| Oxygen concentration in natural waters is usually affected by uncertain natural factors such as temperature,season,weather,and human factors like eutrophication and high stocking density.The dramatically decreased oxygen often causes hypoxic condition of fish,which is also one of the main reasons of “flooding ponds”.Severe acute hypoxia can cause a massive suffocation and death of fish in a short time.In teleost complex physiological and biochemical systems have been developed to response the hypoxia stress.As a good model for the study of hypoxia stress,understanding the physiological mechanism of hypoxia in teleost will facilitate the healthy long-term sustainable development of aquaculture industry.The hybrid yellow catfish "Huangyou-1" is produced by Pelteobagrus fulvidraco(♀)and Pelteobagrus vachelli(♂).It is an important freshwater aquaculture species in China due to the better taste,fewer intermuscular fishbones and high nutritional value.Many studies on antibacterial infections,temperature tolerance and nutrition evaluation of hybrid yellow catfish "Huangyou-1" have been carried out.However,little is known about its antihypoxia mechanism.In this study,the gene expression pattern and physiological and biochemical indexes under acute hypoxia stress(0 h,1.5 h,4 h,6.5 h;0.7 ± 0.05 mg/L)and normoxia conditions(1.5 h,4 h,6.5 h;7.29 ± 0.40 mg/L)were calculated in hybrid yellow catfish "Huangyou-1".This study would provide theoretical basis for the future breeding of hypoxia resistant in teleost.The results are as follows:(1)Effects of hypoxia stress and recovery on oxygen-sensing genes in hybrid yellow catfish "Huangyou-1"The floating head and suffocation points of the hybrid yellow catfish "Huangyou-1" were measured at 0.55 mg/L and 0.25 mg/L,respectively.In this study,the hypoxic stress threshold oxygen concentration(0.7 ± 0.05 mg/L)was slightly higher than the floating point.In this concentration,the fish will be in a stable hypoxic state.Six genes,HIF-1α,HIF-2α,HIF-1β,PHD2,Vhl and FIH-1,were used as oxygen sensor genes in this study and their expression distribution was measured by qRT-PCR.The results revealed that all six genes were ubiquitously expressed in eight organs(intestine,liver,muscle,spleen,heart,gill,brain,and head kidney).HIF-1α and PHD2 were predominantly expressed in liver,while HIF-1β,HIF-2α,Vhl and FIH-1 were highly expressed in heart.Comparing the m RNA levels of hybrid yellow catfish "Huangyou-1" in brain and liver,the six above genes were significantly up-regulated upon hypoxia stress(P<0.05)and down-regulated after reoxygenation.Western blot was used to analyze the temporal expression of FIH-1 protein in brain and liver under hypoxic stress and recovery.The results showed that the expression of FIH-1 protein reached peak under hypoxic stress at 6.5 h(P<0.01)and recovered to normal levels during reoxygenation.It can be speculated that hypoxia stress induced the expression of oxygen sensor-related genes of the hybrid yellow catfish "Huangyou-1",which could be down-regulated by reoxygenation.(2)Effects of hypoxia stress and recovery on respiratory metabolism genes in hybrid yellow catfish "Huangyou-1"The expression distribution of respiratory metabolism-related genes in different tissues was measured by qRT-PCR.The results showed that HK1,PFK and PGK1 were remarkably expressed in heart,CS,SDH and PGAM2 were significantly expressed in muscle and LDH were most expressed in head kidney.The expression of respiratory metabolism-related genes in brain and liver was compared and analyzed under hypoxic stress and recovery.It was found that the expressions of HK1,PFK,PGK1,LDH and PGAM2 were significantly higher than the control group at 6.5 h of hypoxia(P<0.05),and returned to normal levels during reoxygenation.The expression of CS,which is related to TCA cycle,was almost unchanged under hypoxic conditions in liver.However,it was significantly increased during reoxygenation and reaching a peak at 6.5 h(P<0.05).Similar was observed in SDH gene.Although the m RNA level of SDH was significantly lower than the control group at 4 h of hypoxia(P<0.05),it was dramatically increased during reoxygenation and reached the peak at 1.5 h(P<0.01).The above results indicate that the hybrid yellow catfish "Huangyou-1" may be able to proceed normal life activities under hypoxia and reoxygenation,and the metabolic mode is changed from aerobic to anaerobic.(3)Effects of hypoxia stress and recovery on oxidative stress genes in hybrid yellow catfish "Huangyou-1"The expression distribution of oxidative stress-related genes in different tissues was measured by qRT-PCR.The results showed that SOD1,SOD2,GSH-Px and CAT were expressed in variety of tissues including intestine,liver,muscle,spleen,heart,gill,brain,and head kidney.The expressions of SOD1 and CAT was highest in the liver,the expression of SOD2 was highest in the muscle,and GSH-Px was most expressed in the spleen.Compararing the gene expression of hybrid yellow catfish "Huangyou-1" in brain and liver,we found that the expression levels of oxidative stress-related genes(SOD1,SOD2,GSH-Px,and CAT)was significantly higher than the control group at 6.5 h under hypoxic conditions(P<0.05),and returned to normal oxygen levels at 4 h of reoxygenation.The peaks of SOD and CAT enzyme activities and MDA level appeared at 1.5 h of reoxygenation in liver(P<0.05),and the peaks of SOD and GSHPx enzyme activities and MDA level appeared at 1.5 h of reoxygenation(P<0.05)in serum.The above results indicate that the liver and serum of the hybrid yellow catfish "Huangyou-1" showed lipid peroxidation at 6.5 h of hypoxia,while the antioxidant system defense was partially activated at 4 h of hypoxia.It is suggested that the fish’s antioxidative capacity was increased in advance during the hypoxic process to deal with the oxidative stress damage caused by reoxygenation.At the same time,the peroxidation and antioxidative capacity were significantly increased during the recovery process.(4)Effects of hypoxia stress and recovery on apoptosis genes in hybrid yellow catfish "Huangyou-1"The expression distribution of apoptosis-related genes in different tissues was measured by qRT-PCR.The results showed that Apaf-1,Bax,Caspase 3,Caspase 9,p53,Bcl-2 and Cyt C were expressed in variety of tissues including intestine,liver,muscle,spleen,heart,gill,brain,and head kidney.Apaf-1,Bax,Caspase 3,Caspase 9,Cyt C and p53 were most expressed in the spleen,while Bcl-2 was highest in the head kidney.Under hypoxic conditions,the expressions of Caspase 3,Caspase 9,Apaf-1,Bax,and p53 were significantly increased compared with the control group(P<0.05)in liver and brain.While the expressions of Cyt C and Bcl-2 were reduced(P<0.05).The expression of Bax protein in brain reached the peak at 6.5 h of hypoxia(P<0.01).The expression level of Bax protein in liver was significantly different withthe control group at 4 h of hypoxia(P<0.01)and is still higher than the control group after reoxygenation.In summary,during the process of hypoxia and recovery,tissues will produce peroxidation leading to oxidative stress.At the same time,the aerobic metabolism of the mitochondria will be blocked,which will affect the normal cell function and cause the initiation of apoptosis. |
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