Study On The Physiological,biochemical And Morphological Characteristics Of The Peripheral Blood Cells And Immune-related Genes Expressions In Sebastes Schlegelii

Sebastes schlegelii mainly live in the near marine reef zone of the northwest Pacific Ocean.It is a vital mariculture fish in China.However,the farming industry of S.schlegelii has suffered from seawater pollution and frequent diseases infections with production decline.In this thesis,S.schlegelii was used as the experimental object to study the morphological and cytochemical characterizations of the peripheral blood cells,the effect of the water-soluble fraction of diesel oil（WSD）on the serum biochemical indicators,histological changes,and immune responses,the effect of Vibrio parahemolyticus infection on the hematological varieties,immune responses and spleen transcriptome.This study was expected to provide basic data for health assessments and disease diagnosis of S.schlegelii,and establish the foundation for the immune-related researches of S.schlegelii.The specific research contents and results were as follows:1.Morphological and cytochemical characterization of peripheral blood cells in S.schlegelii.The morphology,morphometry and cytochemistry of peripheral blood cells in S.schlegelii were investigated by the routine Wright-Giemsa staining,transmission electron microscopy（TEM）and various of cytochemical staining（periodic acid Schiff PAS,Sudan black B SBB,acid phosphatase ACP,alkaline phosphatase ALP,peroxidase POX and naphthol acetate NAE）methods.Erythrocytes,monocytes,lymphocytes,neutrophils and thrombocytes were observed in the peripheral blood.Erythrocytes were the most abundant blood cells,lymphocytes were the most abundant leucocytes,followed by neutrophils and monocytes.The size of the monocytes（9.89±0.70）μm×（8.72±0.68）μm were the largest.The mature erythrocytes were ovally shaped with smooth edges,and the nucleus was round or oval locating in the centre of the cell.The monocytes were round,with uneven edges and the large nucleus was located on one side or in the middle of the cell.The lymphocytes were usually round,with less cytoplasm.The nucleus of neutrophils was small,often located on one side of the cell with irregular shapes,such as long elliptic,rod-shaped,or divided into two leaves.Thrombocytes had various shapes,round or rod-shaped.Mitochondrium and golgi complex were found in the erythrocytes with TEM.Monocytes and neutrophils were possessing vacuoles and endoplasmic.Meanwhile,the neutrophils contained intracytoplasmic granules.Lymphocytes and thrombocytes had scarce organelles,and only mitochondria were observed.The results of cytochemical staining showed that erythrocytes and thrombocytes were all negative for the six reactions.The monocytes were strong positive for PAS and ACP,and positive for SBB,ALP and POX.Except positive for ACP,the cytochemical staining pattern of neutrophils were the same as those of monocytes.The lymphocytes were positive for PAS and ALP,and negative for SBB,ACP,POX and NAE,PAS negative also present.2.Effects of the water-soluble fraction of diesel oil on the serum biochemical indicators,histological changes,and immune responses of S.schlegelii.Significant differences in some serum biochemical indicators were observed in S.schlegelii after exposure WSD.The alkaline phosphatase（ALP）,glucose（GLU）and globulin（GLOB）were reduced 3.51-fold,3.12-fold,and1.58-fold,respectively,however,K⁺was increased 3.55-fold.The results of HE staining showed that the pathological changes of interstitial congestion were observed in the liver tissue;the secondary lamellae deformity,epithelial lifting,hyperplasia of the secondary lamellae,hyperplasia of the primary lamellae and aneurism were observed.The length of the secondary lamellae shortened significantly after WSD exposure（P<0.05）.The results of AB-PAS staining showed that there were three different types of mucous cells in the gills,and a significant increase in the number of all the three types of mucous cells were observed after WSD exposure（P<0.05）.In addition,the relative m RNA expressions in liver of 11 immune-related genes were investigated.The results showed that compared with the LPS stimulation group,the relative expressions of IL-1β,IL-8,TNF receptor,BAFF,C1s,C1r and My D88 in the WSD exposure LPS stimulation group were significantly higher than those in the LPS stimulation group（P<0.05）,and the relative expression of caspase 10 was significantly lower than that in the LPS stimulation group（P<0.05）,while no significant differences were observed in the relative expressions of IL-1,TNFαand C1inh between two groups（P>0.05）.3.Hematological varieties,histological changes,and immune responses in the early stage of infection with Vibrio parahemolyticus in S.schlegelii This study found that hyperemia,hemorrhage and ascites were common during the V.parahaemolyticus infection.Inflammation,degeneration and necrosis in the liver could be observed.The epithelial mucosal integrity,the intestine epidermis thickness,the villus heights,and the number of mucous cells in the intestine were also decreased after infection.Obvious differences were observed in most of the hematological parameters between the control group and experimental group,such the corpuscular volume（CV）and Na Cl concentrations of the beginning hemolysis were decreased extremely（P<0.01）after infection.However,the white blood cell count（WBC）,lysozyme（LZM）,malondialdehyde（MDA）,K⁺,Cl^-and Ca²⁺were increased significantly（P<0.01）after infection.In addition,the results of m RNA expressions in four tissues（gills,liver,blood and spleen）of 11immune-related genes showed that C1r,C1s,and C1inh expression varied considerably among tissues.Caspase 10 expression was reduced in four tissues after infection,however,My D88,TNF receptor,TNFα,BAFF,IL-1β,IL-8,and IL-10 showed increased expression profiles within 24hours.Particularly,IL-1βexpressions in the liver and spleen increased 123.67 and 104.94-fold at6 hpi,respectively.4.Transcriptome analysis of the spleen of S.schlegelii infection with V.parahemolyticus.Transcriptome response of S.schlegelii in the spleen infected with V.parahemolyticus was examined using paired end Illumina sequencing to understand the immune response.The de novo assembly generated 114,260 unigenes ranging from 301 to 32,331 bp in length and an average length of 1,094 bp.The gene ontology annotations resulted a total of 25,999 term assignments to the annotated transcriptome including 52.47%allocations from the biological process;22.77%from molecular function and24.76%from cellular components.Compared with the 0 hpi group,the number of differential genes in 12 hpi,48 hpi,72 hpi,and 96 hpi treatment was 1,826（1276up-regulated;550 down-regulated）,1,196（827 up-regulated;369 down-regulated）,532（414 up-regulated;118 down-regulated）,701（547 up-regulated;154 down-regulated）.Differentially expressed genes were mainly involved in Toll-like receptor（TLR）and NOD-like receptor（NLR）,RNA sensor RIG-I-like receptor（RLR）and cytoplasmic nucleic acid sensor Cytosolic DNA sensors（CDS）.There were 20 figures,10 tables and 206 references in this thesis.