Comparative metabolomic analysis reveals sex differences and heat stress responses in germ cells of Sipunculus nudus

Abstract

The sipunculid worm Sipunculus nudus is a dioecious marine invertebrate with ecological and aquaculture importance. To investigate the metabolic features underlying gamete specialization and thermal response, we performed untargeted metabolomic analysis of spermatozeugmata and oocytes under control conditions, as well as oocytes exposed to acute heat stress (37 °C for 24 h; n = 6 males, 6 females, and 4 heat-stressed females) using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Combined multivariate and univariate statistical analyses, applied with fold-change and significance thresholds, revealed 894 putatively annotated metabolites that differed between oocytes and spermatozeugmata, and 1749 that changed significantly under heat stress. Oocytes were enriched in lipid species (e.g., phospholipids such as PI and LPC) and nucleotide derivatives (e.g., putatively annotated uridine monophosphate, deoxynucleosides), whereas spermatozeugmata showed higher levels of lipids (e.g., PE and PC species), amino acid derivatives (e.g., putatively annotated N-acetyl-DL-serine), and small peptides. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that metabolites mapped to neurotransmitter and energy metabolism pathways were more prominent in oocytes, while metabolites associated with antioxidant and nucleotide biosynthesis pathways were comparatively enriched in spermatozeugmata. Under heat stress, oocytes exhibited significant metabolic changes, with increased organic acids and lipid metabolites (fatty acids and glycerophospholipids) and decreased nucleotide- and amino acid-related compounds. Most KEGG-mapped metabolic pathways showed an overall reduction in associated metabolites, including amino sugar, nucleotide, and amino acid metabolism, whereas metabolites mapped to Fc epsilon RI signaling and bile secretion KEGG pathways were relatively enriched. This study provides the first comprehensive metabolomic profile of S. nudus germ cells, revealing sex-specific metabolic patterns and heat-induced reprogramming of oocyte metabolism. These findings enhance our understanding of gamete physiology and acute thermal stress responses in marine invertebrates and may contribute to the development of reproductive health biomarkers.

Keywords: Germ cells; Heat stress; Marine invertebrates; Metabolomics; Sexual dimorphism; Sipunculus nudus.