Accurate Analysis of Tricarboxylic Acid Cycle Metabolites and Anion Components in Hemocytes by IC-CD/ESI-MS for Quantifying Insecticide Impairment on Cellular Immunity in Mythimna separata.

Insecticides are more broadly known to affect insect cellular immunity, but the components in hemocytes and their response to insecticide stress are still unknown. In this paper, a method based on trifluoroacetic acid extraction, followed by IC-CD/ESI-MS analysis, was developed to simultaneously determine tricarboxylic acid (TCA) cycle metabolites and anion components in hemocytes from Mythimna separata larvae. Validation gave excellent selectivity, recovery (88.7-107.6%), linear correlation (r2 > 0.9961), precision (<3.89%), LOD (0.002-0.006 mg/L), LOQ (0.006-0.020 mg/L), and a short chromatographic run. The method was verified by administration of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl 3-(1,3-dioxoiso-indolin-2-yl) propanoate (QDP) or emamectin benzoate (EMB) to hemocytes in vitro and larvae in vivo. TCA metabolites including citrate, α-ketoglutarate, fumarate, malate, and oxaloacetate, and anions including acetate, oxalate, chloride, carbonate, and sulfate were identified and clearly separated. QDP and EMB showed a biphasic dose effect on TCA metabolites, and the contrary hormesis paralleled the different actions of QDP and EMB. The inhibition or improvement of cellular immunity depended on the QDP concentration. In conclusion, a highly sensitive, reliable, and robust method was developed, enabling the monitoring of hemocyte immunity by the quantification of TCA metabolites and anion components in minute hemocyte samples.

Fungicidal Effect of Pyraclostrobin against Botrytis cinerea in Relation to Its Crystal Structure.

Pyraclostrobin (PYR) is a commonly used strobilurin fungicide, which inhibits mitochondrial respiration at the ubiquinol oxidation center site of the cytochrome bc1 complex. Little information is available regarding the crystal structure of PYR on its fungicidal effect. In this study, the crystal structures of eight PYRs (PYR-A to H) from different sources are determined by using high-resolution X-ray powder diffraction (XRPD) and model construction with the Pawley refinement module. The effects of PYRs on mycelium growth, the kinetics of mycelial growth, conidial germination, and tube elongation of conidia of Botrytis cinerea from tomato are compared. The level of organic acids in the mitochondrial tricarboxylic acid cycle of PYR-treated B. cinerea is analyzed. The results show that PYR-A to PYR-H have their own unique character of XRPD patterns, but the crystal morphology of eight PYRs presents in the triclinic crystal system and space group P1̅. PYR-D with the eclipsed conformation and rational edge angles α (72.599°) and ß (98.612°) in the crystal cell shows the highest inhibitory effect against mycelium growth with EC50 as 3.383 µg mL-1, the best time-dependent effects on the mycelium growth kinetics, and the strongest inhibition on tube elongation of conidia, whereas PYR-E with anticonformation is the worst. Moreover, a significant accumulation of fumarate, malate, and oxalate in the PYR-D-treated mycelium is observed. These findings reinforce the need for a definite crystal structure of PYR to limit usage and mitigate future selection pressure for gray mold management.

Safety assessment of a new benzoylphenylurea TXH09 and its efficacy against two borers Ostrinia furnacalis and Grapholitha molesta in field.

Benzoylphenylureas as an important type of insect growth regulators, acting on the moulting stage in immature insects, are highly effective and low toxic. The new benzoylphenylurea TXH09 [N-((2,6-dimethyl-4-(heptafluoropropyl-2-yl)phenyl)carbamoyl)-2,6-difluorobenzamide] has high efficacy against chewing insect pests harming vegetables and rice. In this paper, the efficacy of TXH09 against two intractable borers Ostrinia furnacalis and Grapholitha molesta were evaluated in field, and safety assessment by exploring the characteristics of photodegradation, cytotoxicity, micronucleus generation and chromosome aberration was performed. The results showed that TXH09 had good capability in preventing infested corn and reducing the population of O. furnacalis larvae, and maintained high efficacy on shoot protection and peach conservation against G. molesta larvae. There were no significant differences between the control effects of TXH09 and that of hexaflumuron or diflubenzuron at the same active dose. TXH09 photolysis in solvents N,N-dimethylformamide, toluene and methanol yielded two major products, and the photodegradation of TXH09 was more prone to occur in N,N-dimethylformamide. TXH09 and the mixture of its photoproducts showed higher cytotoxicity on insect Sf-9 cells than on human Hek293 cells. Moreover, TXH09 didn't show significant effects in inducing micronucleated cells in both male and female mice and chromosomal aberrations in mouse spermatocytes by its own. In conclusion, TXH09, as an effective insecticide, has good environmental safety performance against O. furnacalis and G. molesta in field.

