Effects Of Starvation Stress On Hematological Physiology And Immune Function Of Sinibrama Taeniatus

Sichuan bream(Sinibrama taeniatus)is a rare and endemic small economic fish in the upper reaches of the Yangtze River,with a narrow distribution range and reduced resources.S.taeniatus has a stable 14-day reproductive cycle under breeding conditions,which breaks the breeding cycle of only twice a year in the wild environment.However,at present,research on the basic biology and scientific artificial breeding of S.taeniatus has not yet been carried out.Starvation stress is one of the most common environmental stress factors faced by fish,affecting its growth and development,hematological physiology,tissue morphology,enzyme activity and immune function.Whereas,the complex physiological mechanism between nutrient metabolism and the immune system is still being explored.Therefore,the study used a variety of experimental techniques to clarify the basic composition of the immune system of S.taeniatus and to explore deeply the effect of starvation stress on the immune function of S.taeniatus.The main research results are as follows:Determination of peripheral blood composition types and hematocytes development sites of S.taeniatus.Smears of peripheral blood and imprints including pronephros,body kidney,liver and spleen were stained with Wright-Giemsa staining.After that,the morphological characteristics and distribution of hematocytes in peripheral blood and hematopoietic organs were observed,and the sizes and proportion of different types of hematocytes in the detected four organ prints were statistically analyzed as well.The results showed that the hematocytes of S.taeniatus was mainly comprised of erythrocytes and leukocytes.Leukocytes can be divided into several types including lymphocytes,granulocytes,monocytes,thrombocytes and so on.In the peripheral blood smears,as the main group,lymphocytes accounted for the largest proportion of total leukocytes(65.79± 0.10 %),followed by thrombocytes and neutro-phils,accounting for 16.76 ± 0.07 %and 13.92 ± 0.09 % respectively.The number of monocytes was the least,only 3.33 ±0.01 %.The hematopoiesis of S.taeniatus was mainly divided into four lineages:erythroid,lymphoid,monocytoid and granuloid,and each lineage can be further divided into three stages called primitive,immature and mature ones.The second stages of all three kinds varied depending on types.Immature erythrocytes can just be divided into early and late periods,while that of granulocytes consisted early,middle and late periods.Among these four hematopoietic organs,the head kidney and trunk kidney were the central places of hematocytes development,and were important immune organs and lymphoid organs of S.taeniatus,while the spleen maintained the ability of hematopoiesis of various hematocytes,and the liver can only host the development of lymphocytes.The effects of starvation stress on hematological parameters,antioxidant indexes and immune parameters of S.taeniatus were investigated by means of tissue morphology observation,hematological analysis,physiological and biochemical indexes detection.After 28 days of starvation stress treatment on adult male S.taeniatus,it was found that RBC in peripheral blood increased significantly in a time-dependent manner(P< 0.01),and WBC increased significantly in the early stage of starvation and then decreased.The results of white blood cell differential count showed that starvation stress significantly decreased the proportion of lymphocytes(P< 0.001),on the contrary,the proportion of thrombus cells increased significantly(P< 0.01).The proportion of neutrophils increased on the 7th and 28 th day of starvation(P< 0.01),and the proportion of monocytes increased in the early stage of starvation(P< 0.01),and then decreased gradually with the extension of starvation time.The activity of antioxidant enzyme CAT was consistently significantly higher than that of the control group(P< 0.05),and SOD activity was significantly increased after the 14 th day of starvation(P< 0.05).The levels of innate immunity indexes LZM and C3 increased significantly after short-term starvation(7 days)(P< 0.01),and LZM increased in a time-dependent manner.TNF-α was higher than control group at day14 of starvation(P< 0.05).Nevertheless,plasma Ig M content represented adaptive immunity decreased significantly after starvation(P< 0.01).Transcriptome sequencing was performed on the kidney tissues of S.taeniatus in three different starvation periods(7,14,28 days).GO and KEGG pathway analysis,GSEA analysis,and WGCNA analysis combined with plasma LZM and Ig M phenotypic traits were performed to explore the effects of starvation stress on the immune function of S.taeniatus and its internal mechanism.The results showed that starvation stress could cause transcriptome changes in S.taeniatus.The differentially expressed genes were mainly significantly enriched in regulation of complement activation and lectin pathway,phagosome,peroxisome and other related pathways,which were consistent with phenotypic changes.These genes played a key role in activating the antioxidant system and inducing innate immune responses,including antioxidant-related genes such as glul,gstk1,gpx4,innate immune factor-related genes such as c3,il1r1,ccl19,a2 m and innate immune negative regulators such as nlrc3 and nlrp7.The genes related to mediating adaptive immune response,immunoglobulin synthesis,primordial hematopoietic cell proliferation and development,as well as the gene set of specific immune response physiological processes of tissues or organs,were all down-regulated under starvation stress,suggesting that starvation stress had an inhibitory effect on the adaptive immunity of S.taeniatus.In summary,on the premise of clarifying the composition of its immune system,the effects of starvation stress on the immune function of S.taeniatus and its internal mechanism were further explored based on the analysis of hematological physiological indexes.According to the results of the study,it is speculated that appropriate starvation stress can activate the antioxidant system and induce innate immune response.However,at the same time,starvation stress may inhibit the adaptive immune function of S.taeniatus by inhibiting the expression of key genes in the process of mediating adaptive immune response and hindering the proliferation of lymphocytes and reducing the synthesis and secretion of immunoglobulin.The research provided basic information for the conservation biology,scientific artificial breeding and health assessment of C.abramis,as well as information and scientific reference for the further study of immunometabolism mechanism.