Comparative transcriptomic analysis primarily explores the molecular mechanism of compound eye formation in Neocaridina denticulata sinensis

Abstract

Compound eyes formation in decapod crustaceans occurs after the nauplius stage. However, the key genes and regulatory mechanisms of compound eye development during crustacean embryonic development have not yet been clarified. In this study, RNA-seq was used to investigate the gene expression profiles of Neocaridina denticulata sinensis from nauplius to zoea stage. Based on RNA-seq data analysis, the phototransduction and insect hormone biosynthesis pathways were enriched, and molting-related neuropeptides were highly expressed. There was strong cell proliferation in the embryo prior to compound eye development. The formation of the visual system and the hormonal regulation of hatching were the dominant biological events during compound eye development. The functional analysis of DEGs across all four developmental stages showed that cuticle formation, muscle growth and the establishment of immune system occurred from nauplius to zoea stage. Key genes related to eye development were discovered, including those involved in the determination and differentiation of the eye field, eye-color formation, and visual signal transduction. In conclusion, the results increase the understanding of the molecular mechanism of eye formation in crustacean embryonic stage.

Keywords: Neocaridina denticulata sinensis; Comparative transcriptomic analysis; Compound eye; Differentially expressed genes; Embryonic development.

Fig. 1

Late stage of embryonic development of N. denticulata sinensis. (a)-(d): Nauplius stage, Compound eye pigment formation I, Compound eye pigment formation II, Zoea stage

Fig. 2

Summary of gene annotation. (a) The COG annotation of assembled genes. The x-axis indicates content of each category of COG and the y-axis indicates number of genes annotated in each category. (b) The GO annotation of assembled genes. the x-axis indicates number of genes annotated in each term and the y-axis indicates names of each term. (c) The KEGG classification of assembled genes

Fig. 3

Venn diagram of expressed genes across four groups

Fig. 4

Summary of differential gene analysis. The average FPKM values were used to plot heatmaps. (a) Statistics of differentially expressed genes (DEGs) between adjacent groups. Blue columns represent the total number of differentially expressed genes, red columns represent up-regulated genes and green columns represent down-regulated genes. (b) Overlap of DEGs between comparison groups. (c) The clustering analysis of DEGs. The figure consists of a boxplot, heatmap and KEGG annotation information. The boxplot and heatmap shows the gene expression in each cluster, while the pathways are shown on the right side of the heatmap. For the heatmap, red indicates high relative expression level and blue indicates low relative expression level. (d) Heatmap of genes related to molting. CHH, crustacean hyperglycemic hormones; MIH, molt-inhibiting hormone; CCAP, crustacean cardioactive peptide; JHEH, juvenile hormone epoxide hydrolase; JHAMT, juvenile hormone acid O-methyltransferase

Fig. 5

Enrichment analysis of DEGs. (a) GO enrichment analysis from N to CE1. X-axis indicates -log₁₀ p-value, and the Y-axis indicates the enriched GO terms. Three different colors of the columns represent three basic classifications of GO term. Each category displays five GO terms. (b) GO enrichment analysis from CE1 to CE2 stage. (c) GO enrichment analysis from CE2 to Z stage. (d) KEGG enrichment of DEGs. X-axis indicates the comparison groups and Y-axis indicates the pathway names. The different colors of the dots represent the p-value, and the number of DEGs in each pathway is represented by the size of the dots

Fig. 6

The summary of the phototransduction - fly signaling pathway. The average FPKM values were used to plot heatmaps. (a)The KEEG pathway of the phototransduction - fly signaling pathway. The green background indicates the unigenes annotated on the pathway. (b) The expression patterns of DEGs enriched in phototransduction - fly signaling pathway

Fig. 7

Clustering of DEGs identified in all three comparative groups. The average FPKM values were used to plot heatmaps. (a) Heat map of DEGs related to cuticle formation. Purple label indicates the genes related to chitin metabolism pathway, pink indicates putative cuticle gene, and green indicates the bursicon gene (b) Heatmap of DEGs related to muscle growth. (c) Heatmap of DEGs related to immune system. (d) Heatmap of crustacyanin genes. (e-f) Heatmap of stage-specific genes associated with chitin binding and structural constituent of cuticle

Fig. 8

Analysis of key genes related to eye development. (a) Phylogenetic tree of visual opsins. The opsins of N. denticulata sinensis is marked in the red typeface. All accession numbers of opsin sequences used in the phylogenetic tree are shown in Table S1. (b) Heatmap of putative visual opsins of N. denticulata sinensis. The average FPKM values were used to plot the heatmap. (c) Putative genes related to eye determination and eye color of N. denticulata sinensis