| T-2 toxin,which belongs to trichothecenes,is a toxic secondary metabolite produced by Fusarium.As one of the most toxic A-type mycotoxins,T-2 toxin widely exists in moldy feed and raw materials.T-2 toxin can cause adverse effects such as slow growth and immunosuppression on animals,resulting in the reduction of animal production and economic benefits.At the same time,T-2 toxin residues will also pose a potential threat to food safety.Chinese mitten crab(Eriocheir sinensis)is popular for its unique flavor and high nutritional value in China.The output of Chinese mitten crab has increased from 4800 tons in 1990 to 750000 tons in 2019.Raw materials like corn and wheat in formula feed of crabs are easily contaminated by mycotoxins such as T-2toxin.However,there are no reports on the toxicity of T-2 toxin to juvenile Chinese mitten crab.In this study,the effects of T-2 toxin on the growth and immune function of juvenile Chinese mitten crab were investigated by means of molecular biology and nutrition,and the methods to alleviate the toxicity of T-2 toxin were discussed depending on its toxicity characteristics.From the previous studies,physical adsorption method is the best method to remove mycotoxins with low price.In this study,yeast cell wall was used as a feed additive to explore its alleviating effect on T-2 toxin induced-toxicity of juvenile crabs,but it did not achieve the expected effect.Combined with in vitro experiments,it was found that the toxicity of T-2 toxin was related to the disturbance of redox homeostasis.Therefore,N-acetylcysteine(NAC)and curcumin were selected as functional feed additives to evaluate their alleviating effects on the toxicity of T-2 toxin in juvenile E.sinensis.The main results and conclusions in the present study are presented as follows:1.Toxic effects of T-2 toxin on juvenile E.sinensisIn order to explore the effects of T-2 toxin on the growth and immune function of juvenile Chinese mitten crab,The isonitrogenous and isoenergetic diets were prepared by adding 0(control group),0.6(T1),2.5(T2)and 5.0(T3)mg / kg T-2 toxin to the diets respectively and used to feed juvenile crabs for 8 weeks.The results showed that T-2 toxin significantly reduced the weight gain and specific growth rate of juvenile E.sinensis.With the increase of T-2 toxin content in feed,the survival of juvenile crabs decreased in a dose-dependent manner.The survival of crabs in T3 group was significantly lower than that of the control.T-2 toxin significantly increased the content of malonaldehyde(MDA)in hepatopancreas,decreased the activities of total superoxide dismutase(T-SOD)and total antioxidant capacity(T-AOC).The activity of glutathione peroxidase(GSH-Px)in T3 group was obviously lower than that in the control.T-2 toxin decreased total blood cell count(THC),respiratory burst,phenol oxidase(PO)in hemolymph,the activities of acid phosphatase(ACP),alkaline phosphatase(AKP)and PO in hepatopancreas compared with the control,.The expression of anti-lipopolysaccharide factors(ALF1 and ALF2)firstly increased,then decreased with the increase of T-2 toxin content in diet.The expression level was the highest in T-2 group.4.6 mg / kg T-2 toxin significantly increased the expressions of lipopolysaccharide-induced TNF factor(LITAF)and Relish.T-2 toxin significantly upregulated the expressions of caspase-3,caspase-8 and Bax,and the ratio of Bax / Bcl-2in the hepatopancreas.Crabs fed diets with T-2 toxin have the lower expression of Bcl-2.The genes related to detoxication including cytochrome P450 gene superfamily(CYP2,CYP3,CYP4)and glutathione-S-transferase(GST)were induced in low concentration of T-2 toxin,then decreased in high concentration.In addition,chronic exposure to T-2 toxin caused the damage of hepatopancreas structure,especially as seen in the detached basal membrane of hepatopancreatic tubules in juvenile E.sinensis.These results indicate that chronic exposure to T-2 toxin can reduce growth performance,deteriorate health status and cause hepatopancreas dysfunction in crabs characterized by oxidative stress and apoptosis of hepatopancreas in E.sinensis.The intestinal tract not only has the function of digestion and absorption,but also is one of the important immune organs of crustaceans.Maintaining the intestinal health of crustaceans helps to improve their growth and obtain higher economic value.Intestinal tract