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The joint toxicity of chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, and sinigrin, a natural plant defense compound from brassicaceous vegetables, to the larvae of Spodoptera exigua (Hübner) was determined in this paper. Additionally, the joint effects of the two compounds on cytochrome P450 enzyme activity and on the expression levels of mRNA of three P450 genes (including CYP9A9, CYP6B, and CYP4G37) and an NADPH cytochrome P450 reductase gene (HQ852049) were investigated. The toxicity of the mixture of chlorantraniliprole and sinigrin to fourth-instar S. exigua larvae was 1.60-fold higher than the toxicity of the chlorantraniliprole-only treatment after 24 h. Induced by chlorantraniliprole and sinigrin, the specific activity of the P450 O-deethylase was affected in a time-, dose-, and organ-specific manner in fifth-instar S. exigua larvae. The effects were more pronounced in the midgut than in the fat body. The specific activity of the P450 O-deethylase in almost all treatments increased at 12, 24, and 36 h posttreatment compared with that in the control. Based on real-time PCR analyses, the expression levels of the P450 genes CYP9A9, CYP6B, and CYP4G37 and the NADPH cytochrome P450 reductase gene HQ852049 in fifth-instar S. exigua larvae were induced by chlorantraniliprole and sinigrin, and the trends were similar to the specific activity of the P450 O-deethylase. Therefore, the CYP9A9, CYP6B, and HQ852049 in the tested genes were the most inducible genes that were expressed when the S. exigua larvae were exposed to chlorantraniliprole and sinigrin.

A triple-quadrupole LC-MS/MS method was developed for the investigation of indoxacarb N -decarbomethoxyllation activity catalyzed by insect crude enzymes. The metabolite, N -decarbomethoxyllated indoxacarb (DCJW), was determined using electrospray ionization in positive ion mode. The quantitative and qualitative analyses were performed in multiple reaction monitoring (MRM) mode. The relative standard deviation (RSD, n = 5) of DCJW was 2.1%, indicating the metabolite had a good chemical stability in 30 h after the sample preparation. Five injections of DCJW standard solution in acetonitrile, five injections of the same sample in one incubation (substrates plus enzyme samples), and five injections of 5 separate metabolic reactions with the whole body of a susceptible strain of Plutella xylostella (L.) were individually injected into LC-MS/MS system. The RSD was 0.7%, 1.1% and 2.7%, respectively. The average recoveries for 440, 880 and 2200 pg of DCJW added to incubation mixtures were 96.1%–102.9%, and the RSD of recoveries of three added levels ranged from 4.8% to 9.4%. The limits of quantification and detection were 0.1 and 0.01 pg, respectively. A good linearity was achieved in the range of 46–2310 pg ( R 2 = 0.9996). This method with high sensitivity and simplicity was applicable to the assay of indoxacarb N -decarbomethoxyllation activity of insect metabolic enzymes. A comparison of indoxacarb N -decarbomethoxyllation activity of an avermectin-resistant strain and a susceptible strain of Plutella xylostella (L.) was carried out using this novel LC-MS/MS method. The results showed that the indoxacarb N -decarbomethoxyllation activity in the resistant strain was 3.4-fold of that in the susceptible strain, implicating that negative cross-resistance might exist between indoxacarb and avermectin in Plutella xylostella (L.).

Suspension array technology enjoys great potential in simultaneous detection of drug residues in the field of food safety. A monoclonal antibody against gentamycin was produced and a bead-based indirect competitive fluorescent immunoassay was developed using the suspension array system for the simultaneous detection of antibiotic residues such as gentamycin and kanamycin in raw milk. The limit of detection (LOD) for kanamycin and gentamycin were 0.5 ng/mL and 4.1 ng/mL in a phosphate-buffered saline (PBS), 2.2 ng/mL and 12.2 ng/mL in milk. The monoclonal antibodies used for the multiplexed assays showed no significant cross-reactivity with other aminoglycoside antibiotics. To validate the method, milk samples spiked with gentamycin or kanamycin were detected using suspension array technology and conventional enzyme-linked immunosorbent assay (ELISA) in parallel. The good correlation between the obtained results showed that the method is feasible in actual application, and it also laid a firm foundation fo...

