After insecticides are used in the field, their toxicity will gradually decrease to sublethal concentration with the passage of time and environmental factors, and some pest individuals will be exposed to sublethal concentrations, which will result in the sublethal effects. Therefore, understanding the sublethal effects of pesticides is the key to examine their efficacy and evaluate pesticide risk management. Panonychus citri McGregor (Acari: Tetranychidae) is a key citrus pest in the world and its control is largely based on the use of acaricides, such as spirobudiclofen In this study, the sublethal effects of spirobudiclofen on population parameters of Panonychus citri adult females were evaluated. The results showed that sublethal concentration of spirobudiclofen significantly reduced in the longevity and fecundity of female adults, and the effects increased with increasing tested concentrations (LC30, LC50). In order to characterise the spirobudiclofen molecular mechanism, the transcriptomes and metabolomics of spirobudiclofen-treated (two sublethal concentrations LC30 and LC50) and untreated adult females were compared using RNA-sequencing and LC-MS. The RNA-seq results revealed that the immune defense, antioxidative system, cuticle formation and lipid metabolic pathway of P. citri were involved in the responses to spirobudiclofen stress. Moreover, based on the disordered lipid metabolic pathways obtained from transcriptomic studies, the sublethal effects of spirobudiclofen on P. citri were examined by LC-MS; the results showed that the tolerance metabolism of P. citri exposed to spirobudiclofen was regulated by enhancing the metabolism of glycerophospholipid and glycine, serine and threonine. In the present study, transcriptomics and metabolomics-based approaches were employed to examine the molecular mechanism of spirobudiclofen-induced sublethal effects on P. citri, and the results provide new insights into the defense mechanisms at the molecular and biochemical level, and valuable information for pest control strategy development.
As the second largest lipid, sphingolipids play an important role in the growth and development of organisms. However, due to the complexity and diversity of their molecular structures, the knowledge about the structure and function of sphingolipids remians unclear for many insects and mites. Ceramidase, as an important sphingolipid substance, maintains cell survival by regulating the dynamic balance between ceramide and sphingosine. In order to study the role of ceramidase in the growth and development of Panonychus citri, the full-length cDNA sequence of ceramidase (CDase) gene was cloned by RT-PCR combined with RACE technology for the first time. The results showed that the full-length of Ceramidase cDNA sequence was 1841 bp , including a 164-bp 5′-UTR, a 1572-bp ORF , and a 105-bp 3-UTR and encodes a deduced protein of 523 amino acids. Amino acid sequence alignment showed that CDase gene had the highest homology with that of Tetranychus urticae and the closest genetic relationship with it. In addition, quantitative analysis using RT-qPCR of the gene expression levels of CDase during different developmental stages demonstrated that the CDase gene was expressed during all developmental stages of P. citri, and the relative expression in larvae was significantly higher than those in other stages. Furthermore, RNAi technology was used to silence CDase gene of P. citri, and the corresponding expression and mortality analysis were observed under different concentration gradients (1500ng/ul, 2000ng/ul, 2500ng/ul) and time gradients (24h, 48h). The dsRNA treatment of CDase gene showed a decrease in gene expression. Compared with the control group, the gene expression of CDase in the dsRNA treatment at 2000 ng/ul decreased by 72%, and the mortality rate of adult females increased by 40%. Furthermore, CDase genes treated with dsRNA also decreased by 78% within 48 hours. In general, the interference effect of dsRNA at 2000 ng/ul and 48h was the best, compared with the control group. Our findings will provide insight into the molecular mechanisms regulating lipid metabolism in P. citri.
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