Early Development And Physiological Mechanism Of Salinity-Alkalinity Adaption Of Triplophysa Yarkandensis(Day)

Triplophysa（Hedinichthys）yarkandensis（Day）,also called Dog-Head Fish locally,belongs in the genus of Cyprinidformes,Cobitidae,Nemachilinae,Triplophysa and Hedinichthys.As one of the fast-growing,big-sized cobitidae,T.yarkandensis is widespread in the Tarim River and,the biggest individual found is 33.0 cm in length and425g in weight.T.yarkandensis is an endemic,indigenous and predominant fish in the Tarim River.At 2015,in the Vertebrate Volume of the China Biodiversity Red List co-edited by the Ministry of Environmental Protection and the Chinese Academy of Sciences,T.yarkandensis is categorized as“Vulnerable（VU）”.In the revision of List on Key Conservation of Aquatic Wildlife in the Xinjiang Uygur Autonomous Region published in 2018,T.yarkandensis is listed as the endangered aquatic wildlife under the second level of state conservation.Over the period from January 2015 to December 2018,2764 specimens of T.yarkandensis were collected from the Tarim River,and 2000samples were cultured in the Aquatic Experimental Station of the Tarim University.Through the test,analysis and process of the samples,we examined the early development and physiological mechanism of saline-alkali tolerance of T.yarkandensis.1.The water temperature was high in the Alar section of the Tarim River,and the Taitema Lake had the lowest temperature about 14℃,which was significantly different from other sampling spots（p<0.05）.Dissolved Oxygen,temperature and pH value was not much different.The salinity and alkalinity in Alar,Taitema Lake and Gaiz River was not so different（p>0.05）.The highest salinity of 8.86±2.9093 was in Hotan River.The highest alkalinity was found in Taitema Lake with value of 6.90±0.2409 g/L and the lowest in Akesu River which was 0.43±1.3056,0.65±0.0714g/L.The 2764 specimens collected from the Tarim River belonged to 6 Orders,10 Families,19 Genuses and 25Species.Cyprinidformes accounted for the majority,Pleurotus 20.48%,indigenous 26.30%and exotic species 73.70%.The majority of the indigenous belonged to the sub-Tarim region in Huaxi District（a mountainous region in Central Asia）.The length and weight data collected from 5 indigenous Triplophysa showed that Triplophysa bombifrons with rised forehead grew at an even speed,while T.yarkandensis and the other species showed allometric growth speed.At salinity 1 to 3,the number of Triplophysa was relatively large,in which Triplophysa microphysa with small bladder in the Aksu River had thegreatest quantity,Triplophysa zamegacephalaz with giant head in the Gaiz River had the second greatest quantity,and Triplophysa tenuis with rised forehead in the Niya River had the smallest quantity.The population of Triplophysa yarkandensis（Day）was positive correlated to salinity（S）and negative correlated to alkalinity（A）.2.The egg of T.yarkandensis was viscous and demersal and the fertilized egg was ovoid with a diameter of 0.060±0.052mm.Under the water temperature of（20±1.0）℃,the whole embryonic development lasted 65 hours and 34 minutes,which comprised of 7physiological phases.The post-embryonic development consisted of 3 phases,namely larva,young-of-the-year（YOY）and juvenile based on the change in yolk sac,body color,bladder and barbel.The mean total length of newly-hatched larva with yolk sac was2.0±0.65 mm.The yolk sac was completely resorbed 7 days after the hatch.When the larva was 16-day old,the bladder on the rear edge of the gill cover was clearly visible.The barbell was also clearly identifiable,body color was deepened and the heart looked red obviously.The body color looked similar to the adult at the time,symbolizing the post-larvae grew fully into the phase of young-of-the-year and the mean total length was8.0±0.45 mm.Upon 30 days of age,the bladder was fully grown,gill cover could be seen opening and shutting clearly,and body was no longer transparent,symbolizing that the young-of-the-year grew fully into the phase of juvenile and the mean total length reached13.0±0.55 mm.At this phase,the morphological and ecological traits were similar to the adult.In the experiment,the relationship between the length of the yolk sac（LY）and the days after the hatch（D）can be expressed by the formula as below,L_Y=0.0286D²-0.0636D+3.1196（R²=0.9050）.The relationship between the length of the yolk sac and（LY）and the length of the larva with yolk sac（LT）can be modeled by the linear formula as below,LY=-1.315LT+5.368（R²=0.8199）.The relationship between the length of the yolk sac（LY）and the days after the hatch（D）can be modeled by the formula as below,LT=-0.0263D²+0.5113D+1.6169（R²=0.9890）.3.Larva began to feed when it aged 3 days old,symbolizing the initiation of the mixed nutrition period.The yolk was completely consumed when the larva was 6 or 7days old and the mixed nutrition period lasted only 3 to 4 days.The first feeding happened 3 days after the hatch and the feeding rate was only 16.7%.The feeding rate peaked on the 7th day after the hatch,which was 90%.The Point of No Return appeared when larva was 8 or 9 days of age.When larva was older than 3 days,starvation affected the yolk absorption rate most significantly（P<0.05）.The feeding larva grew linearly,while the growth of the unfed larva first inclined but then declined.The best time to feed T.yarkandensis was within 4 days after the larva hatched.The size of T.yarkandensis is relatively small,there are jusr a few selections for initial feed and the death rate of newly-hatched larvae is high.Further study needs to be conducted on the initial feed for T.yarkandensis.The weight