The carmine spider mite is the most serious crop mite pests in China. Abamectin has been used to control insects and mites worldwide but carmine spider mites, Tetranychus cinnabarinus , had developed resistance to it. Genetic research on insecticide resistance has been fundamental for understanding the resistance development, studying resistance mechanisms, and designing appropriate resistance management strategies to control insect pests. A resistant colony of T. cinnabarinus , RRG42 , was established to examine the inheritance of abamectin resistance in T. cinnabarinus . The females of T. cinnabarinus were selected for bioassay using a slide dip method. After 42 generations of selection, the RRG42 strain was 8.7-fold resistant to abamectin compared with the susceptible strain (SS). The logarithm (log) concentration–probit response curve for F 1 s from reciprocal crosses, of F 1 RS and F 1 SR, were inclined to that for SS and the degree of dominance ( D ) values for F 1 s were −0.81 and −0.17. There was a significant difference in values of LC 50 and slope of log concentration–probit lines between F 1 RS and F 1 SR. The observed mortalities of BC1 (F 1 RS♀ × RRG42♂) and BC1′ (F 1 SR♀ × SS♂) were significantly different from the expected mortalities based on a monogenic resistance in the chi-square tests. The inheritance of abamectin resistance in T. cinnabarinus is incompletely recessive and may be controlled by more than one gene. The maternal or cytoplasmic effect may exist in the inheritance of resistance to abamectin in T. cinnabarinus .

A novel sensitive method was developed for measuring p -nitroanisole o -demethylation (PNOD) catalyzed by cytochrome P450 enzymes in crude homogenates of housefly, Musca domestica L., using high performance liquid chromatography (HPLC) with diode array detector. This method is based on the use of liquid-liquid extraction and HPLC with reversed-phase C 18 column and gradient ultraviolet detection at 316 nm. The detection limit ( S/N of 3/1) of p -nitrophenol ( p NP) was 0.1 ng. The relative standard deviation (RSD) of peak area response of the HPLC analysis system was 1.0%. The average recoveries of 0.351, 1.755 and 8.755 μg of p NP added to incubation mixtures ranged from 92.3% to 87.6%, and the RSD of recoveries of three added levels ranged 2.64%–5.90%. The relationships between peak areas and concentration were linear in the range of 9.72–486 ng with good coefficient of determination ( r 2 = 0.9995). This method is sensitive, precise, and reproducible and is also applicable to the assay of p -nitroanisole o -demethylases, which have low catalytic activities, present in other insects or organisms. With this method, the comparison of P450 o -demethylation activity of an imidacloprid-resistant strain with a susceptible strain of M. domestica (L.) was conducted to identify the roles of P450 enzymes involved in housefly resistance to imidacloprid. The results indicated that the resistance mechanism of housefly to imidacloprid was intensely assisted with elevated P450 activities.

Tyrosinase is a copper enzyme and plays a key role in normal insect development. We studied the in vitro inhibitory effects of selected insecticides and allelochemicals on the diphenolase activity of tyrosinase in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae). Two pyrethriods (cyfluthrin and deltamethrin) and 3 other insecticides (hexaflumuron, abamectin and imidacloprid) were the least inhibitory, whereas 5 organophosphates (triazophos, malathion, chlorpyrifos, omethoate and profenofos), 1 carbamate (methomyl), 4 pyrethriods (fenpropathrin, beta-cypermethrin, bifenthrin and lambda-cyhalothrin), 1 organochlorine (endosulfan), 2 allelochemicals (tannic acid and 2-tridecanone) and 4 other insecticides (emamectin benzoate, fipronil, acetamiprid and pyridaben) were moderately inhibitory. Three chemicals (quercetin, phenyl thiourea and phoxim) were the most potent inhibitors of the enzymes among all compounds tested and inhibited the diphenolase activity of tyrosinase in vitro in a dose-dependent manner. Furthermore, phenyl thiourea, phoxim and quercetin showed neither typical competitive nor noncompetitive binding to the substrate, with Ki of 0.13 μM, 49.30 μM and 37.71 μM, respectively.

Monochamus alternatus Hope (Coleoptera: Cerambycidae) is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides (organophosphates and pyrethroids), which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro32P-labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate (cAMP), deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin (Ca2+/CaM)-stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.

The small prominent, Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae), is an important pest of poplar in China. Glutathione S-transferases are known to be responsible for adaptation mechanisms of M. troglodyta. Thus, the tissue distribution and kinetic constants of glutathione S-transferase activity in the small prominent were studied. Significant differences in glutathione S-transferase (GST) activity and distribution percentages of GST activity and kinetic characteristics were observed among 4 tissues (head, midgut, fat body and integument). Furthermore, the inhibition of glutathione S-transferase activity in 4 tissues by 21 inhibitors was conducted. The results showed the inhibition of GST activity of different tissues by 21 inhibitors is different. For GST activity in heads, chlorpyrifos, profenofos, lambda-cyhalothrin, fipronil and quercetin were the best inhibitors tested. Tannic acid was the most potent inhibitor of midgut GST activity. In the fat body, GST activity was inhibited most by tannic acid, chlorpyrifos and profenofos. The inhibitory effect of profenofos and phoxim was highest for GST activity in the integument. Our results showed that glutathione S-transferases in different tissues are qualitatively different in isozyme composition and thus different in sensitivity to inhibitors.

