3.6 Identification of cold-associated genes
Compared with the control group (28 °C), the eyestalk tissue exhibited 720 (LT vs. CT, 343 upregulated and 377 downregulated) and 2,347 (LT vs. CT, 1,275 upregulated and 1,072 downregulated) DEGs at 12 h and 48 h under 10 °C exposure, respectively (|Log2 fold-change| >1 and p -value <0.05; Table S8). According to GO analysis, the DEGs from 12 h LT vs. CT were enriched mainly in chitin metabolic process (GO:0006030), extracellular region (GO:0005576), and chitin binding (GO:0008061) in the biological process, cellular component, molecular function GO categories, respectively. We observed the enrichment of certain GO terms involved in the neuroendocrine system, such as endopeptidase activity (GO:0004175), endopeptidase inhibitor activity (GO:0004866), and endopeptidase regulator activity (GO:0061135) (Table S9, Figure S5A). In the DEGs from 48 h LT vs . CT group, GO term analysis showed that enrichment mainly in organo-nitrogen compound catabolic process (GO:1901565), proteasome core complex (GO:0005839), and threonine-type endopeptidase activity (GO:0004298), in the biological process, cellular component, and molecular function GO categories respectively. We observed the enrichment of certain GO terms involved in the neuroendocrine system, such as cellular catabolic process (GO:0044248), heterocycle catabolic process (GO:0046700), threonine-type endopeptidase activity (GO:0004298), threonine-type peptidase activity (GO:0070003), and endopeptidase activity (GO:0004175) (Table S9, Figure S5B).
We identified the top 20 significantly enriched KEGG pathways in the eyestalk. In the 12 h LT vs . CT group comparison, spliceosome (dme03040), proteasome (dme03050), and lysosome (dme04142) were the main clusters. We also observed enrichment of certain KEGG pathways associated with the neuroendocrine system, such as longevity regulating pathway‑multiple species (dme04213), phosphatidylinositol signaling system (dme04070), and endocytosis (dme04144) (Table S10, Figure S6A). As for the 48 h LT vs. CT group, proteasome (dme03050), drug metabolism-other enzymes (dme00983), and drug metabolism-cytochrome P450 (dme00982) were the main clusters. We also observed enrichment of certain KEGG pathways associated with the neuroendocrine system, such as ubiquitin mediated proteolysis (dme04120), phosphatidylinositol signaling system (dme04070), neuroactive ligand-receptor interaction (dme04080), and SNARE interactions in vesicular transport (dme04130) (Table S10, Figure S6B).
We observed that under low temperature stress, many DEGs were upregulated significantly, most of which were associated closely with the neuroendocrine system, amino acid transport and metabolism, carbohydrate transport, lipid transport and metabolism, HSPs, and hemocyanin. Thus, these DEGs might participate in the M. japonicus cold tolerance mechanism (Fig. 8).