Glutathione S-transferase genes in the epsilon group were reported to function in insecticide resistance. SlGSTE12 was validated to be overexpressed in pyrethroid-and organophosphate-resistant populations of Spodoptera litura compared to a susceptible population. A functional study of heterologously expressed SlGSTE12 showed that K m and V max for 1-chloro-2,4dinitrobenzene (CDNB) conjugating activity were 0.70 ± 0.18 mmol L −1 and 90.6 ± 9.4 nmol mg −1 min −1 , respectively. β-Cypermethrin and cyhalothrin showed much weaker inhibition of SlGSTE12 activity to CDNB conjugation than fenvalerate, chlorpyrifos, and phoxim. Ultrahigh-performance liquid chromatography analysis showed that SlGSTE12 had significant metabolism activity to fenvalerate and phoxim both in vitro and in Escherichia coli, especially to chlorpyrifos, and slight metabolism activity toward cyhalothrin only in vitro. Silencing of SlGSTE12 by RNAi increased the mortality to fenvalerate, cyhalothrin, and chlorpyrifos significantly. SlGSTE12 also had a significant antioxidant ability against cumene hydroperoxide. Our study suggested that SlGSTE12 could metabolize phoxim, fenvalerate, cyhalothrin, and especially chlorpyrifos. SlGSTE12 might also participate in pyrethroid and organophosphate resistance by antioxidant activity.

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Citation: Liu J., Li P., Lu L., Xie L., Chen X., Zhang B. (2019): Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua. Plant Protect. Sci., 55: 61-71.Abstract: Eight commonly used candidate reference genes, 18S ribosomal RNA (rRNA) (18S), 28S rRNA (28S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1α), ribosomal protein L7 (RPL7), Alpha-tubulin (α-TUB), and TATA box binding protein-associated factor (TBP), were evaluated under various experimental conditions to assess their suitability in different developmental stages, tissues and herbicide treatments in Avena fatua. The results indicated the most suitable reference genes for the different experimental conditions. For developmental stages, 28S and EF1α were the optimal reference genes, both EF1α and 28S were suitable for experiments of different tissues, whereas for herbicide treatments, GAPDH and ACT were suitable for normalizations of expression data. In addition, GAPDH and EF1α were the suitable reference genes.

BACKGROUND: Spodoptera litura is an important agricultural pest and has developed serious resistance to multiple insecticides. The resistance level to several insecticides is reported to be unstable, but the mechanism is less reported.RESULTS: Chlorpyrifos and phoxim resistance level in a field-collected population of S. litura declined continuously from the first to the tenth generation and remained stable at the 11th and 12th generations without insecticide exposure. Synergist experiment showed that diethyl maleate and piperonyl butoxide significantly increased mortality to chlorpyrifos and phoxim in the first and sixth generations, but not in the 12th generation. The expression of 31 identified glutathione S-transferase (GST) genes in the third-instar larvae of S. litura in the first, sixth and 12th generations was determined, and eight genes were seen to decrease significantly in the sixth and 12th generations compared with the first generation. SlGSTe9 was selected for further functional study as it had higher abundance and significantly higher expression in the chlorpyrifos-resistant population than in the susceptible population. The recombinant protein of SlGSTE9 showed metabolism activity to chlorpyrifos in vitro and in Escherichia coli, but not to phoxim. Silencing of SlGSTe9 increased the cumulative mortality to chlorpyrifos significantly. SlGSTE9 also showed antioxidant activity to cumene hydroperoxide. CONCLUSION: Our results suggest that SlGSTe9 is directly involved in chlorpyrifos resistance stability, but not in phoxim. SlGSTE9 may also participate in insecticides resistance by relieving the oxidase stress induced by insecticides.

Rhyzopertha dominica (Fabricius) has developed extensive pesticide resistance in the last several decades. We have developed a supercritical fluid extraction method for Trigonella foenum-graecum L. (TFG) and studied the contact toxicities of the extracts to R. dominica. The extraction method was designed with orthogonal experiments to preserve and collect all the possible active components. Contact toxicity and efficiency of extraction were used as standard values to optimize extraction conditions, which were achieved at 55°C under 25 Mpa of pressure. The extraction efficiency for 200 g of dry sample reached 6.21% with 30 ml of 95% alcohol. Extracts loaded on filter paper showed dose and time dependent toxicities to adult R. dominica with a LC 50 value of 65.02 lg/cm 2 after 3 days post treatment. Our extensive in vivo studies indicated the extracts from Trigonella foenum-graecum seeds have high efficacy against pesticide resistant R. dominica. The active ingredient(s) from the extract shows promise as a novel pesticide candidate.

