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

Materials and reagents

Grayanotoxin I was procured from Sichuan Biocrick Biotech Co. Ltd (4720-09-6, Chengdu, China). The free fatty acid assay kit was obtained from Jiancheng Co. Ltd. (Nanjing, China), while the hematoxylin-eosin (H & E) staining solution was obtained from Beyotime Biotechnology (Shanghai, China). The Vazyme^® HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) and Vazyme ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme Corporation (Nanjing, China). The primers were synthesized by Takara (Dalian, China). Further, Lipase (JM-00078O1), 3-hydroxyacyl-CoA dehydrogenase (JM-00048O1), and Acetyl-CoA carboxylase (JM-00064O1) ELISA kits were procured from Jingmei Biotechnology (Jiangsu, China).

Spodoptera litura culture, treatment, and sample collection

The larvae of S. litura were obtained from Keyun Biopesticide Co. Ltd in Henan, China. These larvae were sourced from fields free from heavy metal pollution with no prior application of chemical insecticides. Optimal laboratory culture conditions of a temperature of 25 ± 2 °C, humidity of 75%–85%, and a light cycle of light/dark: 14 h/10 h were employed for the rearing of the larvae. Only the second instar larvae with uniform size and normal development were selected for further testing.

To investigate the effects of grayanotoxin I on S. litura, the plant-derived insecticide, matrine was used as the positive control. Matrine is an alkaloid derived from plants belonging to the Sophora genus. As a naturally occurring plant-based pesticide, matrine generally poses low toxicity to humans. Matrine operates as a broad-spectrum insecticide, effectively targeting pests through both contact and ingestion mechanisms. The second instar larvae were randomly divided into the normal diet, different concentrations of grayanotoxin I-containing diet, or matrine-containing diet group. The diets were prepared by adding 7 mL of ddH₂O, 1.25–6.25 mg/L grayanotoxin I, or 0.4% matrine solution to 5 g diet. The survival rates were calculated at 24-hour, 48-hour, and 72-hour treatments. The midgut of S. litura larvae fed on a 1.25% grayanotoxin I-contained diet or normal diet (ddH₂O) for 72 h was collected for RNA-Seq.

For analysis of body weight and developmental time, sublethal concentrations (0.62–1.25 mg/L) of grayanotoxin I were used to treat S. litura larvae. The diets were prepared by adding 7 mL of grayanotoxin I solution to 5 g of normal diet. The wet body weight of each larvae was collected at each instar stage until pupation, and the data was recorded.

Hematoxylin and Eosin (H & E) staining of fat body

The growth rate of insects is largely regulated by the fat body (Yuan et al., 2020). To assess the development of this crucial tissue, we utilized the H & E staining method, as previously described (Yamahama, Seno & Hariyama, 2008). The specimens were subjected to a 5-hour incubation at 5 °C in 10% sucrose in 0.01 M phosphate-buffered saline (PBS, pH 7.4), with sucrose concentration gradually increased to 20%. The samples were then embedded in an optimal cutting temperature (OCT) compound and instantaneously frozen with dry ice. Further, frozen samples were sectioned at 10 µm and stained by the H & E method to obtain images. The images were examined under a microscope to evaluate the development of the fat body.

RNA extraction and RNA-sequencing

To further explore the impact of grayanotoxin I on the expression of lipid metabolism-related genes, RNA-Sequencing using Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was carried out at Shanghai Personal Biotechnology Cp., Ltd (Shanghai, China). The methodology was consistent with previously published studies (Bao et al., 2016a; Bao et al., 2016b). Briefly, total RNA was extracted using the Trizol reagent. The quality and quantity of total RNA were assessed by measuring the absorbance on wavelengths of 260 nm and 280 nm by NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). After the removal of rRNA by using poly-T oligo-attached magnetic beads, the total RNA was fragmented by using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. The first strand cDNA was synthesized using random oligonucleotides and Super Script II. Subsequently, the second strand cDNA synthesis was performed by using DNA Polymerase I and RNase H. For hybridization preparation, the DNA fragments’ 3′ ends were adenylated, followed by ligation of Illumina PE adapter oligonucleotides. To obtain cDNA fragments of the desired length (400–500 bp), the library fragments were purified using the AMPure XP system (Beckman Coulter, Pasadena, CA, USA). DNA fragments possessing adapter molecules on both ends were selectively enriched through a 15-cycle PCR reaction with the Illumina PCR Primer Cocktail. The resulting products were purified using the AMPure XP system and the quantity was measured using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). Finally, the sequencing library was sequenced on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA) by Shanghai Personal Biotechnology Cp. Ltd.