is also an important target organ of T-2 toxin,which is damaged firstly by T-2 toxin when animals are fed the diet contaminated with T-2 toxin.This study was to explore the effects of T-2 toxin on the intestinal health and intestinal microbial composition of juvenile Chinese mitten crab.The results showed that the contents of MDA in intestinal tissue increased with the increase of T-2 toxin content in feed.Moreover,the content of MDA in T2 and T3 group was significantly higher than that in control group,while the activities of GSH-Px and T-AOC increased firstly and then decreased with the increase of T-2 toxin.The activity of antioxidant enzymes in T2 group was significantly higher than that in control group.T-2 toxin significantly decreased the expression of antimicrobial peptides(ALF1,ALF3,crustin-1 and crustin-2)and peritrophic membrane factors(PM1 and PM2),and increased the expression of inflammation related genes(TLR,Myd88,LITAF and relish).The expression of MAPKs(p38,JNK and ERK)significantly increased with the increase of T-2 toxin content.T-2 toxin significantly increased the expression of caspase-3,caspase-8,Bax gene and the ratio of Bax / Bcl-2,and decreased the expression of Bcl-2 gene.In addition,4.6 mg / kg T-2 toxin changed the richness and diversity of intestinal microbiota.At the phylum level,Bacteroidetes,Firmicutes,tenericetes and Proteobacteria were the dominant bacteria in the intestine of E.sinensis.T-2 toxin significantly decreased the abundance of Bacteroidetes and increased the abundance of Firmicutes,Tenericutes and Proteobacteria.At the genus level,T-2 toxin decreased the abundance of beneficial bacteria(Dysgonomonas and Romboutsia),and increased the abundance of pathogenic bacteria(Candidatus Bacilloplasma,Chryseobacterium and Streptococcus).In conclusion,T-2 toxin in feed can cause oxidative damage,apoptosis and immunosuppression,increase disorder of gut microbiota of intestine in juvenile Chinese mitten crab,thus affecting the integrity and health of intestinal tract.2.Improvement of yeast cell wall(YCW)on T-2 toxin-induced toxicity in juvenile E.sinensisFish meal,casein and gelatin were used as protein sources.Four diets were prepared by adding T-2 toxin and YCW: 0(control,without T-2 toxin and YCW),2.5mg/kg T-2 toxin(T-2 group,without YCW),2.5mg/kg T-2 toxin + 1% YCW(YCW-1 group)and 2.5mg/kg T-2 toxin + 2% YCW(YCW-2 group).800 juvenile crabs(0.74± 0.01)g were randomly divided into 4 groups.Crabs were fed with the four diets for8 weeks to explore the alleviating effects of yeast cell wall on T-2 toxin-induced toxicity in juvenile Chinese mitten crabs.The results showed that T-2 toxin significantly reduced the growth and survival of juvenile E.sinensis.Compared with T-2 group,supplement of yeast cell wall in diets could improve the growth of crabs,but there was no significant difference.When diet was supplemented with 2% yeast cell wall,the survival of crabs was significantly higher than that of crabs in the T-2 group.T-2 toxin significantly increased the contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in hemolymph of juvenile crabs.However the addition of yeast cell wall could not reverse the content of ALT in hemolymph,while 2% yeast cell wall could significantly reverse the content of AST in hemolymph.Compared with the control,the contents of MDA in hemolymph and hepatopancreas of crabs in T-2 group significantly increased,while the activities of GSH-Px in hemolymph,GSH-Px and TAOC in hepatopancreas significantly decreased.Supplementation of 1% yeast cell wall in diet significantly increased the activities of GSH-Px and T-AOC in hepatopancreas,while 2% yeast cell wall significantly decreased the content of MDA in hepatopancreas and increased the activity of GSH-Px in hemolymph and hepatopancreas.T-2 toxin could inhibit the immune function of hepatopancreas and increase the expression of inflammatory regulatory factors.Compared with T-2 group,supplementation of yeast cell wall significantly reduced the expressions of TLR and My D88 genes.1% yeast cell wall enhanced the activities of ACP and AKP in hepatopancreas.In addition,T-2 toxin induced hepatopancreas apoptosis in juvenile crabs,but supplement of yeast cell wall did not improve hepatopancreas apoptosis induced by T-2 toxin.In conclusion,T-2toxin can inhibit the growth and immune function of juvenile E.sinensis,but yeast