population, when α-NA, α-NB and β-NA were used as a substrate, respectively. Kinetic studies of general esterase showed that Vmax values of general esterases hydrolyzing α-NA,α-NB and β-NA in the Daixian population were 2.15-, 1.12-, and 1.47-fold, respectively, higher than those in the Fanshi population. The AChE activity of the Fanshi population was 1.54-fold higher than that of the Daixian population. Kinetic analysis of AChE showed that significant differences were presented between the two populations in the Km values; and the Vmax value in the Fanshi population was higher than that in the Daixian population. Inhibition studies of AChE indicated that AChE from the Daixian population was 2.56-, 2.80-, and 2.29-fold less sensitive to inhibition by paraoxon, chlorpyrifos-oxon, and demeton-S-methyl, respectively, than that from the Fanshi population. These biochemical characterizations of general esterases and AChE were consistent with malathion bioassay in the two populations. It is inferred that the reduced sensitivity of altered AChE and increased general esterase activities play an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.

Isolation, identification, derivatization and phytotoxic activity of secondary metabolites produced by Cladosporium oxysporum DH14, a locust-associated fungus

Three main phytotoxic compounds including lunatoic acid A （1）, 5Z-7-oxozeaenol （2） and zeaenol （3） were isolated from the fermentation broth of Cladosporium oxysporum DH14, a fungus residing in the locust （Oxya chinensis ） gut. Two additional derivative compounds, compound la and lb, were synthesized by methylation and chlorination of compound 1, respectively. The structures of such compounds were identified on the basis of spectroscopic analysis and by comparison of the corresponding data to those previously reported in the literature. Compounds 1-3 exhibited significantly phytotoxic activities against the radicle growth of Amaranthus retroflexus L. with the 50% inhibitory concentrations （ICso values） of 4.51, 4.80 and 8.16 μg· mL^-1, respectively, which is comparable to that positive control 2,4-dichlorophenoxyacetic acid （IC50=1.95 μg· mL^-1）. Furthermore, the compound 1 showed selective phytotoxic activity with the inhibition rate of less than 22% against the crops of Brassica rapa L., Sorghum durra, Brassica campestris L,, Capsicum annucm and Raphanus safivus L. under the concentration of 100 μg· mL^-1 Both derivatives of compound 1 had moderate phytotoxic activity against the radicle growth of A. retroflexus L. The findings of our present study suggest that these compounds provide new promising candidates for the potential management strategies of weeds.