Physiology And Molecular Biology Of Phoxinus Lagowskii Under Diel-Cycling And Sustained Hypoxia

As a small cold-water fish widely distributed in high latitudes of China,Phoxinus lagowskii is a dominant species for analyzing diel-cycling hypoxia（DCH）and sustained hypoxia（SH）due to its small body and hypoxia tolerance.In order to further understand the regulation mechanism of P.lagowskii under different hypoxic conditions,this paper investigated the oxygen consumption rate（MO₂）,changes in hematological parameters,gill and liver morphology,glycolipid metabolizing enzymes in the liver and changes in clock genes after diel-cycling hypoxia（DCH）and sustained hypoxia（SH）in P.lagowskii.Meanwhile,gene cloning and tissue expression combined with fluorescence in situ hybridization were used to analyze the P.lagowskii tumor suppressor gene P53.The results can provide a theoretical basis for studying the mechanism of anoxia tolerance in P.lagowskii,fish resilience analysis,and aquaculture application.The main research results are as follows:（1）Oxygen consumption rate（MO₂）analysis was performed after 10 days of hypoxia.This analysis revealed that the hypoxia tolerance of P.lagowskii was significantly improved after 10 days of either sustained or diel-cycling hypoxia,with the improvement being more pronounced in the DCH.（2）An increase in the number of red blood cells（RBCs）was observed in P.lagowskii during both hypoxic conditions,indicating an increased ability of the blood to carry oxygen.Hemoglobin（Hb）concentrations increased on the fourth day of diel-cycling hypoxia,confirming that physiological and metabolic adaptation to hypoxia is based on the increased duration of exposure.（3）Histological results of gills showed that the surface area of gill lamellae increased more in SH than in DCH,indicating that P.lagowskii increased oxygen acquisition by increasing the gas exchange area of gills under hypoxic conditions and that sustained hypoxia had a more significant effect on gills.Histological changes in the liver showed a higher frequency of cytoplasmic vacuolization in the liver exposed to diel-cycling hypoxia.The results of enzyme activity measurements in the liver showed that pyruvate kinase（PK）was increased in the SH,suggesting that P.lagowskii can use different sources of energy under sustained hypoxic conditions,while the DCH mainly used triglyceride（TG）as an energy source.（4）Significant upregulation of Cry1b gene could be observed under sustained hypoxic conditions.The expression of three of the eight clock genes studied was increased under diel-cycling hypoxic conditions,including Per1a,Clocka and Cry1b,suggesting that clock genes play different roles under different hypoxic stresses.（5）The c DNA of P53 from the P.lagowskii was cloned by the combination of homology cloning and rapid amplification of c DNA ends（RACE）approaches.The full-length c DNA of Pl-P53 was 1878 bp,including an open reading frame（ORF）of 1116 bp encoding a polypeptide of 371 amino acids with a predicted molecular weight of 41.22k Da and a theoretical isoelectric point of 7.38.Pl-P53 was commonly expressed in eight tissues（gill,eye,brain,heart,liver,spleen,muscle,and intestine）of the P.lagowskii,with the highest expression in the heart.In addition,the expression of Pl-P53 in five tissues under different hypoxic conditions was investigated.In the SH group,there was a significant increase in Pl-P53 expression in the brain and spleen with prolonged time,while the expression decreased in the heart,muscle and liver.Pl-P53 expression was decreased in both brain and heart in the DCH group,and significantly increased in spleen at 10 days DCH.