Ma Enbo

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). 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 Cd(2+). Fifth-nymphs of O. chinensis insects were injected with Cd(2+) at different concentrations (0, 0.55×10(-4), 1.10×10(-4), 1.65×10(-4), 2.20×10(-4), and 2.75×10(-4)gg(-1)). An increase in SOD activity in O. chinensis was observed at 1.10×10(-4) to 2.75×10(-4)gg(-1) Cd(2+). The SOD activity was lower at 2.20×10(-4)and 2.75×10(-4)gg(-1) than that at 1.10×10(-4) and 1.65×10(-4)gg(-1). It appears that SOD had a positive protective effect at low Cd(2+) concentrations, and that this effect disappeared at high Cd(2+) concentrations. CAT activity was accelerated to varying degrees at 1.10×10(-4) to 2.75×10(-4)gg(-1) for males and at 1.10×10(-4), 2.20×10(-4), and 2.75×10(-4)gg(-1) for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd(2+) concentration with all treatments for males and at 2.20×10(-4) and 2.65×10(-4)gg(-1) for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd(2+) for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.

Phylogeny of Limenitidinae Butterflies （Lepidoptera： Nymphalidae） Inferred from Mitochondrial Cytochrome Oxidase I Gene Sequences

Abstract The phylogenetic analyses of the subfamily Limenitidinae are performed based on 1 471 bp of mtDNA cytochrome oxidase subunit I (COI) gene sequence data which were obtained from 21 individuals spanning 9 genera, along with those of 17 species obtained from GenBank, using Apatura iris, Aglais urticae, and Polyura dolon as outgroup species. Although the transitions at the third codon positions of the COI data set were highly saturated, they were still retained for analysis as they contain the majority of the phylogenetic information, and thus, the maximum pasimony (MP) under different weighting schemes and maximum likelihood (ML) trees were reconstructed in this study. The results showed that within this subfamily, the results based on the COI gene sequences are approximately identical to the traditional classification results. However, the clustering of Lexias pardalis and Tanaecia julii within the genus Euthalia as well as the clustering of Phaedyma aspasia within the genus Neptis with weak support are different from that of the current classification scheme made by Chinese scholars. The genus Limenitis is splited into two subclusters in the trees constructed by using MP and ML methods. These results support one of the strongest hypotheses for the tribe relationships within Limenitidinae.

Molecular Phylogenetic Analysis of the Main Lineages of Nymphalinae (Nymphalidae: Lepidoptera) Based on the Partial Mitochondrial COI Gene

The phylogenetic relationships of the subfamily Nymphalinae (sensu Chou 1994) were analyzed based on 1 488 bp of mtDNA cytochrome oxidase subunit I (COI) gene sequence data obtained from 24 individuals, along with those of eight species obtained from GenBank. The base compositions of this COI fragment varied among the individuals as follows: T 39.9%, C 14.6%, A 32.2%, and G 13.4%, with a strong AT bias (72.1%), as usually found in insect mitochondrial genomes. The A + T contents of the third, second, and first codon positions of the COI fragments in this study was 92.4, 62.2, and 61.4%, respectively. The phylogenetic trees were reconstructed by neighbor-joining (NJ), maximum likelihood (ML), and Bayesian methods by using Byblia anvatara as outgroup. Phylogenetic analyses based on the COI gene sequence data created very similar topologies, which were producing trees with two main clades A and B, and five subclades. The data indicated that the tribes Nymphalini and Hypolimni (sensu Chou 1994) are not monophyletic groups, and the genus Junonia should be removed from Nymphalini to Hypolimni (= Junoniini). On the basis of the data, the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML, and Bayesian methods, namely ((((Nymphalis + Kaniska) + Polygonia) + Aglais) + Vanessa) + (Symbrenthia + Araschnia). This investigation provides a little novel information for Chinese researches of butterflies.

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 chinensiswere studied. Fourth-instar nymphs of O. chinensis were treated with different doses of ALA (A1, 250 mM; A2, 450 mM; A3, 750 mM; A4, 1000 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-l body weight (95% confidence interval). Biochemical studies showed that the activities of AChE and OPx 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 OSTs activities of O. chinensis enhanced with increasing dose of ALA. The OSTs 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 OPx, which induced nerve transmission blocking and the capability to defend oxidation damage declining. Meanwhile, a high dose of ALA could activate OSTs, which caused a feedback inhibition of the insect to the phototoxic substance.

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

Abstract 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%o in the 1st instar nymphs, 61.4%o in the 2nd instar nymphs, 71.4%o in the 3rd instar nymphs, and 64.4%o 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 α-NA, α-NB and β-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 inhibitois 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.

Characterization of Carboxylesterase Associated with Malathion Insensitivity in the Field Population of the Oriental Migratory Locust

Abstract Carboxylesterases (CarEs) from two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population (WD) were 1.75- and 1.50-fold significantly higher than those in Huangliu population (HL) when α-naphthyl acetate (α-NA) and β-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the α-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L . migratoria manilensis . Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.

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 (LD50: 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).

A new species of Caryanda Stål and its chromosome C-banding karyotype (Orthoptera: Acridoidea)

Abstract A new species of Acrididae, namely Caryanda rufofemorata is described from P. R. China. Its chromosome C-banding karyotype is analyzed.

Expression and Characterization of a Sigma-Class Glutathione S-transferase of the Oriental Migratory Locust, Locusta migratoria manilensis (Meyen)

Abstract A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis (LmGSTsl), was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame (204 amino acid polypeptide), which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein (LmGSTsl) was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTsl was able to catalyze the biotranslation of glutathione with l-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with/?-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30°C. Incubation for 30 min at temperatures below 50°C scarcely affected the activity. The I 50 of reactive blue (RB) was 18.5 umol L- 1 . In the presence of 0.05 mmol L- 1 ethacrynic acid (ECA), LmGSTsl showed (81±3)% of the original activities.