Genome-wide identification of G-protein coupled receptors (GPCRs) and their expression profile in response to ß-cypermethrin stress in Zeugodacus cucurbitae.

G-protein coupled receptors (GPCRs) are the largest and most diverse transmembrane receptor family in the cell. They are involved in regulating a wide range of biological processes, including behavior, reproduction, and development. However, GPCRs have not yet been identified in Zeugodacus cucurbitae. The current study focuses on the GPCRs identification, classification, distribution, and their expression analysis under ß-cypermethrin stress to uncover novel targets for pest management and assist in the development of effective strategies for controlling the melon fly population. We identified 80 GPCRs genes including 50 GPCRs identified in family A, 17 GPCRs identified in family B, 8 identified in family C, and 5 identified in family F. Z. cucurbitae GPCRs showed significant differences in both the number of genes in families or subfamilies, as well as the sequencing of the genes. Interestingly, newly identified GPCRs genes are expressed differently at various developmental stages of Z. cucurbitae. Further, we evaluated these 80 GPCRs using Realtime quantitative PCR to confirm their expression between ß-cypermethrin-resistant (RS) strain and susceptible strain (SS) of Z. cucurbitae. We identified 50 GPCR genes were highly overexpressed in a RS. Among these genes, eight genes were strongly induced by the 30% lethal concentration (LC) while two genes were significantly increased by the 50% LC of ß-cypermethrin. This first genome-wide profiling and characterization of GPCRs could lay foundation for unraveling detoxification mechanism and target site modifications which may improve the insect resistance and could be effective insecticide targets for Z. cucurbitae management.

Transcriptome Analysis Revealed Genes Related to γ-Irradiation Induced Emergence Failure in Third-Instar Larvae of Bactrocera dorsalis.

The oriental fruit fly is a polyphagous and highly invasive economically important pest in the world. We proposed the hypothesis that radiation treatment influence RNA expression in the larvae and leads to emergence failure. Therefore, transcriptome analyses of third-instar larvae of B. dorsalis ionizing, irradiated with 60Co-γ at 116Gy, were conducted and compared with the controls; a total of 608 DEGs were identified, including 348 up-regulated genes and 260 down-regulated ones. In addition, 130 SNPs in 125 unigenes were identified. For the DEGs, the most significantly enriched GO item was hemolymph coagulation, and some of the enriched pathways were involved in digestive processes. The subsequent validation experiment confirmed the differential expression of six genes, including sqd, ENPEP, Jhe, mth, Notch, and Ugt. Additionally, the 3401:G->A SNP in the Notch gene was also successfully validated. According to previous research, this was the first comparative transcriptome study to discover the candidate genes involved in insect molt to pupae. These results not only deepen our understanding of the emerging mechanism of B. dorsalis but also provide new insights into the research of biomarkers for quarantine insect treatment with the appropriate dose of radiation.

MicroRNA-1-3p affects lung adenocarcinoma progression through E2F8 and regulating NF-кB pathway.

E2F8 can modulate development and progression of various cancers including cervical cancer, breast cancer and hepatocellular carcinoma. But its mechanism in lung adenocarcinoma (LUAD) remains underexplored. In this study, we conducted a series of experiments including qRT-PCR, western blot, CCK-8, scratch healing assay, Transwell, and flow cytometry. Through these assays, we confirmed the notable overexpression of E2F8 in LUAD and its promoting effects on LUAD cell proliferation, migration and invasion. Subsequently, microRNA-1-3p that was negatively associated with E2F8 expression was identified through bioinformatics analysis. qRT-PCR was then carried out for quantification of microRNA-1-3p expression, which displayed low microRNA-1-3p expression in LUAD cells. In addition, dual-luciferase reporter gene assay was utilized for validating the targeted relationship between microRNA-1-3p and E2F8. The results denoted that microRNA-1-3p could bind to the promoter region of E2F8. Finally, the results of rescue experiment revealed that microRNA-1-3p negatively modulated E2F8 level. It regulated NF-κB pathway to repress LUAD cell proliferative, migratory, and invasive properties, lead to cell cycle arrest in G0/G1 phase, and enhance cell apoptosis level. This study unraveled that microRNA-1-3p/E2F8 constrained LUAD malignant progression through NF-κB pathway, which may provide possible targets for LUAD diagnosis and treatment.

