Changes In Immune Gene Expression In Microcystin-LR Injected Grass Carp (Ctenopharyngodon Idellus) And Zebrafish (Denio Rerio) And In Ultrastructure Of Immune Organs In Grass Carp

It has been well documented that microcystin-LR(MC-LR) may have adverse effect on behaviour,embryo development and growth of fish,and pathological damages are also reported in liver and kidney of fish exposed to MC-LR,with observation of changes in physiological and biochemical parameters.However,the toxic effect of MC-LR on immune systems of fish has not yet been investigated.The present study was therefore designed to examine the pathological effect of MC-LR on immune organs of grass carp Ctenopharyngodon idella by using transmission electron microscopy,and on immune gene expression in grass carp and zebrafish Danio rerio by using real-time PCR and gene microarray,respectively.Pathological changes were observed in liver of grass carp injected intraperitoneally with MC-LR(purity＞98%) at the dose of 50μg/kg,and also in immune organs such as the head kidney and spleen of this fish.The increased intercellular space,cholestasis,mitochondria and endoplasmic reticulum edema in hepatocytes were the major symptoms of early toxic effect by MC-LR,however,these symptoms were not observed in hepatocytes 21 days post intraperitoneal injection (dpi).In head kidney and spleen of MC-LR injected grass carp,lymphocytes were the major cell type affected.In head kidney,edema was the major pathological symptom for mitochondria and endoplasmic reticulum of lymphocytes,however,this symptom was not observed 21 dpi.In spleen,apoptotic lymphocytes were found,with a large number of lymphocytes undergoing apoptosis at 2 dpi,but apoptotic lymphocytes were hardly detected at 21 dpi.The expression of genes in relation to the MC-LR effect was investigated at two levels,i.e.by using real-time PCR to investigate quantitatively the expression of certain kinds of immune genes in grass carp,and by using gene microarray to examine differentially expressed genes in zebrafish.Spleen,head kidney and liver were dissected out in grass carp injected intraperitoneally with MC-LR at a dose of 50μg/kg(purity＞98%),and the alteration in expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),interferonⅠ(IFNⅠ),peptidoglycan recognition protein-L (PGRP-L),immunoglobulin M(IgM) and major histocompatibility complex classⅠ(MHC-Ⅰ) was examined.The expression of IL-1βwas significantly suppressed in the head kidney(P＜0.05),and the expression of TNF-α,IFN I and PGRP-L was also significantly suppressed in liver,spleen and head kidney at all sampling intervals(P＜0.05).Significant suppression was also observed for the expression of IgM and MHC-I in liver at all sampling intervals(P＜0.05).It is thus indicated that MC-LR can inhibit the immune function of fish.Differentially expressed genes in liver of zebrafish treated with MC-LR(purity＞95%,400μg/kg) were analyzed by microarray method.Ten fish were sampled 12, 48 and 96 hours post intraperitoneal injection for experimental and control groups, and then total RNA was extracted and mixed with equal amount for the analysis of Affymetrix Zebrafish GeneChips.Genes≥2-fold or≤0.5-fold changes were considered as differentially expressed.A total of 273 genes(10.13%) were of this category,among which 243 genes were up-regulated and 30 down-regulated. Differentially expressed genes can be classified into different categories according to their putative functions.The up-regulated genes include cytoskeleton structures, extracellular matrix components,apoptosis,tumorigenesis,stress factors,signal transduction,cell cycle,growth factors,carriers and transporters,transcription factors, metabolism genes and immune factors,but the down-regulated genes were mainly of immune factors.According to GOstat analysis,MC-LR mainly influenced the cell cycle and MAPK signaling pathway.The results obtained in this microarray study suggest that tumor might be induced by MC-LR in fish.Furthermore,the obvious decrease in expression of several immune-related genes,especially complement and coagulation factors,indicate that MC-LR might induce suppression of immune functions in zebrafish,as observed in grass carp.