Illumina sequencing
Using the Illumina NovaSeq 6000 sequencing platform, H. bacteriophora H06 treated with or without DMSO was sequenced to identify genes involved in the nematode development. Total RNA was collected from 8 independent samples (4 samples treated with DMSO before or after egg formation, along with corresponding water-treated controls). This RNA was used to characterize the transcriptome and measure differential gene expression. RNA sequencing generated a minimum of approximately 42.56 million reads per sample, with at least 94.67% of reads in the control group and at least 94.70% in the treatment group aligning to reference sequences assembled using Trinity (Supplementary Table 4). On average, the filtered clean reads amounted to 51,941,386.5 and 50,702,032.5 for the treatments and controls, respectively. Details of the Unigene alignment results were provided in Supplementary Table 5. The correlation coefficients between biological replicate samples were all greater than 0.96, as depicted in Fig. 1, indicating high reproducibility.
To gain a deeper understanding of the differences in H. bacteriophora H06 under DMSO induction at different time points, pairwise comparisons were conducted (|log2(Fold Change)| > 1 and q value < 0.05). Specifically, in the comparison of the nematodes before egg formation under DMSO or water treatment, 306 genes were identified, including 11 up-regulated genes and 295 down-regulated genes. In the comparison of the nematodes after egg formation under DMSO or water treatment), 97 genes were identified, including 28 up-regulated genes and 69 down-regulated genes. So more downregulated genes were detected. Therefore, subsequent research and analysis focused on the downregulated genes(Table 1).
Functional differentially expressed genes (DEGs) by GO and KEGG
The 4875 DEGs were annotated with GO terms and assigned to three functional categories using Blast2GO (Fig. 1). In biological processes, annotations were primarily associated with metabolic processes, developmental processes, response to stimulus, metabolic processes, and biological regulation. In cellular components, annotations were mainly enriched in intracellular components, cytoplasmic lipid, organelles, and molecular complexes. Additionally, in molecular function, differential expression was mainly enriched in catalytic activity, nucleic acid binding, and protein binding.
To systematically analyze metabolic pathways and gene functions within the cell, the results of KEGG pathway annotations were categorized and summarized. A total of 32 pathways were significantly enriched across five categories, including cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. Among these, the top five pathways were signal transduction, endocrine system (409 DEGs), amino acid metabolism (383 DEGs), translation (373 DEGs), and transport and metabolism (331 DEGs) (Fig. 1).
Analysis of differentially expressed genes in IJs response to DMSO
For the H06 nematodes on LA medium with DMSO or without DMSO, 3 days before complete egg formation, GO enrichment was classified into three functional categories (Fig. 2): biological processes (132 downregulated), molecular functions (75 downregulated), and cellular components (85 downregulated). Biological processes included terms such as molting cycle (GO:0018996, GO:0042303), collagen and cornification development (GO:0042338, GO:0040002, GO:0042335). Cellular components involved extracellular matrix (GO:0060102, GO:0031012) and collagen and cornification (GO:0060103, GO:0060106, GO:0060111). Molecular functions encompassed DNA binding (GO:0001012, GO:0044212, GO:0043565), nucleic acid binding (GO:0001067), and structural constituents of collagen and cornification (GO:0042329, GO:0042302), mostly comprising downregulated genes within this group. In KEGG enrichment analysis, by using the KOBAS (v3.0) software, the top 20 significantly enriched pathways were selected, and statistics on the number of enriched genes in each pathway was conducted (Fig. 2). Associations were primarily linked to metabolic pathways (ko01100) and secondary metabolites (ko01110). For the H06 nematodes on LA medium with DMSO or without DMSO, 3 days after complete egg formation, biological processes were mainly associated with ribonucleoside metabolism (GO:0009144, GO:0009126, GO:0009259). Cellular components were related to mitochondrial inner membrane (GO:0031966), while molecular functions were associated with catalytic activity (GO:0003824) and oxidoreductase activity (GO:0003824).
RNA-seq verification by qRT-PCR
To confirm the expression changes observed through differential expression analysis using RNA-seq data, qRT-PCR was performed on the selected genes. The genes showing significant differential expression (p < 0.05) were chosen for verification, as listed in Supplementary Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as the reference gene (Supplementary Table 2). Relative expression was calculated using the 2−ΔΔCt method with transcriptome data in the reference gene. The qRT-PCR results were consistent with the trends observed in the RNA-seq analysis (Fig. 3).
Functional verification of the key DEGs by RNAi
To further elucidate the role of genes associated with the increased IJ yield under DMSO treatment, downregulated genes were selected to identify their functions (Supplementary Table 3, Supplementary Table 5), based on the criteria of |log2(Fold Change)| > 1 and q value < 0.05, along with the conditions related to enriched pathways.
During the cultivation of nematodes in the 96-well plate, three genes exhibited noticeable phenotypes (Fig. 4). There was no significant difference in the egg numbers of H06 hermaphrodites in the medium containing DMSO or dsHint, however, significant difference in was found between the egg numbers of the hermaphrodites treated by dsHint and dsEgfp (Fig. 4A). The similar pattern was found in the IJ yields after 15 days as in egg numbers (Fig. 4D). For the PAN gene, although no significant difference was detected in the egg numbers of the hermaphrodites treated by dsPAN and dsEgfp (Fig. 4B), or by dsDyp-13 and dsEgfp (Fig. 4C), the IJ yields were significant different from the medium containing dsPAN (608 IJs per well in the plate) and dsEgfp (224 IJs per well) (Fig. 4E), and dsDyp-13 and dsEgfp (Fig. 4F).
Knockdown of these three genes (dsHint, dsPAN and dsDPY-13) indicated that the expression suppression of specific genes can effectively enhance the reproductive and productive performance of H. bacteriophora H06, which is similar to the addition of DMSO in the medium.
Gene expression after RNAi
No significant differences in the expressions of three genes treated with dsEgfp and pure water were found by qRT-PCR analysis. However, after the introduction of dsRNA, the expression levels of Hint, PAN, and DPY-13 genes decreased significantly by 60%, 61%, and 56%, respectively, compared to the introduction of pure water. These results indicated successful knockdown of these genes, which is consistent with expression levels observed under DMSO treatment (Fig. 5).