Antifungal effects of 3-(2-pyridyl)methyl-2-(4-chlorphenyl) iminothiazolidine against Sclerotinia sclerotiorum.

BACKGROUND: Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum threatens oilseed rape cultivation, and the emergence of fungicide-resistant strains has led to control failures worldwide. Identifying novel chemical alternatives with different modes of action and high antifungal activities is thus crucial. Herein we evaluated the antifungal effects of 3-(2-pyridyl)methyl-2-(4-chlorphenyl)imino- thiazolidine (PMAS) on S. sclerotiorum to determine its efficacy for SSR management. RESULTS: PMAS had an inhibitory effect on mycelial growth; the EC50 values were 17.83 and 21.15 µg mL-1 for the carbendazim-susceptible strain Ss01 and carbendazim-resistant strain Hm25, respectively. PMAS treatment changed the color of inhibited mycelia to green, and the hyphae were sustained in the undifferentiated stage. Cysteine supplementation made this green color disappear, whereas methionine enhanced the color. Moreover, PMAS treatment markedly inhibited oxalic acid biogenesis, increased free thiol content in mycelia, and weakened the activities of oxaloacetase and malate dehydrogenase, but had little effect on the activity of glyoxylate dehydrogenase. Cysteine could reverse the inhibitory effects of PMAS on mycelial morphogenesis and biochemical constituents, except thiol production. In the pot-culture experiment, PMAS showed a good protective effect, with the control efficacy being >91% on SSR. CONCLUSION: PMAS appears to be an effective fungicide for SSR management. © 2020 Society of Chemical Industry.

Comparative cytotoxic effects of five commonly used triazole alcohol fungicides on human cells of different tissue types.

The widespread application of triazole fungicides makes people attach great concern over its adverse effects in mammalian. In this paper, cytotoxic effects of triazole alcohol fungicides (TAFs) were assessed on human HeLa, A549, HCT116 and K562 cells, and the potential mechanism of TAFs cytotoxicity was studied preliminarily. Results showed that TAFs had cytotoxicity on human cells with different level and cytotoxic selectivity. TAFs cytotoxicity was resonated with a typical hormetic biphasic dose action that produced a complex pattern of stimulatory or inhibitory effects on cell viability. Among the five TAFs, diniconazole revealed a widest range of cytotoxicity to inhibit the viability of the adherent and the suspension cells, causing HeLa cells shrinkage, A549 cells shrunken, and K562 cells collapse, and showed stronger cytotoxicity than hexaconazole. Moreover, the involvement of ROS generation in the cytotoxicity of TAFs on human cells was observed, and the apoptosis of HeLa cells and the formation of apoptotic body in K562 cells induced by diniconazole were characterized. The results indicated the cytotoxicity of TAFs with different structures on human cells was depended on their own property and cell specificity, K562 cells were the most susceptible to TAFs and diniconazole was the strongest toxic.

Cytotoxic selectivity and apoptosis induction of piericidin A contributes potentially to its insecticidal effect against Mythimna separata (Lepidoptera: Noctuidae) larvae.

