Understanding The Diet Composition Of Mesopelagic Fishes And Cold Seep Mussels In The South China Sea By Biomarker Methods

Mesopelagic fishes are widely distributed in open ocean except the arctic,and they are the most abundant fishes in the ocean with the largest biomass.Most mesopelagic fishes undergo diel vertical migration（DVM）,accelerating the burial of surface particulate organic carbon to the middle and deep ocean.This phenomenon forms a unique active biological pump mechanism and provides an adequate food sources for deep-sea ecosystems.However,it is reported that mesopelagic fishes varied in their migration behaviors and feeding habits,and our understanding of mesopelagic fishes’s food web is still limited.The cold seep is a window to study the deep ocean biosphere and has an important influence on global climate change.A high productivity ecosystem based on chemical energy is formed in dark cold seep.Mussels with chemical symbiosis are the representative of organisms in cold seeps.However,few studies have been conducted on the carbon,nitrogen cycle and energy flow of mussels with chemical symbiosis.Here,we combined the multiple tools(e.g.δ¹³C,δ¹⁵N,fatty acids,δ¹³C of fatty acids andδ¹⁵N of amino acids)to study the mesopelagic fishes and symbiotic mussels in the South China Sea（SCS）.The study revealed the complex food web of mesopelagic fishes in the SCS and gave a preliminary analysis on contribution of different nitrogen sources to the symbiotic mussels and the matter（fatty acids and amino acids）synthesis process in the symbiotic mussels.In the traditional method ofδ¹⁵N of individual amino acids,a large amount of toxic organic reagents were required for derivatization,and an unique GC-C-IRMS system is needed with concentrated samples.In order to avoid the use of highly toxic organic reagents and reduce the amount of samples,we used IC（ion chromatography）to separate and collect amino acids,and then converted amino acids to N₂O by"Strecker"method for the measure ofδ¹⁵N isotopes.In the process of converting NO₂^-to N₂O,the ratio of hydroxylamine to nitrite needed to be adjusted to 15:1 to complete conversion of nitrite.The lipid content of DVM mesopelagic fishes were higher than that of other types of fishes.Thus,the variation ofδ¹³C caused by high lipid content should be considered to explore the food sources of mesopelagic fishes.Mathematical correction formula was applied in lipid correction of fishes.In this study,there was an obvious linear correlation between C/N_mass andΔδ¹³C(the differentδ¹³C before and after lipid extraction).However,we observed that variations in geopraphical location and growth stage might result in the change of the linear calibration curve,and theδ¹³C of high lipid organs had existed obvious nonlinear correlation with dorsal muscle.The mesopelagic fishes in the SCS had various migration behaviors and feeding habits.The high lipid contents of migrant planktivorous fishes limited the application ofδ¹³C_bulk(bulk-tissueδ¹³C)as diet indicator,insteadδ¹³C_extraction(the lipid extractedδ¹³C)values were applied successfully to reflect dietary sources.The low temperature in the middle water impacted the metabolic rate of the mesopelagic fishes,which affected the enrichment process ofδ¹⁵N.The ratio of DHA（22:6n-3）to EPA（20:5n-3）mirrored the potential carnivory in the mesopelagic fishes,which replaced the ofδ¹⁵N.Theδ¹³C of ARA（20:4n-6）indicated that deep zooplankton was an important diet source of non-migrant planktivorous fishes.The combination ofδ¹³C of ARA andδ¹⁵N of glutamic acid revealed more details of food web in non-migrant piscivorous fishes,and it suggested that the enrichment of trophic amino acids in the mesopelagic fishes was 4‰.In the midwestern SCS,theδ¹⁵N of mesopelagic fishes in the upwelling area were2‰higher than that in the non-upwelling area,which was caused by theδ¹⁵N of plankton.The input of Mekong river was the main source of nutrients in the upwelling area and resulted in theδ¹⁵N of plankton in the upwelling area was 2‰higher than that in the non-upwelling area.Meanwhile,the fatty acid composition of the mesopelagic fishes in the upwelling area and the non-upwelling area also has obvious differences caused by the influence of river input.By the principal component analysis of fatty acid andδ¹³C of fatty acids of mesopelagic fishes with known living habits,the mesopelagic fishes with unknown habits could be preliminarily judged.The energy of various mussels in cold seep were supplied by different symbiotic bacteria.Theδ¹³C of mussels with methanotrophic bacteria（Bathymodiolus platifrons）was very negative,indicating that methane is their main energy source.Theδ¹³C of mussels with sulfur oxidizing bacteria（Bathymodiolus aduloides）indicated that CO₂was their main carbon source.Based on the two-end member mixed model ofδ¹⁵N in mussels and environment（NO₂^-and organic matter in the sediment）,the inorganic nitrogen was the main nitrogen source of the B.platifrons,while organic nitrogen was the main source of the B.aduloides,which might link to their different living environments of the two mussels.Based on theδ¹³C of fatty acids andδ¹⁵N of amino acids,we revealed the different synthetic pathways of specific fatty acids and amino acids（e.g.Glu,Phe,Leu,Asp,etc）.By the study of mesopelagic fishes and symbiotic mussels in the SCS,we found that the traditional methods of carbon and nitrogen isotopes had the limitation to directly be applied to analyze the food web of mesopelagic fishes.δ¹³C_extraction can better reflect the diet sources of mesopelagic fishes,the ratio of DHA to EPA replace theδ¹⁵N to morror the trophic level.The combine ofδ¹³C of 22:4n-6 andδ¹⁵N of glutamic acid will reveal more detailed food sources of mesopelagic fishes.Due to the difference in living environment,the symbiotic mussels have contrasting contributions of inorganic and organic nitrogen.The different sources of carbon and nitrogen also lead the different synthesis pathways of fatty acids and amino acids in symbiotic mussels.