The sea cucumber Holothuria leucospilota, a nutritive and commercial marine species, has a high protein and low lipid content. To date, the mechanisms underlying gender determination and differentiation in sea cucumbers remain unclear. Identifying gender-specific molecular markers is an effective method of revealing the genetic basis of gender determination and differentiation. The inability to distinguish between male and female individuals causes reproductive efficiency to decline in aquaculture. In this study, we used the gonads of the sea cucumber H. leucospilota as samples to conduct the experiment. The differentially abundant metabolites (DAMs) detected by liquid chromatography-mass spectrometry were enriched in pathways associated with prolactin metabolism, insulin metabolism, hypoxia-inducible factor-1 signaling, and calcium signaling. At the transcriptome level, Illumina sequencing was performed on H. leucospilota, demonstrating that gender-specific expression genes were enriched in the retinoic acid-inducible gene I-like receptor signaling pathway, C-type lectin receptor signaling pathway, alpha-linolenic acid metabolism, and ether lipid metabolism by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. By analyzing the common pathways between DAMs and differentially expressed genes, we found that gender-related genes of H. leucospilota were mostly enriched in the necroptosis pathway and the cysteine and methionine metabolism pathways. According to the common pathways, uch-sc1 and uch-sc2 are male-specific expression genes, and uch-sc3 and bhmt are female-specific expression genes at the mRNA level. These results provide information on gender differences in H. leucospilota.

The oyster Crassostrea hongkongensis is eaten raw and whole, so the endogenous peptides carried by its tissue reach the intestinal lumen unprocessed; functional work on food-derived peptides has concentrated on cationic sequences. This study asked whether the endogenous peptide population of this species is anionically skewed and whether a preparation of that population alters the cytoskeletal gene programme of human intestinal epithelial cells. The published plasma peptidome of this species (PXD025247) was re-analysed, an endogenous peptide fraction below 10 kDa was prepared from oyster soft tissues without adding any protease, and the preparation was applied to differentiated Caco-2 monolayers at 0.1–2.0 mg/mL for 6–24 h; nine transcripts were measured by quantitative PCR against vehicle, protease-digested and digestion-only controls. Of 333 peptides, 157 (47.1%) carried a net charge of −1 or lower at pH 7, 148 (44.4%) were near-neutral and 28 (8.4%) carried +2 or above, and anionic peptides outnumbered cationic peptides in all four detection strata (36.4–58.3% against 4.8–9.1%). Actin-family proteins were the largest precursor source (186 peptides, 55.9%; 68.3% coverage of actin). The preparation was recovered at 2.41 ± 0.23 mg/g of wet tissues and, at 2.0 mg/mL, increased transcription of the SRF targets CCN1 (2.91-fold), VIL1 (2.29-fold), MYH9 (2.17-fold) and ACTN1 (1.83-fold) (p < 0.01 for each) and of TMSB4X, CFL1 and PFN1 (1.90-, 1.75- and 1.55-fold; q < 0.05), whereas SRF and MRTFA transcripts were unchanged; proteinase K digestion abolished the response. An endogenous peptide preparation from oyster soft tissues therefore acts on the cytoskeletal gene programme of human intestinal epithelial cells, and the anionically skewed majority of this peptidome is the fraction that warrants direct functional characterisation.

Background/Objectives: Long non-coding RNA (lncRNA) was structurally similar to mRNAs, yet they could not be translated into proteins. While an increasing number of reports have systematically identified and described lncRNA in model species, information about non-model species remains scarce. Sea cucumber Holothuria leucospilota could be used for both medicinal and food purposes, which have high economic value, gradually attracting the attention of researchers. Methods: In this research, we constructed lncRNA library and compared the difference in lncRNA expression profiles between testis and ovary of sea cucumber H. leucospilota. To elucidate the molecular interactions between lncRNA and mRNA, we computationally predicted potential complementary binding sites through analysis of both cis- and trans-acting antisense mechanisms. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that the identified target genes are potentially involved in the regulatory pathways governing gonad development. Results: Quantitative reverse transcription PCR analysis showed that MSTRG.32831.1-sox9 and MSTRG.57315.1-mthfr exhibited a high expression pattern in testis; while MSTRG.11041.1-mafa and MSTRG.11074.1-macf1 showed a high expression pattern in the ovary. Conclusions: Deciphering lncRNA–mRNA expression patterns may uncover fundamental principles governing reproductive regulation in marine invertebrates. This discovery not only deepens understanding in this field but also provides valuable comparative insights for developmental biology.

MicroRNAs are defined as a class of short noncoding RNAs, which play important function in cell differentiation, developmental process and gender determination. The sea cucumber Holothuria leucospilota is a member of the Echinodermata, known for its excellent nutritional and economic value. Breeding efficiency of sea cucumbers is low due to the difficulty in distinguishing between male and female individuals. Discovering molecular markers that distinguish between genders will provide a robust strategy for elucidating the genetic mechanisms governing gender determination and differentiation. In this investigation, high-throughput sequencing was utilized to compare miRNA expression patterns in the gonadal tissues of male and female specimens of H. leucospilota. A total of 584 known miRNAs and 404 novel miRNAs were identified, of which 97 miRNAs were identified as significantly differentially expressed miRNAs (DEMs) between male and female sea cucumbers. These findings enabled the construction of a miRNA-mRNA regulatory network. Pathway analysis of target genes highlighted the potential roles of neuroactive ligand-receptor interactions, calcium signaling, Salmonella infection, endocytosis, and MAPK signaling in governing gender differentiation processes. The expression results of high-throughput sequencing were validated in sc-miR121, sc-miR41, sc-miR67, sc-miR90, sc-miR104 and sc-miR189 by quantitative real-time quantity polymerase chain reaction (RT-qPCR). In summary, our results will provide a foundation for future studies of miRNA regulation in gender differentiation of sea cucumber H. leucospilota.