Effects Of Nitrite And Microplastic Stress On The Physiological Functions Of Penaeus Vanname

Litopenaeus vannamei is an important aquaculture species in China.In the process of intensive shrimp culture,the accumulation of animal residual bait feces and poor water circulation will cause the increase of nitrite concentration in aquaculture water,affect the growth,survival and immunity of shrimp,increase the susceptibility to pathogens,and endanger the economic benefits of aquaculture.In addition to environmental factors,shrimp farming is also affected by exogenous pollutants,such as microplastics.Existing studies have confirmed that there is a risk of microplastic pollution in the shrimp aquaculture environment.In addition to its own toxicity,microplastics are easy to adsorb other pollutants in the environment due to their small particle size and large specific surface area,and then cause secondary damage to aquatic animals.However,the current research on the toxic effects of nitrite or microplastics on L.vannamei is mainly focused on a single factor,but the combined effects of multiple factors in the aquaculture system are rarely considered.In this study,the healthy shrimp were divided into four groups:control group,20 mg·L^-1nitrite stress group,10μg·L^-1 microplastic stress group,20 mg·L^-1nitrite and 10μg·L^-1 microplastic combined stress group.On the 14th day,the changes of histological morphology and physiological indexes of the gills,hepatopancreas and intestine of shrimp were measured,and the changes of the gill and hepatopancreas metabolome,and intestinal microbial community were analyzed,in order to reveal the physiological response mechanism of L.vannamei to nitrite and microplastic stress.These results can provide theoretical reference for the healthy breeding of shrimp.The main results are as follows:1.The effects of nitrite and microplastic stress on the physiological function in the gills of L.vannameiThe effects of nitrite and microplastic stress on the histological morphology,physiological indexes,and metabolic function in the gills of L.vannamei were investigated.The results showed that:1)The gills tissue showed damage characteristics such as deformation and cavitation.2)The contents of malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）,and the activity of antioxidant enzymes such as superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GPx）were changed in different degrees after stress.3)The relative expression levels of immune indexes such as cytochrome P450 gene（CYP450）and glutathione S-transferase gene（GST）were disordered in different degrees after stress.The relative expression level of apoptosis factor gene（CASP-3）was significantly decreased in the three stress groups（P<0.05）.4)Osmotic regulation indicators such as ion transporter vacuolar ATPase（VATP）,Na⁺/H⁺exchanger 7（NHE7）,Na⁺/K⁺-ATPase subunitα（NKA-α）,Na⁺/K⁺-ATPase subunitβ（NKA-β）,carbonic anhydrase（CA）and aquaporin TIP4-1（TIP4）,calcium channel protein 1（CCP）,chloride channel protein 2（CLC）,aquaporin（AQP）gene relative expression levels were different degrees of disorder after stress.5)Metabolic function,especially PPAR signaling pathway,amino acid metabolism and lipid metabolism were highly affected,and 14 metabolites were identified.The results showed that nitrite and microplastic stress could induce the changes of gill tissue morphology,oxidative stress,immunity,detoxification metabolism and osmotic regulation of L.vannamei,resulting in metabolic dysfunction and affecting its normal physiological function.2.The effects of nitrite and microplastic stress on the physiological function in the hepatopancreas of L.vannameiThe effects of nitrite and microplastic stress on the morphology,physiological indexes and metabolic function of hepatopancreas of L.vannamei were investigated.The results showed that:1)The stress caused the hepatopancreas tissue damage characteristics,such as vacuole rupture of hepatic tubular atrophy deformed cells.2)Oxidative damage indicators such as superoxide anion（O₂^-）,H₂O₂,MDA and lipid peroxidase（LPO）content,and antioxidant enzyme activities such as CAT and SOD in the stress group were changed.3)The relative gene expression levels of immune indexes such as reactive oxygen species regulator-1（ROMO1）,SOD,GPx,thioredoxin（TRX）,cytochrome oxidase（Cyt C）,apoptosis factor Caspase-3（CASP-3）,antimicrobial peptide（Crus）and prophenoloxidase（pro PO）were disordered after stress.Endoplasmic reticulum stress-related indicators such as binding immunoglobulin（Bip）,eukaryotic initiation factor 2（e IF2α）,beclin1autophagy gene（Beclin1）,and ring amplifier-dependent transcription factor ATF6α（ATF6）showed different degrees of fluctuation.4)Metabolic function such as“arginine and proline metabolism”,“ABC transporter”,amino acid and lipid metabolism were highly affected,and a total of 17 metabolic markers were identified.The results showed that nitrite and microplastic stress could induce the changes of hepatopancreatic tissue morphology,oxidative stress,immunity,oxidative stress,detoxification metabolism,endoplasmic reticulum stress and other related indexes of L.vannamei,which affected the metabolic function of hepatopancreas and the normal physiological function of L.vannamei.3.The effects of nitrite and microplastic stress on the physiological function in the intestinal of L.vannameiThe effects of nitrite and microplastic stress on intestinal morphology,physiological indexes and microbial community of L.vannamei were investigated.The results showed that:1)After stress,the intestinal villus height was shortened,the epithelial cells were necrotic,and cells were vacuolated or ruptured.2)Physiological and biochemical indexes such as CAT,GPx,peroxidase（POD）,lipase,andα-amylase activity changed in different degrees after stress.3)The relative expression levels of immune indexes such as CAT,SOD,GPx,heat shock protein 90（Hsp90）,pro PO,CASP-3,and endoplasmic reticulum stress-related genes glucose-regulated protein 94（GRP94）and X-box binding protein 1（XBP1）were disordered after stress.4)Stress induced changes in the intestinal microbial diversity of the shrimp.The relative abundances of Bacteroides,Bifidobacterium,Escherichia-Shigella,Pandoraea,Streptococcus and Weissella were increased,while the relative abundances of Lactobacillus,Pseudoalteromonas and Vibrio were decreased.5)The functions of“antigen processing and presentation”and“NOD-like receptor signal transduction”of intestinal microbial were increased in the three stress groups,but the function of“N-glycan biosynthesis”was decreased.The results showed that nitrite and microplastic stress could induce changes in intestinal tissue morphology,oxidative stress,immunity,endoplasmic reticulum stress and other related indicators of L.vannamei,resulting in the variation of intestinal microbial diversity and composition of L.vannamei.