cell wall supplementation can not relieve the growth inhibition caused by T-2 toxin,and can only partially improve antioxidant capacity and immune function of crabs.Therefore,yeast cell wall supplementation in feed can not completely eliminate the adverse effects of T-2 toxin.3.Toxic effects of T-2 toxin on primary hemolymph cellPrevious studies have shown that oxidative stress is the key mechanism of mycotoxin toxicity.While Cnc C / keap1 pathway can improve the antioxidant capacity and maintain the redox homeostasis to mitigate the toxicity induced by mycotoxins.However,there are no reports on the structure and expression characteristics of Cnc C and keap1 genes in Chinese mitten crab.In order to explore the toxic mechanism of T-2 toxin in E.sinensis,the full-length c DNA of Cnc C and keap1 genes were cloned by RT-PCR and RACE based on the transcriptome data of E.sinensis.Bioinformatics analysis showed that the full length of Cnc C gene in E.sinensis is 6993 bp.The open reading frame length is 2604 bp,encoding 867 amino acids.The theoretical molecular weight of the protein is 95.18 k Da,and the theoretical isoelectric point is 5.23.The results of amino acid sequence alignment showed that the amino acid sequence of Cnc C in E.sinensis had the highest identity with sequenece of Cnc family in Litopenaeus vannamei(80-86.48%),followed by that of Tribolium castaneum(40.68%).By analyzing the expression of Cnc C gene in different tissues,it was found that the highest expression of Cnc C was found in hepatopancreas,followed by haemolymph cell and intestine.The full length of keap1 gene in E.sinensis is 4609 bp.with an open reading frame of 1860 bp.It encodes 619 amino acids.The theoretical molecular weight of the protein is about 69.23 k Da,and the theoretical isoelectric point is 6.42.The results of amino acid sequence alignment showed that the amino acid sequence of keap1 in E.sinensis had the highest similarity with snow crab(92.15%),followed by Penaeus monodon(85.62%)and L.vannamei(85.46%).The expression analysis of keap1 gene in different tissues showed that the expression of keap1 was higher in hepatopancreas,followed by brain.Based on the coding region sequence of Cnc C obtained in this study,the si RNA sequence of Cnc C gene was designed and synthesized.Through RNA interference technology,the toxic mechanism of T-2 toxin on primary hemolymph cell of Chinese mitten crab was explored.The results showed that different concentrations of T-2 toxin significantly reduced the cell viability and mitochondrial potential,and increased the levels of ROS of primary hemolymph cells.The cytotoxicity of T-2 toxin in primary hemolymph cells was dose-dependent.100 ng / ml T-2 toxin inhibited the antioxidant capacity and immune function of the cells.In addition,the interference of Cnc C in primary hemolymph cells increased the inhibition of anti-oxidation ability and the immunotoxicity induced by T-2 toxin.In conclusion,the oxidative stress induced by T-2 toxin is related to the antioxidant pathway of Cnc C / Keap1.Antioxidants enhancing the antioxidant capacity of cell may be a potential measure to intervene the toxicity of T-2 toxin.4.Improvement of antioxidants on the toxicity of T-2 toxin in juvenile Chinese mitten crab(E.sinensis)Based on the above studies,it was found that the toxicity of T-2 toxin was closely related to oxidative stress.Therefore,two different antioxidants were selected in this study to explore the improvement effect of dietary antioxidants on the toxicity of T-2toxin in juvenile Chinese mitten crab.NAC is an acetylated cysteine derivative and can synthesize glutathione belonging to non enzymatic antioxidants to improve antioxidant capacity.It is commonly used in the treatment of oxidative stress-related diseases in clinic.A total of 800 crabs(0.27 ± 0.00 g)were randomly divided into 4 groups with 5replicates and were fed 4 diets: 0(control,without T-2 toxin and NAC),2.5mg/kg T-2toxin(T-2 group,without NAC),2.5mg/kg T-2 toxin + 0.05% NAC(0.05 N group)and2.5mg/kg T-2 toxin + 0.1% NAC(0.1N group)for 8 weeks to explore the effect of NAC on the toxicity of T-2 toxin in juvenile Chinese mitten crab.The results showed that T-2 toxin significantly inhibited the growth and immune function,and caused hepatopancreatic oxidative stress and apoptosis in juvenile crabs compared with the control.The diet supplemented with NAC significantly increased the weight gain,specific growth rate and hepatosomatic index(HIS)of juvenile Chinese mitten crab fed with T-2 toxin.0.1% NAC significantly increased the survival of juvenile Chinese mitten crab.0.1% NAC significantly reduced the contents of AST and ALT in hemolymph of juvenile Chinese mitten crab,and significantly increased the oxygen consumption rate of juvenile Chinese mitten crab compared with T-2 group.Dietary NAC significantly decreased the contents of ROS in hemolymph and MDA in hemolymph and hepatopancreas.While NAC supplementation significantly increased the activities of GSH-Px,T-AOC,?