increased more remarkably when the feeding increased to 6times a day.In absolute growth rate（AGR）,relative growth rate（RGR）and instantaneous growth rate（SGR）,the difference was most remarkable with the 6 feeding times（p<0.01）.The daily feeding with microsome（WL）,that was different from that with other materials（p<0.05）.In AGR,RGR and SGR,difference was very big with different illumination（p<0.01）.Growth was greatest in natural light.4.The hatchability rate of fertilized eggs,survival rate,and vitality coefficiency of larvae of T.yarkandensis showed the highest value at salinity 2⁶ and at alkalinity 0.5¹.5 g/L.With the elevation of salinity and alkalinity,when the value exceeded the threshold,the hatchability rate and survival rate decreased.SAI and hatchability rate was linearly correlated and could be expressed in the form as below,Y=-0.0053X²+0.3027X-3.3947（R²=0.9999）.Under different salinity and alkalinity,the larvae and the young of the year of T.yarkandensis exhibited mania,rapid movement and then died of exhaustion.The surface of the body secreted more slime,and red spots appeared on the head and the abdomen.The median lethal concentration(LC₅₀)of salinity for larvae and young of the year in 24h,48h,72h and 96h were 7.7334、6.4007、5.6797 and 4.3908 respectively,and the safe concentration was 1.5893.The LC₅₀ of alkalinity for young of the year in 24h,48h,72h and 96h were 3.9194 g/L、2.7417 g/L、1.7618 g/L and 1.0281 g/L,and the safe concentration was 0.5754 g/L.The LC₅₀0 of salinity for adult in 24h,48h,72h and 96h were 13.9790、12.9200、12.1170 and 10.7700,and the safe concentration（SC）was 3.785 2.The LC₅₀ of alkalinity for adult in 24h,48h,72h and 96h were 5.1645 g/L、4.0047 g/L、3.6017 g/L and 2.9524 g/L,and the safe concentration（SC）was 0.9316 g/L.At salinity 4 and alkalinity 0.5g/L,larvae and young of the year of T.yarkandensis indicated the most obvious growth strength in total length,weight,absolute growth rate and instantaneous growth rate.At salinity 2 and alkalinity0.5g/L,juvenile growth rate a little different.At alkalinity 0.5 g/L,the value of total length,weight,absolute growth rate,relative growth rate and instantaneous growth rate was greater than that at other alkalinity concentration.For adult,the value of growth rate at salinity 4 and alkalinity 0.5 g/L show remarkable difference（p>0.05）.5.Under different salinity-alkalinity stress,the sodium and chlorine concentration in blood was considerably high and very different from the control group.In 24h,48h,72h and 96h,biochemical index of total protein（TP）,globulin（GLP）,albumin（ALB）,total cholesterol（CHOL）,alanine aminotransferase（ALT）,aspartate transaminase（AST）,alkaline phosphatase（AKP）and Iactate dehydrogenase（LDH）were different.Most indices changed between 48h and 72h.NKA showed insignificant difference in 72h（p>0.05）and low vitality in high or low salinity-alkalinity environment.Gill lamella（GL）of T.yarkandensis became shortened and bent.With the elevation of salinity,gill lamellae became more shortened and bent and appeared vacuolated with epithelial cells exfoliated,thinned and dissolved.At the same time,chloride cells（CC）increased inconspicuously and the cell body enlarged.When exposed under higher salinity stress,the space between gill lamellae increased continuously until exfoliated and rotten which resulted in inability to breathe and suffocation.Glomeruli were developed but the majority was of poor plumpness.Bowman’s capsule consisted of flat cells,and the epithelial cells of renal tubule presented cylindrical shape.As salinity and alkalinity elevated with time,glomerulus（G）atrophied,Bowman’s capsule（BC）inflated,renal tubule shrank and epithelial cells swollen and denatured,distal convoluted tubule（PI）and proximal convoluted tubule（PⅡ）withered badly,and collecting tubule（CS）and neck（NE）worse than the former.When salinity and alkalinity elevated with time,the single cylindrical epithelial cells（SCE）on intestinal villus had signs of thickening and the goblet cells（GC）contained reduced significantly in quantity and differed obviously in size,and meanwhile the striated border damaged severely.A total of 1182224012 raw reads were yielded.After filtering the ambiguous nucleotides and low-quality sequences,a total of 1123448964 clean reads（95.03%of raw reads）were obtained.The clean reads were assembled into 177271 unigenes,ranging from 201,the average length was 1703 bp,the unigenes were clustered in>2000 bp,accounting for 29.76%of all unigenes.These GO terms were categorized into three major categories of cellular component.As for the category of biological process,cellular process 19.76%was the most domain group.Within category of molecular function,45.63%of unigenes were assigned to binding.We first compared the DEGs among concentration gradients within salinity and alkalinity,including 1794 up regulated unigenes and 1180 down regulated unigenes;including 2495 up regulated unigenes and2463 down regulated unigenes.Comparing the pathways enriched from salinity and alkalinity,up regulated pathways such as transcriptional misregulation in cancer,collecting duct acid secretion,fatty acid elongation and protein export were shared in both salinity and alkalinity treatment;down regulated pathways like NOD-like receptor signaling pathway,pathways in cancer,Ras signaling pathway,and viral carcinogenesis were shared by both treatments.At S4 and A0.5,the trend of five differentially expressed genes,such as fubp3,glud,galphai2,gbas,slco2b1,pdlim5,prkci,zfyve16 and abcc1,was significantly higher than that of other saline-alkali groups,while prkci,zfyve16 and abcc1 3 genes showed the opposite trend.