Metabolomic Profiles of Limulus Polyphemus Eggs

Limulus polyphemus, the Atlantic horseshoe crab (HSC) is a keystone estuarian species used in the production of pharmaceutical endotoxin assay reagents, making it an important species both in human economic activities and maintaining coastal biodiversity. To understand the natural product and chemical composition of the eggs of this animal, which play a critical ecological role, we examined the metabolite profile of HSC egg extracts thoroughly using NMR and multiple mass spectrometric techniques. In the NMR analysis, maltose-type oligosaccharides, homarine (1), and glycine betaine (2) emerged as a source of the glycogen biosynthetic pathway. Nine amino acids were also identified, and five of them, threonine (4), leucine (7), valine (9), lysine (10), and arginine (11), were essential amino acids used by marine life. By using GC-MS to analyze fatty acid contents, palmitic acid and stearic acid (15) were identified as the major saturated fatty acids in the extracts, and palmitic acid was higher in surface eggs while stearic acid was higher in buried egg clutches. The unsaturated fatty acid profile was similar in the two types of eggs, including elaidic acid (13), oleic acid (14), and EPA. An LC-MS/MS analysis revealed that PC was found to be the primary membrane component of HSC eggs. The elemental composition of HSC egg powder was evaluated using ICP-MS and EA-IRMS. From ICP-MS, the metals Cd, Co, Fe, Mn, Ni, and Pb were significantly higher in surface eggs, and the EA-IRMS analysis showed the carbon and nitrogen contents consistent with a detritivore lifestyle. Through the combination of NMR and MS techniques, we acquired a comprehensive metabolite profile of HSC eggs, which is important for advancing the developmental and conservation biology of HSC and understanding the ecological role of this keystone species.