-GCS and the content of GSH in hepatopancreas.NAC supplementation enhanced respiratory burst,total protein content in hemolymph and activity of ACP in hepatopancreas.The number of hemocytes,the activities of ACP in hemolymph and AKP in hepatopancreas of crabs from 0.1N group were significantly higher than those of T-2 group.Compared with T-2 group,0.1% NAC significantly increased the expressions of antimicrobial peptides(ALF1,ALF2,crustin-1 and crustin-2)and peroxinectin genes in hepatopancreas.Meanwhile,the expressions of Relish and LITAF in hepatopancreas from 0.1N group decreased.In addition,the expression of Caspase-3,Caspase-8,Bax and p53 genes and the ratio of Bax / Bcl-2significantly decreased in crabs fed diets with NAC.2.5 mg / kg T-2 toxin can lead to growth inhibition and health damage of juvenile E.sinensis.Supplement of 0.1% NAC in the diet can significantly enhance the antioxidant capacity of juveniles by increasing the content of GSH,improve the growth and immunosuppression induced by T-2 toxin,and alleviate oxidative stress and apoptosis.CncC / keap1 is a key nuclear transcription factor that regulates the expression of detoxification enzymes and antioxidant enzymes in animals.It is considered as a key target to prevent the imbalance of redox.Curcumin can induce the activation of Cnc C,enhance the activity of antioxidant enzymes to improve antioxidant capacity,which is considered that has antioxidant,anti-inflammatory and anti apoptotic functions.In this experiment,fish meal,casein and gelatin were used as the main protein sources.T-2toxin and curcumin were added to prepare four isonitrogenous and isoenergetic diets:C(control,without T-2 toxin and curcumin),T-2 group(2.5 mg / kg T-2 toxin),0.5C group(2.5 mg / kg T-2 toxin + 0.5% curcumin)and 1C group(2.5 mg / kg T-2 toxin +1% curcumin).Crabs were fed with the four diets for 8 weeks to explore the improvement effect of curcumin on T-2 toxin-induced toxicity in juvenile E.sinensis.The results showed that T-2 toxin significantly reduced the weight gain,specific growth rate and HSI of juvenile E.sinensis.The growth and survival of juvenile E.sinensis in0.5C group significantly improved.Compared with the control group,the motion distanse and oxygen consumption rate of crabs in T-2 group significantly decreased,while curcumin could significantly reverse the adverse effects.0.5% curcumin significantly reduced the content of reactive oxygen species in hemolymph and the content of MDA in hemolymph and hepatopancreas compared with T-2 toxin group.The activities of GSH-Px and T-AOC,and the expression of Cnc C gene in 0.5C group were higher than those in T-2 group.T-2 toxin inhibited the immune function of juvenile E.sinensis and enhanced the expressions of inflammatory genes.The expressions of antimicrobial peptides(ALF1,ALF2,ALF3 and clustin-1)and peroxinectin genes in0.5C group significantly increased,and the expressions of Relish and LITAF significantly decreased 0.5C group compared with those in T-2 group.In addition,T-2toxin promoted the apoptosis of hepatopancreas.Curcumin supplementation significantly decreased the expressions of p53,Bax,caspase-8 and caspase-3 genes and the ratio of Bax / Bcl-2.The expression of Bcl-2 gene increased with curcumin.The results showed that 0.5% curcumin could improve the growth inhibition,immunosuppression,the oxidative damage and apoptosis of hepatopancreas caused by T-2 toxin by activating the expression of Cnc C / keap1 gene in hepatopancreas to improve the antioxidant capacity of juvenile crab.In conclusion,dietary supplementation of antioxidants is helpful to improve the growth inhibition and health damage of juvenile E.sinensis caused by T-2 toxin. |
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