Differentially expressed genes (DEGs) identification

The reference genome used in the present transcriptome was ASM270686v3. The sequencing data was filtered to get high-quality sequences by using Cutadapt (v1.15) software. The filtered data were mapped to the reference genome using HISAT2 (v2.0.5). The analysis of S. litura mRNA expression was performed using HTSeq (0.9.1) statistics. The original expressed read count value per gene was normalized via the FPKM method. DESeq (1.30.0) was employed to analyze differences in mRNA expression levels. RNAs with |log2FoldChange| > 1.0 and P-value < 0.05 were identified as differentially expressed. To perform heatmap clustering, MeV 4.9.0 software was used. Using this method, differentially expressed lipid metabolism-related genes were selected and heatmap clustering was conducted.

RT-qPCR verification of lipid metabolism-related DEGs

To verify the expression of four differentially expressed lipid metabolism-related genes, we utilized RT-qPCR as described previously  (Bao et al., 2018). Total RNAs were extracted using Trizol reagent, followed by reverse transcription to cDNA utilizing the Vazyme HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). PCR reactions were carried out using the Vazyme ChamQ Universal SYBR qPCR Master Mix kit on the Applied Biosystems Quantstudio 5 system. The qPCR program was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The GAPDH and β-actin were used as reference genes. The primers were presented in Table 1. The non-transcribed RNA was used as a negative control. The melting curve analysis was performed to verify the specificity of PCR products. All samples were run in triplicate and analyzed using the 2^(−ΔΔCt) method.

Detection of FFA, lipase, ACC, and HOAD

Lipase, HOAD, and ACC are enzymes that play key roles in the metabolism of fatty acids. To investigate the impact of grayanotoxin I on the lipid metabolism of S. litura, we employed an ELISA-based approach to measure the levels of lipase, HOAD, and ACC in the hemolymph of 5th instar larvae. Briefly, hemolymph samples were collected in a 1.5 ml tube with 0.1% dithiothreitol (DTT), and centrifuged for 5 min (10,000 rpm) at 4 °C. The supernatant was stored at −80 °C for further use (Bai & Grewal, 2007). The ELISA analysis was conducted according to the manufacturer’s instructions. Specifically, 50 µL of serum samples were added to enzyme-linked immunosorbent plates, mixed with enzyme labeling reagents, and incubated at 37 °C for 60 min. The liquid was then removed, and each well was washed five times with washing solution before adding chromogenic reagent and mixing. The mixture was incubated for 15 min at 37 °C in the dark, after which the reaction was halted using a stop solution. The absorbance value was then measured to determine the levels of lipase, HOAD, and ACC.

FFA was measured by using the fatty acid assay kit purchased from Jiancheng Co. Ltd. (Nanjing, China) according to the manufacturer’s instructions. The assay kit is based on the principle that FFA reacts with copper ions to form fatty acid copper salts, which are soluble in chloroform. By using the copper reagent method to determine the copper ion content, the content of FFA can be estimated by colorimetric assay.

Gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) and protein-protein interaction (PPI) analysis of lipid metabolism-related differentially expressed genes

To investigate the functions of differentially expressed genes related to lipid metabolism, we conducted GO enrichment analysis and KEGG pathway analysis. This analysis was carried out using the online tool DAVID (https://david.ncifcrf.gov/) (Yu et al., 2022; Xu et al., 2020a). The top 10 terms from biological process (BP), cell components (CC), molecular function (MF), and KEGG pathway were visualized, and a P-value < 0.05 was considered significant for both GO terms and KEGG pathways.

To further examine the interactions between lipid metabolism-related DEGs, we utilized the online tool STRING (https://string-db.org/). As S. litura data was not available in STRING, we used Bombyx mori data as an alternative. We also performed a further analysis of the signal pathways of the lipid metabolism-related DEGs on the KEGG pathway (https://www.genome.jp/kegg/).

Statistical analysis

All the statistics were presented in the form of mean ± S.D. The significance of the differences was analyzed by ANOVA followed by the Newman-Student-Keuls test. A value of P < 0.05 was considered statistically significant.

Influence of grayanotoxin I on S. litura growth and development

To investigate the impact of grayanotoxin I on S. litura, we monitored the survival rate, growth, and development of the insects after being subjected to grayanotoxin I-contained, matrine-contained, or normal diet. As depicted in Fig. 1A, the application of a positive control, 0.4% matrine, reduced the survival rate to 18.8% after a 72-hour treatment. While 72-hour treatment with 6.25 mg/L grayanotoxin I reduced the survival rate to 40.0%, as compared to the normal diet (ddH₂O, survival rate of 96.7%). Additionally, lower concentrations of grayanotoxin I (0.62–1.25 mg/L) significantly hindered the growth of S. litura (Figs. 1B–1C). Compared to the ddH₂O group, the 0.2% matrine hindered the 95.3% body weight of S. litura on day 14. The suppression rate was 90.65% for 1.25 mg/L grayanotoxin I, and 56.29% for 0.65 mg/L grayanotoxin I after 14-day treatment (Figs. 1B–1C). Furthermore, we observed a significant delay in the pupation time of S. litura because of grayanotoxin I (Fig. 1D). The average pupation time for the ddH₂O group was 14.72 days. While it was 20.23 days for 1.25 mg/L grayanotoxin I treatment and 18.25 days for 0.62 mg/L grayanotoxin I treatment (Fig. 1D).