Descurainia sophia L. is a notorious weed in winter wheat field and has serious resistance to tribenuron-methyl. Xinjiang is a main wheat production region in China with no information on D. sophia resistance to tribenuron-methyl. Here, resistance levels of D. sophia populations to tribenuron-methyl from Xinjiang and Henan were investigated. In addition, homozygous mutation subpopulations of high resistant D. sophia populations from Xinjiang and Henan were generated and then cross-resistance and fitness cost were determined. Results showed that 5 out of 31 populations from Xinjiang developed resistance to tribenuron-methyl, including two high resistant populations (X30 and X31). While 10 out of 11 populations from Henan showed resistance to tribenuron-methyl, including three high resistant populations (H5, H6 and H7). X30 and X31 shared the same mutation type of Pro197Thr in ALS1, while the mutation type of ALS1 in H5, H6 and H7 were Pro197Ser, Pro197His and Pro197Ala, respectively. The homozygous mutation subpopulations (SX30, SX31, SH5, SH6, SH7) showed cross-resistance to flucarbazone-sodium, bensulfuron methyl and flumetsulam. Under monoculture condition, relative growth rates of SX30, SX31 were higher than susceptible population (SX13), while that in SH5, SH6, SH7 were almost same with SX13. When mix planted with SX13, SX30 and SX31 displayed weaker competitiveness than SX13, while SH5, SH6, SH7 showed stronger competitiveness than SX13. The results suggested that D. sophia from Xinjiang had low resistance frequency to tribenuron-methyl and the high resistant populations had fitness costs.

Backgroud: MicroRNAs (miRNAs), which are as short single-stranded non-coding RNAs, coudl regulate the expression of target genes, especially regulation or metabolism of endogenous or xenobiotic compounds. Results: The de novo assembly of the transcriptomes was obtained through Illumina short-read sequencing technology in Sitobion avenae. 57 miRNAs, of which 36 were known and 21 were novel were identified. Quantitative expression levels of miRNA showed that the expression of 5 miRNAs were significant up-regulation, and the expression of 11 miRNAs were significant down-regulation in the nymph of S. avenae treated by imidacloprid in comparison with the control, respectively. The candidate transcript target genes in S. avenae that could be regulated by these miRNAs were also carried out. The functions of the miRNAs, which could potentially regulate the target genes participated in the metabolism, regulatory or detoxification of S. avenae were clarified based on Gene Ontology and KEGG pathway. The effects of the miRNAs identified api-miR-1000, api-miR-316, and api-miR-iab-4 on susceptibility of S. avenae to imidacloprid was determined. The abundance of api-miR-1000, api-miR-316, and api-miR-iab-4 modulated by the addition of their own inhibitors to the artificial diet could change significantly the susceptibility of S. avenae to imidacloprid, which further proved that the regulatory affect of these miRNAs on regulation or metabolism of insecticides. Conclusion: It suggested that differentially expressed microRNAs under the stress of imidacloprid could play critical regulatory role in the resistance of S. avenae to imidacloprid.

Background MicroRNAs (miRNAs), which are short single-stranded non-coding RNAs, regulate the expression of target genes, especially those involved in the regulation or metabolism of endogenous or xenobiotic compounds.Results De novo assemblies of the transcriptome of Sitobion avenae Fabricius under control conditions and under imidacloprid treatment were obtained using Illumina short-read sequencing technology. Fifty-seven miRNAs, of which 36 were known and 21 were novel, were identified. Quantitative analysis of miRNA levels showed that five miRNAs were significantly up-regulated, and 11 miRNAs were significantly down-regulated in the nymphs of S. avenae treated with imidacloprid in comparison with those of the control. Analysis of the candidate target genes in S. avenae that could be regulated by these miRNAs were also carried out. The functions of the miRNAs, which could potentially regulate target genes that participate in metabolism, regulatory or detoxification pathways in S. avenae , were clarified based on Gene Ontology and KEGG pathway analysis. The effects of the miRNAs api-miR-1000, api-miR-316, and api-miR-iab-4 on susceptibility of S. avenae to imidacloprid was determined. Modulation of the abundances of api-miR-1000, api-miR-316, and api-miR-iab-4 by the addition of the correspondign inhibitors to the artificial diet significantly changed the susceptibility of S. avenae to imidacloprid, which further demonstrated the effect of these miRNAs on the regulation or metabolism of insecticides.Conclusion The results of this study suggested that miRNAs differentially expressed in response to imidacloprid could play a critical regulatory role in the resistance of S. avenae to imidacloprid.