Both Micromelalopha troglodyta (Graeser) and Clostera anastomosis (L.), are important pests of poplar, and usually coexist during the period of poplar growth in China. The carboxylesterases have been recognized to play an important role in the detoxification of xenobiotics in the two Notodontidae species. In vitro inhibitory effects by insecticides and allelochemicals on carboxylesterase activity in M. troglodyta and C. anastomosis were studied. The results showed that three organophosphates (chlorpyrifos, phoxim and profenofos) were the best inhibitors of the enzymes among all compounds tested in M. troglodyta and C. anastomosis. Chlorpyrifos inhibited carboxylesterase activity in M. troglodyta and C. anastomosis to a similar degree, but phoxim and profenofos inhibited to different degrees. Furthermore, kinetic analyses of carboxylesterase inhibition by phoxim, chlorpyrifos and profenofos were also investigated. The results showed that phoxim inhibited carboxylesterase activity with respect to α...

The effects of methamidophos and tri- o -cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ- 32 P]ATP as phosphate donor. Tri- o -cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.

Glutathione S-transferases are a group of enzymes catalyzing the conjugation of reduced glutathione (GSH) with a wide range of xenobiotics bearing electrophilic sites. In insects, GSTs are involved in resistance to insecticides and allelochemicals. In vitro inhibitory effects of several insecticides and allelochemicals on glutathione S-transferases activity in the cotton bollworm, Helicoverpa armigera Hübner, were studied. All organophosphate, carbamate and pyrethroid insecticides tested demonstrated moderate or low inhibiting GST activity toward 1-chlorine-2, 4-ditrobenzo (CDNB). Three allelochemicals (quercetin, tannic acid and rutin) were the most potent inhibitors of the enzymes among all compounds tested. Tannic acid was competitive with CDNB, quercetin was noncompetitive, and rutin was neither competitive nor noncompetitive.

The possibility of communication between plants was proposed over 25 years ago, although previous demonstrations have suffered from methodological problems and have not been widely accepted. Micromelalopha troglodyta (Graeser) and Clostera anastomosis (L.) (both Lepidoptera: Notodontidae) are important leaf-feeding insects on poplars (Populus spp.; Salicaceae). There is no report of M. troglodyta- or C. anastomosis-induced plant–plant communication up to now. To understand the regulatory mechanism of poplar defense responses, the specific activities of the presumed anti-herbivore enzymes phenylalanine ammonia lyase (PAL), lipoxygenase (LOX), polyphenol oxidase (PPO), and peroxidase (POD) were determined in poplar seedlings treated with herbivore infestation and volatiles, artificially damaged seedlings, and intact healthy poplar seedlings growing nearby. The expression levels of PPO, POD, PAL, and LOX were elevated in poplar seedlings treated by herbivore infestation, mechanical wounding, methyl jasmonate, methyl salicylate, or benzothiazole. Furthermore, plants neighboring the treated poplar seedlings also showed increased expression levels of PPO, POD, PAL, and LOX relative to the control. These induced responses against feeding injury, mechanical wounding, and volatile treatment in expression levels of PPO, POD, PAL, and LOX suggest interplant communication between poplar seedlings.

Seven synthetic herbivore-induced plant volatiles (HIPVs) and a mixture of nonanal + (Z)-3-hexen-1-ol were field tested for their ability to attract beneficial insects in an open cotton field. Eleven species of the main natural enemies of insect pests in cotton fields were studied. Significantly greater numbers of the ladybird beetle Coccinella septempunctata were trapped on (Z)-3-hexenyl acetate-baited cards than on others that were HIPV baited or the control cards. Erigonidium graminicolum was attracted to traps baited with nonanal, (Z)-3-hexenyl acetate, and methyl salicylate (MeSA). The predatory bug Deraeocoris punctulatus was only attracted to traps baited with octanal. The predatory bug Orius similis responded to traps baited with 3,7-dimethyl,1,3,6-octatriene, nonanal, (Z)-3-hexenyl acetate, nonanal + (Z)-3-hexen-1-ol, and MeSA. Dimethyl octatriene, nonanal + (Z)-3-hexen-1-ol, and octanal significantly attracted the syrphid fly Paragus quadrifasciatus. The ladybird beetle Propylaea japonica, the green lacewing Chrysopa sinica, the bigeyed bug Geocoris pallidipennis, the syrphid fly Epistrophe balteata, and the parasitic wasp Campoletis chlorideae did not respond to any of the HIPVs tested. These results are discussed with regard to the potential of HIPVs as tools for recruiting natural enemies into cotton fields.