Meiling Yang

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.

Genomics‐based approaches to screening carboxylesterase‐like genes potentially involved in malathion resistance in oriental migratory locust (Locusta migratoria manilensis)

BACKGROUND Previous studies have indicated that increased carboxylesterase (CarE) activity is a major mechanism of malathion resistance in field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), in China. The aim of the present study was to screen CarE-like genes from a large locust expressed sequence tag (EST) database and to assess their potential roles in malathion resistance. RESULTS Twenty-five ESTs derived from different CarE-like genes in the locust EST database were identified, and 12 candidate genes with significantly increased expressions, ranging from 2.6- to 11.6-fold in a field-derived resistant (FR) colony of the locust, were found. These candidate genes were constitutively expressed in all nymph and adult stages, and most of them were predominantly expressed in the gastric caeca and the midgut. Among the 12 genes, two representative genes (LmCarE9 and LmCarE25) were chosen for RNAi followed by malathion bioassay. The nymph mortalities increased from 34.3 to 65.2 and 54.2% respectively after LmCarE9 and LmcarE25 were silenced. These results indicated significant roles of these CarE-like genes in conferring malathion resistance in the locust. CONCLUSION Multiple CarE-like genes were involved in malathion resistance in the locust. As validated by RNAi followed by malathion bioassay, LmCarE9 and LmcarE25 played a significant role in conferring malathion resistance.

Chronic accumulation of cadmium and its effects on antioxidant enzymes and malondialdehyde in Oxya chinensis (Orthoptera: Acridoidea)

The accumulation of cadmium (Cd) and its effects on antioxidant enzyme activities and malondialdehyde (MDA) levels of Chinese rice grasshopper (Oxya chinensis) were evaluated under the laboratory conditions. Our results showed that Cd accumulation in O. chinensis exhibited a concentration-dependent increase in both males and females under Cd pollution. Environmental Cd can lead to the absorption of large quantities of Cd, which induces oxidative damage in insects by altering antioxidant defense enzyme systems. Our results demonstrated that Cd stress caused a significant decrease in glutathione peroxidase (GPx) levels and a significant increase in superoxide (SOD) dismutase and catalase (CAT) activities. In the grasshoppers, the MDA content was also enhanced, with an increase in Cd concentrations and a positive correlation between them; for females from second instar nymphs to the adult stage, R(2) was 0.6467, 0.9136, 0.6516, 0.942 and 0.7182, whereas for males, it was 0.6467, 0.8239, 0.9302, 0.7861, 0.8632, respectively. We also observed differences in the effects of Cd between grasshoppers of different developmental stages and genders, which suggested that the insects developmental stage and sex should be considered when studying enzyme activity.

Comparative analysis of cytochrome P450-like genes from Locusta migratoria manilensis: expression profiling and response to insecticide exposure

Abstract  The cytochrome P450 monooxygenase (cytochrome P450) gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and other xenobiotics. To date, very little is known about cytochrome P450 genes in the oriental migratory locust, Locusta migratoria manilensis. In this study, we carried out a genome‐wide analysis of cytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures. We identified 15 cytochrome P450‐like genes from a locust expressed sequence tag database (LocustDB). Reverse transcription polymerase chain reaction (RT‐PCR) analysis showed that most cytochrome P450‐like genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgut, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides. Deltamethrin caused significant inductions in 12 h at LD30 (dose to kill 30% of the tested individuals) in the nymphs, whereas malathion and carbaryl did not have significant effect on cytochrome P450 enzyme activity. Further RT‐PCR analysis showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin‐treated locusts. Thus, the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450 genes in response to deltamethrin exposure. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin‐treated insects at various molecular levels.

Comparisons of Malathion Susceptibility, Target Sensitivity, and Detoxification Enzyme Activity in Nine Field Populations of Oxya chinensis (Orthoptera: Acrididae)

Abstract The malathion susceptibility, acetylcholinesterase (AChE) sensitivity, and the activity of selected detoxification enzymes including general esterase (EST) and glutathione S-transferase (GST) were compared among field populations of the grasshopper Oxya chinensis (Thunberg) (Orthoptera: Acrididae) collected from nine regions of China. Bioassay results showed that these populations had various levels of the susceptibility to malathion with the LD50 values ranging from 1.4- to 22.6-fold compared with the most susceptible population (Xiangyuan or XY). The Jinnan (JN) population seemed to be malathion resistant (22.6-fold), whereas other populations exhibited 1.4- to 6.8-fold reduced malathion susceptibility with a rank order of Changan > Baodi > Hanzhong > Xinxiang > Yinchuan > Beidagang > Jinyuan. It seemed that the observed malathion resistance in the JN population was attributed to at least two resistance mechanisms, including increased EST activity (2.2-fold) and reduced sensitivity of AChE to inhibition by malaoxon (4.6-fold) compared with those of the XY population. In contrast, differential malathion susceptibilities in other populations may be due to increased activities of certain detoxification enzymes (e.g., EST and GST), reduced sensitivity of AChE, or other factors, which were not consistent across the populations examined. Such differential susceptibilities to malathion were likely due to different population habitats (e.g., grasslands, rice [Oryza sativa L.]-producing regions) with very different insecticide application histories and pest management practices.

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

Abstract 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 (3.75 μg/g body weight). General esterase‐specific activities in the Daixian population were 1.91, 1.10 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.

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.

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.