Transcriptome analysis unveils the mechanisms of lipid metabolism response to grayanotoxin I stress in Spodoptera litura

Abstract

Background: Spodoptera litura (tobacco caterpillar, S. litura) is a pest of great economic importance due to being a polyphagous and world-distributed agricultural pest. However, agricultural practices involving chemical pesticides have caused resistance, resurgence, and residue problems, highlighting the need for new, environmentally friendly methods to control the spread of S. litura.

Aim: This study aimed to investigate the gut poisoning of grayanotoxin I, an active compound found in Pieris japonica, on S. litura, and to explore the underlying mechanisms of these effects.

Methods: S. litura was cultivated in a laboratory setting, and their survival rate, growth and development, and pupation time were recorded after grayanotoxin I treatment. RNA-Seq was utilized to screen for differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to determine the functions of these DEGs. ELISA was employed to analyze the levels of lipase, 3-hydroxyacyl-CoA dehydrogenase (HOAD), and acetyl-CoA carboxylase (ACC). Hematoxylin and Eosin (H & E) staining was used to detect the development of the fat body.

Results: Grayanotoxin I treatment significantly suppressed the survival rate, growth and development, and pupation of S. litura. RNA-Seq analysis revealed 285 DEGs after grayanotoxin I exposure, with over 16 genes related to lipid metabolism. These 285 DEGs were enriched in the categories of cuticle development, larvae longevity, fat digestion and absorption. Grayanotoxin I treatment also inhibited the levels of FFA, lipase, and HOAD in the hemolymph of S. litura.

Conclusion: The results of this study demonstrated that grayanotoxin I inhibited the growth and development of S. litura. The mechanisms might, at least partly, be related to the interference of lipid synthesis, lipolysis, and fat body development. These findings provide valuable insights into a new, environmentally-friendly plant-derived insecticide, grayanotoxin I, to control the spread of S. litura.

Keywords: Grayanotoxin I; Lipid metabolism; Spodoptera litura.

Figure 1. Effects of grayanotoxin I on survival rate, the growth & development of S. litura.

The second instar larvae of S. litura were fed with the normal diet, grayanotoxin I-containing diet, or matrine-containing diet. The survival rate, body length, body weight, and pupation time were measured. (A) The survival rate of S. litura after ddH₂O, 1.25–6.25 ml/L grayanotoxin I, or 0.4 % matrine treatment in 24, 48, and 72 hours; (B) the body length of S. litura between ddH₂O or 0.62 mg/L grayanotoxin I treatment on day 14; (C) the body weight-time curve after 0.62–1.25 ml/L grayanotoxin I, ddH₂O, or sublethal matrine (0.2 %) treatment; the body weight of each larvae was measured every 2 days. (D) The pupation time after grayanotoxin I, ddH₂O, or sublethal matrine (0.2 %) treatment. All data were presented in mean ± SD, **P < 0.01; *P < 0.05 vs ddH₂O group.

Figure 2. The development of fatty body after treatment of grayanotoxin I.

After treatment with grayanotoxin I for 14 days, the larvae of S. litura specimens were sectioned and stained by Hematoxylin and Eosin. The images were examined under a microscope to evaluate the development of the fat body. A, S. litura treated by ddH₂O; B, S. litura treated by grayanotoxin I.

Figure 3. The transcriptomic analysis, GO enrichment, and KEGG enrichment of differentially expressed genes after grayanotoxin I treatment in S. litura.

(A) The number of upregulated and downregulated genes after grayanotoxin I treatment; (B) the heatmap of all differentially expressed genes after grayanotoxin I treatment; (C) GO enrichment of differentially expressed genes; (D) KEGG enrichment of differentially expressed genes.

Figure 4. Effects of grayanotoxin I on lipid metabolism-related genes, lipid metabolism-related enzyme activities, and FFA levels in S. litura.

The second instar larvae of S. litura were treated with ddH₂O (control group) or 1.25 mg/L grayanotoxin I-containing diet for 72 h following which the midgut of S. litura was collected for RNA-Seq. (A) The heatmap of differentially expressed lipid metabolism-related genes; (B) qPCR verification of 4 randomly chosen lipid metabolism-related genes; (C–F) the level of free fatty acid, lipase, acetyl-CoA carboxylase, and HOAD in the hemolymph of S. litura. All data were presented in mean ± SD, *P < 0.05, **P < 0.01 vs. control group.

Figure 5. The protein-protein interactions and signal pathways of lipid-related DEGs.

(A) The protein–protein interaction of lipid-related DEGs analyzed by STRING online software; (B) the visualization of the PPAR signaling pathway obtained from KEGG pathway online software.