**4. Conclusions**

In conclusion, glucose addition affected the biochemical composition of *N. laevis*. The fucoxanthin and chlorophyll contents were significantly decreased, while EPA was increased. Transcriptomic and metabolomic analysis suggested that the impact of glucoseinduced trophic transition include upregulation of enzymes associated with the pyruvate metabolism, amino acid biosynthesis, and fatty acid elongation. ELOVL and KCS are likely the main contributors to EPA accumulation, and the downregulation of PDS and ZISO is likely to be responsible for the decrease of fucoxanthin content. Moreover, hub genes were identified from two glucose regulatory gene module, GPRM and GNRM respectively, and their functions were associated with cell cycle, carbohydrate metabolism, purine biosynthesis, and lipid metabolism.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md19080426/s1, Figure S1: Transcriptome analysis overview during the transition from autotrophy to mixotrophy. (A) Principal component analysis (PCA) (B) The box plot of the gene expression level (C) Sequence similarity annotation in NR (Non-Redundant Protein Sequence) database. Figure S2: Hierarchical cluster and KEGG classification of most up-regulated and down-regulated differential expressed genes (DEGs) at 3 h. (A) hierarchical cluster analysis of the top 100 most up-regulated DEGs (B) Hierarchical cluster analysis of the top 100 most down-regulated DEGs (C) KEGG classification of the top 100 most up-regulated DEGs (D) KEGG classification of the top 100 most down-regulated. Figure S3: Metabolome analysis overview during the transition from autotrophy to mixotrophy. (A) The volcano plots of metabolites under positive ion mode (ESI+) (B) The volcano plots of metabolites under negative ion mode (ESI-) (C) KEGG classification of all metabolites. Figure S4: The hierarchical cluster analysis of glucose-associated positive regulatory module (GPRM) (A) and glucose-associated negative regulatory module (GNRM) (B). Table S1: The gene expression levels of all unigenes. Table S2: The Pearson correlation coefficients for all gene expression levels. Table S3: KEGG pathway classification of most up-regulated and down-regulated DEGs. Table S4: All peaks of Metabolomics identified and quantified. Table S5 Genes related in carotenoids biosynthesis and fatty acids metabolism.

**Author Contributions:** Conceptualization, X.M., B.L. and F.C.; methodology, X.M. and B.L.; formal analysis, X.M., M.G. and X.W.; investigation, X.M., M.G. and X.W.; data curation, X.M., X.W., X.L., J.Y. and B.L.; writing—original draft preparation, X.M.; writing—review and editing, X.L., J.Y., B.L. and F.C.; visualization, X.M.; supervision, B.L. and F.C.; funding acquisition, B.L. and F.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China, gran<sup>t</sup> number 31901624; Natural Science Foundation of Shandong Province, gran<sup>t</sup> number ZR2019BC068; the Key-Area Research and Development Program of Guangdong Province, gran<sup>t</sup> number 2018B020206001; Guangdong Province Zhujiang Talent Program, gran<sup>t</sup> number 2019ZT08H476; Shenzhen Science and Technology Program, gran<sup>t</sup> number KQTD20180412181334790; and the Innovation Team Project of Universities in Guangdong Province, gran<sup>t</sup> number 2020KCXTD023.

**Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