The glutathione S-transferase (PxGST2L) may contribute to the detoxification metabolism of chlorantraniliprole in Plutella xylostella(L.).

The diamondback moth (Plutella xylostella L.), is an economic pest of cruciferous plants worldwide, which causes great economic loss to cruciferous plants production. However, the pest has developed resistance to insecticides. One of such insecticides is chlorantraniliprole. The study of the mechanisms underlying resistance is key for the effective management of resistance. In this study, a comparative proteomics approach was used to isolate and identify various proteins that differed between chlorantraniliprole-susceptible and -resistant strains of P. xylostella. Eleven proteins were significantly different and were successfully identified by MALDI-TOF-MS. Metabolism-related proteins accounted for the highest proportion among the eleven different proteins. The function of the PxGST2L protein was validated by RNAi. Knockdown of PxGST2L reduced the GST activity and increased the toxicity of chlorantraniliprole to the diamondback moth. The resistance ratio of diamondback moth to chlorantraniliprole was reduced from 1029 to 505. The results indicated that PxGST2L is partly responsible for chlorantraniliprole insecticide resistance in DBM. Our finding contributes to the understanding of the mechanism underlying resistance to chlorantraniliprole in the DBM, to develop effective resistance management tactics.

Baseline Susceptibility of Plutella xylostella (Lepidoptera: Plutellidae) to the Novel Insecticide Spinetoram in China.

Spinetoram is a spinosyn, which is a unique class of natural insecticide. Because of its novel mode of action, spinetoram is more potent and faster acting than other insecticides, even the older spinosyn product, spinosad. On account of being efficient on insect order Lepidoptera, spinetoram provides a new alternative for control of Plutella xylostella (L.) (Lepidoptera: Plutellidae), which are resistant to other chemicals. To determine the current situation of resistance of P. xylostella to spinetoram, the susceptibility of 16 P. xylostella populations from different regions of China or different time in addition to the population from laboratory was assessed using a leaf dip bioassay. The variation in spinetoram susceptibility among the 16 field populations was narrow, with median lethal concentrations (LC50 values) ranging from 0.131 to 1.001 mg/liter. Toxicity ratios (TRs) ranged from 1.5 to 7.6 and were 5.6 and 7.6 for populations SY-2 and FX-1, respectively, indicating some low level of tolerance in these populations. A discriminating concentration (a concentration that can detect the occurrence of resistance in a population) of 10 mg/liter, which was identified based on the pooled toxicological data, caused 100% mortality in all nine tested populations. The baseline susceptibility data reflect the natural variation of the P. xylostella populations to spinetoram rather than variation caused by previous exposure.

Monooxygenase, a novel beta-cypermethrin degrading enzyme from Streptomyces sp.

The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5-8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe(2+), but strongly inhibited by Ag(+), Al(3+), and Cu(2+). The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection.

Transcriptome analysis of chlorantraniliprole resistance development in the diamondback moth Plutella xylostella.

BACKGROUND: The diamondback moth Plutella xyllostella has developed a high level of resistance to the latest insecticide chlorantraniliprole. A better understanding of P. xylostella's resistance mechanism to chlorantraniliprole is needed to develop effective approaches for insecticide resistance management. PRINCIPAL FINDINGS: To provide a comprehensive insight into the resistance mechanisms of P. xylostella to chlorantraniliprole, transcriptome assembly and tag-based digital gene expression (DGE) system were performed using Illumina HiSeq™ 2000. The transcriptome analysis of the susceptible strain (SS) provided 45,231 unigenes (with the size ranging from 200 bp to 13,799 bp), which would be efficient for analyzing the differences in different chlorantraniliprole-resistant P. xylostella stains. DGE analysis indicated that a total of 1215 genes (189 up-regulated and 1026 down-regulated) were gradient differentially expressed among the susceptible strain (SS) and different chlorantraniliprole-resistant P. xylostella strains, including low-level resistance (GXA), moderate resistance (LZA) and high resistance strains (HZA). A detailed analysis of gradient differentially expressed genes elucidated the existence of a phase-dependent divergence of biological investment at the molecular level. The genes related to insecticide resistance, such as P450, GST, the ryanodine receptor, and connectin, had different expression profiles in the different chlorantraniliprole-resistant DGE libraries, suggesting that the genes related to insecticide resistance are involved in P. xylostella resistance development against chlorantraniliprole. To confirm the results from the DGE, the expressional profiles of 4 genes related to insecticide resistance were further validated by qRT-PCR analysis. CONCLUSIONS: The obtained transcriptome information provides large gene resources available for further studying the resistance development of P. xylostella to pesticides. The DGE data provide comprehensive insights into the gene expression profiles of the different chlorantraniliprole-resistant stains. These genes are specifically related to insecticide resistance, with different expressional profiles facilitating the study of the role of each gene in chlorantraniliprole resistance development.

