Study On The Amino Acids Flow In The Food Chain Of Engraulis Japonicus Based On Stable Isotopes

In-depth study of food webs has revealed trophic transfer between biological species to be an important basis for evaluating the trophic quality of food webs.Investigating the trophic transfer between species in food webs under various natural and man-made pressures is therefore of important practical significance for realizing the sustainable utilization of ecological resources.Interspecies relationships within food webs in the Yellow Sea and East China Sea are very complex,and thus it is difficult to investigate the trophic transfers within these food webs.In order to overcome the research bottleneck in food web analysis,it is necessary to adopt a simplified approach.In this thesis,a“simplified food web”method was accordingly used to investigate the mainline trophic transfer in a food chain.Firstly,a simulated mainline food chain was constructed by using the key species at each trophic level as the core species.Variations in nutritional amino acids and stable carbon and nitrogen isotope compositions for species components in the simulated mainline food chain were examined in order to determine the mechanism of nutrient flow in the mainline amino acid trophic transfer for key species in the food webs of the Yellow Sea and East China Sea.The main research content and important conclusions of this thesis can be summarized as follows.1.A simulation study combining controlled ecological experiments and indoor experiments was performed on the key species food chain of"Chlorella spp.?Calanus sinicus?Engraulis japonicus."The food chain simulation experiment mainly included the following parts:?a?cultivation of Chlorella spp.,?b?isolation and purification of living C.sinicus,?c?cultivation of C.sinicus with Chlorella spp.,and?d?cultivation of E.japonicus with living C.sinicus.General nutrition component analysis of Chlorella spp.and C.sinicus,indicated that Chlorella spp.had a high carbohydrate content and relatively low crude protein content.In contrast,C.sinicus had relatively high crude protein and high crude fat contents,but a low carbohydrate content.2.Derivatization of 15 amino acid standard samples was conducted using the N-pivaloyl-isopropyl amino acid ester?NPP?and the N-acetyl propyl amino acid ester?NAP?methods.The stable isotope ratios of carbon and nitrogen in the 15 amino acid standard samples before and after derivatization treatment were then determined by elemental analyzer-isotope ratio mass spectrometry and gas chromatograph-combustion-isotope ratio mass spectrometry,respectively.This enabled comparison of the isotope fractionation effect for amino acids,and assessment of the repeatability of measurements.The results showed that the repeatability of carbon and nitrogen stable isotope measurements using the NPP method was close to that for the NAP method,whereas the fractionation effect of the two isotopes in the NPP method was significantly weaker than that in the NAP method.Hence,the NPP method was considered to be a suitable method for the derivatization of amino acids,and was accordingly used for the preparation of samples in this thesis.3.Amino acid composition analysis results for the species in the food chain indicated that C.sinicus plays a key connecting role in the food chain.C.sinicus not only transforms the vegetable protein in Chlorella spp.into animal protein but also significantly enhances the glutamic acid content,thereby increasing the total amino acid content.E.japonicus,which is the prey of dominant species and fish consumers of main commercial importance in the food webs of the Yellow Sea and East China Sea,has lower non-essential amino acid content but higher essential amino acid content than C.sinicus.There was a clear correlation between the relative amino acid compositions of different trophic levels,and the correlation between essential amino acids was relatively strong.When the feeding habit of E.japonicus changed from synthetic baits to C.sinicus,the muscle amino acid content of E.japonicus varied concomitantly with the change in bait,and was associated more closely with C.sinicus than with Nissin bait.The results also reflected the physiological metabolic processes that determine the amino acid composition of E.japonicus feces.4.The amino acid?¹³C analysis results for biological samples in the food chain indicated that bulk?¹³C was enriched with increasing trophic level.Although little or no?¹³C fractionation occurred for essential amino acids between trophic levels,there was a strongly linear relationship for essential amino acid?¹³C values?r² was close to 1,P<0.05?,indicating that the essential amino acids of food chain consumers were derived directly from bait protein.The?¹³C values of non-essential amino acids differed greatly across trophic levels,and the?¹³C value deviated significantly from 0‰.The?¹³C values for non-essential amino acids showed significant weak correlations between the trophic levels,indicating that the non-essential amino acids in consumers were synthesized de novo from food proteins.The fractionation pattern of non-essential amino acid?¹³C might depend on the carbon pool and the amino acid composition of organisms at a lower trophic level,further confirming the complexity of the trophic transfer of non-essential amino acids in the food chain.5.Nitrogen stable isotope ratio analysis of amino acid components in the food chain species revealed that bulk?¹⁵N and amino acid?¹⁵N values were enriched with increasing trophic level.The?¹⁵N values of amino acids differed greatly across trophic levels,and the?¹⁵N values deviated significantly from 0‰.The?¹⁵N values for essential amino acids showed significant correlations between the three trophic levels?P<0.05?,and the essential amino acid?¹⁵N fractionation patterns were similar,which might indicate that the?¹⁵N of essential amino acids has similar fractionation pathways between trophic levels in the food chain.A comparison of the amino acid?¹⁵N and?¹³C values revealed that essential amino acid?¹³C was more stable than essential amino acid?¹⁵N in the food chain.Compared with the?¹⁵N and?¹³C values of the non-essential amino acids,it is more likely that some non-essential amino acids in C.sinicus were synthesized de novo from Chlorella spp.proteins,whereas E.japonicus showed a clear tendency to directly incorporate non-essential amino acids from C.sinicus.This might indicate a substantial difference in terms of the underlying mechanisms of amino acid metabolism in the species at different trophic levels.