Stage-Specific Transcriptomes of the Mussel Mytilus coruscus Reveals the Developmental Program for the Planktonic to Benthic Transition

Abstract

Many marine invertebrate larvae undergo complex morphological and physiological changes during the planktonic-benthic transition (a.k.a. metamorphosis). In this study, transcriptome analysis of different developmental stages was used to uncover the molecular mechanisms underpinning larval settlement and metamorphosis of the mussel, Mytilus coruscus. Analysis of highly upregulated differentially expressed genes (DEGs) at the pediveliger stage revealed enrichment of immune-related genes. The results may indicate that larvae co-opt molecules of the immune system to sense and respond to external chemical cues and neuroendocrine signaling pathways forecast and trigger the response. The upregulation of adhesive protein genes linked to byssal thread secretion indicates the anchoring capacity required for larval settlement arises prior to metamorphosis. The results of gene expression support a role for the immune and neuroendocrine systems in mussel metamorphosis and provide the basis for future studies to disentangle gene networks and the biology of this important lifecycle transformation.

Keywords: Mytilus coruscus; hard-shelled mussel; larval settlement and metamorphosis; pediveliger larvae; transcriptome.

Figure 1

Gene ontology (GO) term distribution of transcripts. The GO annotation results were based on 20,199 transcripts detected at least in one of the five developmental stages. Gene ontology categories included molecular function, cellular component, and biological process. GO categories for each function were sorted by the increasing number of genes enriched in the process.

Figure 2

The life cycle of the mussel M. coruscus (A) and heat map for the Pearson correlation coefficient for all 20 samples (B). The heat map plots the correlation coefficient score between any two samples. The color of a square represents each coefficient in the correlation matrix. The color scale used is blue to red, with blue representing 0 and red representing 1. The five clusters defined by hierarchical clustering are indicated at the top.

Figure 3

Gene ontology (GO) term enrichment analysis. The GO annotation results were based on the 684 increased DEGs (screened with 2-fold change in the pediveliger stage in comparison with other developmental stages) (A) or the 121 DEGs decreased (B). Gene ontology categories included molecular function, cellular component, and biological process. GO categories for each function were sorted by increasing order of gene numbers, based on the GO enrichment test; p-value < 0.05.

Figure 4

KEGG pathway enrichment scatter plot of the DEGs. The vertical axis represents the path name, and the horizontal axis represents the path factor corresponding to the rich factor. The color of the point represents the size of the p−value. The smaller the p−value, the closer the color is to red. The number of differential genes included in each pathway is presented by the point’s size.

Figure 5

Comparison of gene expression among five developmental stages by RNA-seq and qPCR analysis. Boxplots represent the copy numbers per larvae performed by qPCR. Blue line represents the expression levels of the TPM counts assessed by RNA-seq. Analysis genes were related to neuroendocrine (A), growth and apoptosis (B), immune (C), and others (D). NpffR2: Neuropeptide FF receptor 2; Chrna9: neuronal acetylcholine receptor subunit alpha-9 isoform X1; Chrna10: neuronal acetylcholine receptor subunit alpha-10-like; EsR: estrogen receptor; Notch1: neurogenic locus notch homolog protein 1-like isoform X2; Notch: putative neurogenic locus Notch protein-like; CASP3: caspase-3-like 1 protein, partial; CASP3/7: caspase 3/7-4; TLRW: toll-like receptor W; TLRC: toll-like receptor c; TLR4: toll-like receptor 4 isoform X2; Alox5: arachidonate 5-lipoxygenase isoform X1; Btyp2: byssal tyrosinase-like protein 2. Different letters indicate statistically significantly different copy numbers of the qPCR analysis (p < 0.05).

Figure 6

A schematic depiction of a larval settlement and metamorphosis model based on the genes identified by RNA-seq.