Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis

Abstract

Molting is a critical developmental process for crustaceans, yet the underlying molecular mechanism is unknown. In this study, we used RNA-Seq to investigate transcriptomic profiles of the hepatopancreas and identified differentially expressed genes at four molting stages of Chinese mitten crab (Eriocheir sinensis). A total of 97,398 transcripts were assembled, with 31,900 transcripts annotated. Transcriptomic comparison revealed 1,189 genes differentially expressed amongst different molting stages. We observed a pattern associated with energy metabolism and physiological responses during a molting cycle. In specific, differentially expressed genes enriched in postmolt were linked to energy consumption whereas genes enriched in intermolt were related to carbohydrates, lipids metabolic and biosynthetic processes. In premolt, a preparation stage for upcoming molting and energy consumption, highly expressed genes were enriched in response to steroid hormone stimulus and immune system development. The expression profiles of twelve functional genes detected via RNA-Seq were corroborated through real-time RT-PCR assay. Together, our results, including assembled transcriptomes, annotated functional elements and enriched differentially expressed genes amongst different molting stages, provide novel insights into the functions of the hepatopancreas in energy metabolism and biological processes pertaining to molting in crustaceans.

Figure 1. Energy metabolism and physiological responses in a typical molting cycle in Chinese mitten crab.

(A) Day 2 after molting (postmolt, PoM), soft exoskeleton; (B) Day 10 after molting (intermolt-I, InM-I), hardened exoskeleton and cyanish carapace; (C) Day 20 after molting (intermolt, InM-II), brownish carapace; (D) Day 30 after molting (Premolt, PrM), pleural suture cracks between carapace and abdomen and dark brown carapace. M - molting stage (ecdysis), yellow arrows - molting cycle, green bars - boundaries of different molting stages, black circle - physiological responses, and red circle - energy metabolism pattern in different molting stages.

Figure 2. Assembly, mapping, annotation and comparisons of transcriptomes in different molting stages of Chinese mitten crab.

(A) Total number of de novo assembled transcripts and number of transcripts with FPKM ≥ 1; (B) Total number of reads, number of reads mapped to reference transcriptome and number of reads mapped to stage-specific transcriptome for each molting stage; (C) Number of transcripts annotated using NCBI-NR and UniProt protein database; (D) Venn diagram for the number of annotated genes. PoM: postmolt (Day 2), InM-I: intermolt-I (Day 10), InM-II: intermolt-II (Day 20), and PrM: premolt (Day 30).

Figure 3. Heat map of differentially expressed genes clustered in two ways - molting stages and GO categories.

PoM: postmolt (Day 2), InM-I: intermolt-I (Day 10), InM-II: intermolt-II (Day 20), and PrM: premolt (Day 30). Numbers - replicate samples, C1–C8 - functional clusters of differentially expressed genes, and Color key value - FPKM fold change.

Figure 4. Expression profiles of 12 differentially expressed genes from RNA-Seq (blue) and qRT-PCR (red) with alpha-tubulin as reference gene in different molting stages.