Inhibition effect of grayanotoxin I on S. litura fat body development

In the present study, H & E staining was conducted to investigate the relationship between fat body development and the growth of S. litura. As illustrated in Fig. 2, a noticeable accumulation of fat in the fat body was observed in the ddH₂O control group (Fig. 2A). However, treatment with grayanotoxin I resulted in a significant inhibition of fat body development (Fig. 2B).

Gene expression profiles of S. litura under grayanotoxin I treatment

To investigate the mechanisms of grayanotoxin I, we analyzed the transcriptome alteration after 72-hour 1.25% grayanotoxin I treatment by using the RNA-Seq method. The statistical power of this RNA-Seq data calculated in “RNASeqPower” was 0.855 (sequencing depth: 60, sample size: 3). As a result, 285 DEGs were identified. Among them, 151 were upregulated and 134 were downregulated (Figs. 3A–3B).

GO and KEGG enrichment of differentially expressed lipid metabolism-related genes

To get further insight into the functions of the 285 DEGs, we carried out KEGG pathway enrichment and GO enrichment analysis. In the GO enrichment analysis, these DEGs were mostly enriched in the MF terms related to the structural constituents of chitin-based cuticle; BP terms associated with cuticle development; and CC terms related to the extracellular matrix (as depicted in Fig. 3C). The KEGG analysis (Fig. 3D) revealed that these DEGs were enriched in several pathways including the organismal system terms of longevity regulating pathway, cytosolic DNA-sensing pathway, and fat digestion and absorption pathway; the metabolism terms of cutin, suberin, wax biosynthesis, linoleic acid metabolism, insect hormone biosynthesis, and unsaturated fatty acid synthesis; the cellular process terms of peroxisome.

The effects of grayanotoxin I on lipid metabolism-related gene profile expression, lipid metabolism-related enzyme activities in the hemolymph, and FFA level in S. litura

In our RNA-Seq analysis, we discovered many DEGs related to lipid metabolism. Specifically, we observed an upregulation of genes such as acyl-CoA desaturase, esterase E4, and phospholipase, and downregulated genes such as fatty acid elongase, fatty acid-binding protein, and pancreatic-like lipase following treatment with grayanotoxin I (Fig. 4A). The results of RNA-Seq were verified by qPCR analysis, which was shown in Fig. 4B.

Besides, grayanotoxin I (1.25 mg/L) treatment dramatically decreased the level of FFA in the hemolymph of S. litura (Fig. 4C). Further ELISA analysis revealed a significant decrease in lipase and HOAD mRNA levels after treatment with grayanotoxin I, compared to the normal group (P < 0.05). A slight decrease in ACC mRNA was also found after grayanotoxin I treatment (Figs. 4D–4F).

PPI analysis of lipid metabolism-related DEGs analysis

The PPI of the lipid metabolism-related genes was shown in Fig. 5A. Red circles were upregulated genes in S. litura after grayanotoxin I treatment, while green circles were downregulated genes.

The LOC111354773 (putative fatty acyl-CoA reductase), LOC111355891 (acyl-CoA desaturase 1-like), LOC111350394 (ELOVL fatty acid elongase), LOC111349277 (elongation of very long chain fatty acids protein 7 like), and LOC111360381 (fatty acid-binding protein 2 like) were connected clearly in the network.

Further analysis revealed that LOC111354773 (putative fatty acyl-CoA reductase), LOC111355891 (acyl-CoA desaturase 1-like), LOC111355893 (acyl-CoA desaturase 1-like), LOC111352061 (putative fatty acyl-CoA reductase), and LOC111356581 (fatty acyl-CoA reductase wat-like) were enriched in the longevity regulating pathway and were relevant to the aging of the larvae. The aforementioned genes along with LOC111350394 (ELOVL fatty acid elongase), LOC111348151 (phospholipase A1-like), and LOC111356581 (fatty acyl-CoA reductase wat-like) were found to be associated with lipid metabolism. Additionally, LOC111355891 (acyl-CoA desaturase 1 like), LOC111360381 (fatty acid-binding protein 2 like), and LOC111355893 (acyl-CoA desaturase 1-like) were found to be relevant to the PPAR signaling pathway, as documented in Table 2 and Fig. 5B.