Mechanism On Food Intake Regulation Of Coilia Nasus In The Yangtze River Based On Digestive Enzyme Activity And Multi-omics Techniques

Coilia nasus is the most important fishing and consumption species in the Yangtze River,which usually feeds and overwinters in coastal areas.Every spring,sex matured C.nasus migrate from the sea to the Yangtze River and up to its adjacent lakes to spawn.Previous studies suggested that C.nasus ceases feeding during spawning migration.However,our laboratory investigations showed that a few C.nasus individuals had obvious freshwater shrimp or small fish in their stomachs during upstream migration in the Yangtze River,which conflicted with previous studies.The present study systematically investigated the biological parameters and,spatial and temporal characteristics of C.nasus during spawning migration.This study further elaborated the digestive enzyme activity and studied the variation of feeding-related genes and proteins of C.nasus by using transcriptome and proteomic techniques,in the meantime,to reveal the signaling pathways and key genes involved in feeding regulation.The present research provides insights into the molecular mechanisms underlying the response of C.nasus to food intake and reference for future research on the migratory ecology and physiology of C.nasus.1.Biological parameters and temporal and spatial characteristics of reproductive population in C.nasusIn April and July 2018,1112 samples of C.nasus were collected from the Yangtze River estuary to the Anqing section.The results showed that the average total length of C.nasus population was 27.88±2.81 cm,the average body length was 25.69±2.63 cm,and the average body weight was 63.33±22.12 g.C.nasus individuals tend to miniaturization and fatness decreased significantly compared with the research results of the last century.From the perspective of spatial characteristics,the influence of migration distance on various parameters of female and male C.nasus showed that with the increase of migration distance,body weight and fatness decreased significantly(P<0.05).The reason behind the decline is that migration and gonadal development constantly consumed all kinds of energy reserves such as lipid,causing fish to become thinner and lose weight.It was also found that females are larger than males,which is related to the fact that females request to consume more energy during migration.In terms of time,there is no significant correlation between the total length,body length,body weight,and fatness and migration time,indicating that there is no significant difference in biological parameters of C.nasus populations in different migration time of the same river section.In terms of gonad maturity,the gonads of females and males gradually developed from??? to ???.In addition,intaked food C.nasus during migration,it may be because of the consumption of a large amount of energy in the body,which needs to be supplemented by food intake.2.Effects of migration distance and water temperature on digestive enzyme activity of C.nasus during spawning migrationIn this study,we investigated the activity levels of two major digestive enzymes(pepsin and lipase)in C.nasus during its upstream migration to analyse the digestive physiological responses to starvation and to analyse the influence of the water temperature on enzyme activity.Water temperature had a significant increase in pepsin activity,while long-distance migration resulted in a significant decrease in pepsin activity.In the early stage of migration,pepsin activity remained at a high level.With the increase of distance,pepsin activity in the Zhenjiang region significantly decreased by 12%,and then reached 17% in the Wuhu region,indicating a decrease in the synthesis of digestive enzymes,possibly owing to the lack of food stimulation.As starvation continued,however,a slight increase in pepsin activity between the Wuhu(440 km)and Anqing(620 km)regions may indicate that C.nasus had refeeding behaviour due to its large expenditure of energy reserves.In contrast,lipase activity was not significantly affected by the water temperature,but the effect of migration increased as much as 13% of lipase activity from the Chongming region(20 km)to Anqing region(620 km).After the short distance migration,the lipase activity of C.nasus was reduced by 3.5% compared with that at 20 km,indicating that C.nasus fed less,and the lipase secretion was reduced by hunger stress.Continuing the migration to 290 km,lipase activity increased by 5%,and significantly increased to 19% at 440 km,suggesting that the stored lipids of C.nasus were mobilized to meet energy requirements of upstream migration activity,and gonad development.3.Transcriptome analysis of C.nasusIn order to understand the molecular basis underlying the response to food intake,the stomach transcriptome of C.nasus with feeding group(CSI)and control group(CSN)was analyzed via RNA-Seq.A total of 610 640 864 clean reads were obtained,and annotation to the six databases identified 63 567 unigenes.A total of 149 089 GO functional definitions were obtained through functional prediction,of which 40.1% were involved in biological processes,27.1% involved in cell components,and 32.8% involved molecular functions.Comparative analysis of the gene expression between feeding and control fish identified 1968 differentially expressed genes(P<0.05),including upregulated 1180 genes and down-regulated 788 genes.Our results showed that stomach distention activated the vagal afferent neurons,resulting in satiation.Several of the most relevant appetite genes(e.g.,Leptin receptor(Lep R),Somatostatin(SS),Nucleobindin-2(NUCB2),5-hydroxytryptamine receptor(5-HTR),Growth hormone receptor(GHR))were also involved in the regulation of food intake and led to the termination of feeding.Furthermore,significant differences were observed in the genes encoding key enzymes involved in metabolic pathways such as protein digestion and absorption,TCA cycle pathways,fatty acid synthesis and glycolysis,indicating C.nasus can mobilize energy from carbohydrate and lipid of food for life activities and spawning migration.Real time q PCR confirmed the differential expression in selected genes.4.Proteome analysis of C.nasusWe utilized high-throughput proteomics to display differentially expressed proteins in the stomach tissues of C.nasus with feeding group(CSI)and control group(CSN).In this study,a total of 3279 proteins were identified in three two-dimensional LC-MS/MS analyses.Then we chose the expression differences of 279 proteins(fold change ratio?1.2 and P<0.05),among which 133 were up-regulated and 146 were down-regulated.The enrichment analysis of KEGG pathway showed that a total of 260 pathways were enriched,including PI3K-Akt signaling pathway,protein digestion and absorption,gastric acid secretion,JAK-STAT signaling pathway,carbohydrate digestion and absorption.Among them,SS,SLC26A7,Ca M,PKA and EGF are involved in the secretion of gastric acid;Pepsin,CELA,and CPA are involved in protein digestion and absorption;MHCII,Ig M,Ig D,CD13,and NF-?B are involved in immune response.The results of this study laid a theoretical foundation for further exploration of feeding response and regulation mechanism of C.nasus,and provided the reference for studies on feeding molecular mechanisms of other migratory fish.