Piericidin A (PIA), an active inhibitor of Complex I, is widely used in studies of the anti-bacterial and anti-disease competence, but its physiological and mechanistic effects have rarely been clearly defined in insect individual or insect cells. The present study reveals the considerable insecticidal activity of PIA on Mythimna separata larvae by using a comparison with Aphis craccivora adult, and the cytotoxic selectivity induced by PIA on lepidopteran Tn5B1-4 cells. We demonstrate that the viability of Tn5B1-4 cells is inhibited by PIA in a time- and concentration-dependent manner with IC50 value of 0.061 µM, whilst PIA shows slight inhibitory effect on the viability of HepG2 and Hek293 cells with IC50 value of 233.97 and 228.96 µM, respectively. The inhibitory effect of PIA on the proliferation of Tn5B1-4 cells is significant and persistent, causing a series of morphological changes including cell shrinkage, condensed and fragmented nuclei. Intracellular biochemical assays show that PIA induces apoptosis of Tn5B1-4 cells coincides with a decrease in the mitochondrial membrane potential. PIA in Tn5B1-4 cells can be chelated by EDTA, thereby losing cytotoxicity, whereas exogenous Ca2+ restores the cytotoxicity of PIA by chelating with EDTA in a competitive manner. Our findings highlight the importance of the long-lasting cytotoxicity and the cytoxic selectivity on Tn5B1-4 cells caused by PIA, which ensure the identification of insecticidal effect of PIA against insect pests.

The effect of α-tocopherol and dithiothreitol in ameliorating emamectin benzoate cytotoxicity in human K562 cells involving the modulation of ROS accumulation and NF-κB signaling.

Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (p < 0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.

Photosensitization of Chinese hamster V79 cells to photoactivated alpha-terthienyl involving membrane damage and oxidative stress.

Photosensitization of V79 Chinese hamster lung fibroblasts was tested to investigate if the cells can fit the photoactive effect of alpha-terthienyl for safety application. Using 15-min photoirradiation of a black light (320-400 nm, 40 W), alpha-terthienyl was significantly photoactivated and caused V79 cells to be shrinkage, detachment and necrosis. The photoactivated alpha-terthienyl played a concentration-dependent stress to decrease cell survival and to induce cell death with median inhibitory concentration (IC50) as 4.78 µg/ml. Cell viability in MTT assays also fell down to 10.58% of the control in the treatment of 10.0 µg/ml photoactivated alpha-terthienyl. As the irradiation time prolonged and the concentration of photoactivated alpha-terthienyl increased, cell death increased significantly, the intracellular level of reactive oxygen species (ROS) and the content of extracellular malondialdehyde were gradually increased. The changes of peroxidase, superoxide dismutase and catalase activities in V79 cells were positively responsive to the oxidative stress caused by photoactivated alpha-terthienyl. Moreover, using non-photosensitizing condition, the increased cell death and oxidative stress in the treatment of alpha-terthienyl at >7.0 µg/ml were also observed. The results showed the maladjustment response of V79 cells with membrane damage and cell death, clearly demonstrating the photosensitization of animal cells to the photoactivated cytotoxic effect of alpha-terthienyl.

Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae (Noctuidae, Lepidoptera).

A novel transglutaminase (MsTGase) from Mythimna separata larvae was separated and purified; its biochemical property and enzymatic catalytic activities were investigated. MsTGase was obtained chromatographically by the precipitation of Sephadex G-100 gel and DEAE-Cellulose-52 ion-exchange column with 48-fold purification and a reproducible yield of approximately 12%. Molecular weight of the MsTGase was 63.5 KDa and its N-terminal amino acid sequence was GKIEEG-LVI. Michaelis constant of the MsTGase for the substrate N-CBZ-Gln-Gly was 12.83mM with a Vmax of 7.99U/mL. Optimum conditions for MsTGase activity were at 42°C and pH7.5. The enzyme didn't possess metal ion at its catalytic active site; its activity could be significantly inhibited by Mg2+, but activated by Ca2+. Chlorpyrifos and spinosad showed a strong potential to increase MsTGase activity, supporting the view that MsTGase was a novel target. Moreover, the formation of intermolecular cross-links of casein and bovine serum albumin polymerized by MsTGase in the presence of DTT was observed. These findings pave the way for future studies on the physiological role of MsTGase and the potential impact of its regulation on MsTGase-associated pest management.