Identification of odorant receptors of Tribolium confusum in response to limonene repellent activity.

Tribolium confusum is an important storage pest showing significant resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Present study attempts to explore the molecular mechanism about the repellent activity of limonene. When treatment concentration of limonene was 200.00 µg/cm2, the repellent level remained at grade V after 24 hours. Our study showed that limonene could be distinguished by T. confusum antenna with a maximal electroantennography test value of 0.90 mV. Simultaneously, 88 upregulated and 98 downregulated genes were sequenced in limonene-repellent T. confusum, and RT-qPCR analysis showed that four down-regulated and one up-regulated OR genes play an important role in the response to limonene. The repellent rate was decreased by 22.13% mediated with a knockdown of dsTconOR93, while the EAG value of the female and male adults was reduced to 0.26 mV (49.06%) and 0.20 mV (54.05%), respectively. In conclusion, limonene had a strong repellent activity against T. confusum and TconOR93 gene was determined to be a major effector in perception of limonene. This study provides a basis for the development of limonene as a novel botanical pesticide for the control to storage pests, which will reduce the utilization of chemical pesticides and postpone the development of resistance.

Role of CYP6MS subfamily enzymes in detoxification of Sitophilus zeamais after exposure to terpinen-4-ol and limonene.

Food security is an important basis and guarantee to national safety, the loss caused by storage pests was a serious problem which affects the food security widely. Frequent application of chemical pesticides caused several critical crises including the development of resistance, pesticide residues, environmental pollution, and exposure risk to human or non-target organisms. The utilization of volatile components acts as a natural alternative for controlling storage pests has aroused extensive interest in recent years. It has been reported that terpinene-4-ol and limonene showed significant insecticidal activity against Sitophilus zeamais in our previous studies, which was evaluated to have strong influences to CYP450 genes. To determine the links and roles of related genes, we identified the SzCYP6MS subfamily genes which encoded a putative protein of 493 or 494 amino acids. Then, the expression of four CYP6MS subfamily genes were increased significantly under the fumigation stress by terpinen-4-ol and limonene, which was determined by the RT-qPCR analysis compared with non-fumigated colonies. In addition, we determined that RNAi-mediated CYP6MS genes knockdown significantly increased the sensitivity of S. zeamais to terpinen-4-ol and limonene, the mortality rates of insects with knocked down CYP6MS1, CYP6MS5, CYP6MS6, CYP6MS8, and CYP6MS9 genes increased by 25%, 25%, 16%, 17%, and 4% in terpinen-4-ol treatment groups and by 29%, 25%, 15%, 22%, and 3% in limonene treatment groups compared with that in the control groups, respectively. Finally, it was validated that CYP6MS5 exhibited the most stable binding with terpinen-4-ol that was similar to the result between CYP6MS8 and limonene which were verified by molecular docking analysis. In together, this study demonstrates the potential of terpinen-4-ol and limonene used as novel botanical pesticides to control storage pests, thereby reducing application of chemical pesticides and postponing resistance development.

Genome-Wide Identification of the Long Noncoding RNAs of Tribolium castaneum in Response to Terpinen-4-ol Fumigation.

Long noncoding RNAs (lncRNAs) are important regulatory factors in multiple biological processes, and several lncRNAs are known to respond to insecticides. However, the lncRNA functions that are associated with terpinen-4-ol resistance in the red flour beetle (Tribolium castaneum) have not yet been identified. In this study, we determined the differentially transcribed lncRNAs between fumigated and control experimental groups. In the six libraries that underwent RNA sequencing, 34,546 transcripts were identified, including 8267 novel lncRNAs, 4155 novel mRNAs, 1151 known lncRNAs, and 20,973 known mRNAs. Among these, we found that the expression of 1858 mRNAs and 1663 lncRNAs was significantly different in the fumigated group compared with the control group. Among the differentially transcribed lncRNAs, 453 were up-regulated and 1210 were down-regulated lncRNAs. In addition, we identified the regulatory function targets of the lncRNAs. Functionally, all lncRNAs and target genes associated with terpinen-4-ol metabolism were enriched in several metabolic pathways, like the ATP-binding cassette transporter, pentose interconversion, and glucuronate interconversion. To the best of our knowledge, this study represents the first global identification of lncRNAs and their potential association with terpinen-4-ol metabolism in the red flour beetle. These results will provide reference information for studies on the resistance to terpinen-4-ol and other essential oil compounds and chemical pesticides, as well as an understanding of other biological processes in T. castaneum.