Population dynamics and "outbreaks" of diamondback moth (Lepidoptera: Plutellidae) in Guangdong province, China: climate or failure of management?

Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), became the major pest of Brassica vegetable production in Guangdong, a province in southeastern China, in the late 1980s and has continued to challenge growers, particularly during the spring and autumn. Control has relied on insecticides and, as has happened in other parts of the world, resistance to these has evolved and subsequent field control failures have occurred. We review and summarize the history of diamondback moth management in Guangdong. We show that the geographic distribution of the pest in China is well described by a simple climate niche model. Our model predicts the seasonal phenology and some of the variation in abundance among years in Guangdong. Discrepancies may reflect migration and insecticide use at a landscape level. The scale of the pest problem experienced varies with management practices. Local production breaks, and strict post harvest hygiene are associated with lower pest pressure on large-scale production units. As more and more insecticides become ineffective the need to implement an insecticide resistance management strategy, as well as basic integrated pest management practices, will become more pressing. The potential use and development of a better forecasting system for diamondback moth that will assist these developments is outlined.

Response surface optimization of ultrasound-assisted flavonoids extraction from the flower of Citrus aurantium L. var. amara Engl.

Citrus aurantium L. var. amara Engl is a member of genus Citrus (Rutaceae) and has been used in Chinese medicine with the effectiveness of digestant and expectorant. Ultrasonic-assisted extraction process for maximum flavonoids from the flower of Citrus aurantium L. var. amara Engl was investigated by response surface methodology. Through single factor experiment, ranges of the main variables (including ethanol concentration, solid/liquid ratio, extraction time and temperature) affecting the extraction yield of flavonoids were confirmed. Box-Behnken central composite design consisting of 24 experimental runs and 5 replicates at zero point was then applied and a regress model was obtained to predict the optimal extraction yield. The ANOVA indicated that the regression equation fits very well with the actual situation, reflecting the relationship between the extraction yield of flavonoids and extraction conditions. The optimal conditions were as follows: extraction temperature 72.11 degrees C, time 51.89 min, ethanol concentration 51.19% and liquid/solid ratio of 40:10. Under the optimal conditions, the maximum response value of yield (1.88%) was consistent with the experimental value (1.87%), indicating the feasibility and validation of response surface methodology in optimizing the extraction of flavonoids from the flower of Citrus aurantium L. var. amara Engl.

Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from Semen Ziziphus jujube.

Semen Ziziphus jujube (SZJ), the seeds of Ziziphus jujuba Mill. var. spinosa, is a kind of traditional Chinese medicine used for its action on insomnia. In order to analyze the effective component, we investigated and compared the sedative and hypnotic effects of three kinds of compounds, flavonoids, saponins, and polysaccharides. Flavonoids, saponins, and polysaccharides were extracted from SZJ and orally administered to mice separately at 17 g kg(-1) per day for certain days before animal tests. Spontaneous motility and coordinated movement tests were used to observe the effects of the three kinds of compounds on the mouse behavior, and sodium barbital-induced sleeping time of mouse were tested to analyze the effects of the three kinds of compounds on the sleep of mouse. Results show that flavonoids and saponins caused a significant reduction of walking time and coordinated movement ability of mouse, significantly prolonged its sleeping time at 40 mg kg(-1), ip, subthreshold dose and increased the sleeping number of animals at 50 mg kg(-1), ip, superthreshold dose induced by coeliac injection of sodium barbital. Polysaccharides did not show any significance in all animal tests. Comparative analysis showed that saponins had a more effective sedative and hypnotic function than that of flavonoids, polysaccharides did not show a sedative and hypnotic effect.