DNA methylation regulates growth traits by influencing metabolic pathways in Pacific white shrimp (Litopenaeus vannamei)

Abstract

Background: DNA methylation is a critical epigenetic modification that dynamically regulates gene expression associated with economic traits. Pacific white shrimp (Litopenaeus vannamei) is one of the most important aquatic species for culturing, and growth trait is one of the most important economic traits for its production. However, research on DNA methylation regulation of growth traits is still at an early stage. This study explored DNA methylome dynamics and their associations with the regulatory mechanism behind growth traits using full-subfamily individuals with discrepant growth performance.

Results: The DNA methylation-related genes in L. vannamei were identified, and the expression of DNA methylation genes showed significantly higher levels in the slow growth (SG) group compared to the fast-growing (FG) individuals. The Whole Genome Bisulfite Sequencing (WGBS) analysis revealed that the methylation levels in the muscles of shrimp were notably decreased in SG individuals compared to FG individuals. A total of 532 differentially methylated promoters and 2,067 differentially methylated regions were identified. Through integrative analysis of DNA methylation and transcriptomic data from SG and FG group shrimp, a total of 47 genes were screened out with differential methylation levels (DMGs) and expression levels (DEGs). Functional enrichment analysis revealed that the overlapping DEGs/DMGs were enriched mainly in metabolic pathways, starch and sucrose metabolism, linoleic acid metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversions.

Conclusions: DNA methylation plays a role in the regulation of growth traits in L. vannamei. The level of DNA methylation was found to be negatively correlated with growth traits. Through comprehensive analysis, it was discovered that DNA methylation predominantly affects growth performance by up-regulating the expression of genes involved in metabolic pathways, such as glucose metabolism and amino acid metabolism in L. vannamei. This suggests a higher metabolism activity in SG individuals derived DNA methylation to cope with some unknown internal stress or environmental stress rather than being allocated for growth.

Keywords: Litopenaeus vannamei; DNA methylation; Gene expression; Growth trait.

Fig. 1

Differential growth performance of L. vannamei shrimp from a full-subfamily. A, Differential body sizes. B: Significant difference in body weight between 6 randomly selected FG individuals and 6 SG individuals. C: Genomic DNA methylation levels in the muscles of FG and SG shrimp individuals

Fig. 2

The gene structures and domain compositions of methylation-related genes. A: (a) Phylogenetic analysis of methylation-related genes in L. vannamei and other five arthropod species (Procambarus clarkii, Penaeus monodon, Litopenaeus chinensis, Portunus trituberculatus, Eriocheir sinensis). (b) The gene structures of methylation-related genes. The different boxes represented different motifs, respectively. (c) The domain compositions of methylation-related genes encoded proteins. The different boxes represented different motifs, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). B: mRNA expression levels of two DNA methylation-related genes (dnmt1 and dnmt3a) mRNA in the FG and the SG shrimp individuals (n = 3). C: tissues distribution of two DNA methylation-related genes (dnmt1, dnmt3a) (n = 3). Br: brine, Es: eyestalk, Gi: gill, He: hemocyte, Hp: hepatopancreas, Ht: heart, In: intestine, Ms: muscle, Ov: ovary, St: stomach, Tn, thoracic nerve, Ts: testy, Vn: ventral nerve. Figure C and D were the expression of dnmt3a, and dnmt1; and the Fig. E and F were the tissue distribution of dnmt3a, and dnmt1

Fig. 3

Functional enrichment analysis of DEGs in muscles of the FG and the SG. A, Volcano plot showing DEGs; B, heatmap of DEG hierarchical cluster analysis; C, GO enrichment of DEGs; D, KEGG pathway enrichment of DEGs. E, GO enrichment of upregulated genes between the two groups. F, KEGG enrichment of upregulated genes between the two groups

Fig. 4

Comparative methylome analysis of the muscles of the FG and the SG. A, B, and C represent the Circos plots of the methylation levels of the three types (CG/CHG/CHH), among which the outer circle represents the FG group (designated A), the inner circle represents the SG group (designated B), and the central circle represents the differential methylation levels between the two groups. D, The muscle genome-wide methylation landscape of the three sequence contexts (mCG, mCHG, mCHH); E, density plot of methylation levels in different functional gene elements; F, density plot of methylation levels on different functional gene elements; G, landscape of three cytosine sequence contexts between the FG group and the SG group; H, Venn diagram of the DMR genes; I, distribution of hypomethylated and hypermethylated DMR numbers between the FG and the SG group in different functional gene elements; J, GO analysis of DMR target DMGs; K, KEGG analysis of DMR target DMGs. Note: DNA methylation is found in three different sequence contexts: CG (or CpG), CHG or CHH (where H correspond to A, T or C). The mCG, mCGH, and mCHH means the methylated CG (or CpG), CHG or CHH

Fig. 5

Integrative analysis of WGBS and transcriptomics in the muscles of the FG and SG groups. A, Volcano plot of DEG expression levels and DMR methylation levels. Venn diagram of the DEGs, DMRs and DMPs in the muscles of the two groups. B, Venn diagram of DEG expression and DMR target DMGs in muscles of the FG group and SG group. C: Relationships between the methylation levels and expression levels of methylation-related genes. D: Venn diagram of DEG expression and DMP target DMGs in muscles between the FG group and SG group

Fig. 6

A, GO enrichment of the overlapping DMR-targeted DMGs with CG types; B, KEGG analysis of the overlapping DMR-targeted DMGs with CG types. The items and pathways at a significance of p < 0.05 were highlighted with a asterisk