The Effect Of Environmental Factors On The Ability Of Takifugu Obscurus To Tolerate Salinity And Its Differences At The Transcriptomics Level

Obscure Puffer(Takifugu obscurus),belonging to Tetraodontiformes,is an important economic fish in China and east Asian countries.The major habitats of T.obscurus distributed in the East China Sea,the Yellow Sea,the Bohai Sea,and the middle and lower Yangtze River in China.In spring,T.obscurus migrate to freshwater rivers along the Yangtze River to reproduce.Newly hatched larvae remain in freshwater for fattening and return to the ocean in the end of the year or the next spring.In recent years,due to environmental pollution and overfishing,the wild population of T.obscurus is endangered.The success in artificial reproduction of T.obscurus prevented their population from getting worse.However,no systematic study has yet been found on the basic biological functions especially osmoregulation during migration,and many scientific problems still demand solution.With the ability to adjust internal osmotic pressure and the long tolerant range of salinity,T.obscurus can be used as a typical model for the study on osmoregulation of migration species.In the course of its migration,it will also be challenged by the deterioration of water environment.In the early life stage of T.obscurus,the growth and development as well as the tolerance to salinity are affected significantly by temperature.Excessive heavy metal in water and enrichment in the organism can cause damage to the body,whther it will affect its osmoregulation?Water eutrophication often results in the high nitrite which causes fish to die of hypoxia,and how does it affect the fish in different salinity?As a typical anadromous fish,studies on the osmoregulation mechanisms of T.obscurus,especially at the molecular level,have not received sufficient attention.This study will focus on the above scientific questions,combining with various experimental methods and indicators,and emphatically discuss the influence of environmental factors on the salinity tolerance of T.obscurus and its osmoregulatory mechanism.The major results of the present study included 6 aspects as below:1.The effect of temperature on the salinity tolerance and development of embryo T.obscurusIn this experiment,the embryos were placed under different temperature(16,19,22,25,28,and 31 ?)and salinities(0,5,10,13,16,and 18 ppt),the experiment lasted until 24 h post hatch.The results showed that the development rate of embryos increased with the increasing temperature.In appropriate temperature range(19-25 ?),the salinity tolerance of embryo was increased with the increase of temperature.At low temperature(16 ?),embryo could not develop normally and could not tolerate higher salinity(? 13 ppt).At high temperature(28 and 31 ?),embryo could hatched out rapidly under high salinity(18 ppt),but the viability of larvae was poor and the abnormal rate was very high.Salinity also affect the accumulated temperature and development threshold temperature:with the increase of salinity,the development threshold temperature was higher and the accumulated temperature was lower.2.Combined effects of temperature and salinity on the osmoregulation and oxidative stress response of newly hatched larvae T.obscurusThe newly hatched larvae were placed under different temperature(19,25,and 31 ?)and salinities(0,10,and 20 ppt).After 96 h,their survival rates and related enzyme activities were measured.The results confirmed that larvae could not tolerate the temperature more than 31 ?,and could not tolerate higher salinity in the high temperature.This may due to that the growth of larvae was more rapid at high temperature,but also required more energy,so that there was no additional capacity to maintain the osmotic balance.With the increase of temperature,the levels of MDA,SOD and Na+/K+-ATPase were increased at high salinity and reached the maximum in advance.The increase of both temperature and salinity had a great effect on the survival of larvae and induced severe oxidative stress response.3.The effect of heavy metal cadmium on salinity tolerance and oxidative stress of juvenile T.obscurusThe present study aimed to detect the combined effects of different concentrations of Cd(0,5,10,20 and 50 mg L-1)and salinity(0,15 and 30 ppt)on juvenile T.obscurus.Results showed the juveniles could survive well at different salinities;however,under the Cd exposure,the survival rates significantly decreased at 0 and 30 ppt.Cd exposure clearly induced oxidative stress,and the responses among different tissues were qualitatively similar.Salinity acted as a protective factor which could reduce the reactive oxygen species and malondialdehyde levels.Thus,the role of salinity should be considered when evaluating the effect of heavy metals on migration fishes.We also suggested that T.obscurus is an effective animal model,which can be used to evaluate the risk of different pollutants on migratory fish or estuarine fishes under different salinity.4.The effect of nitrite on salinity tolerance of juvenile T.obscurus and their physiological-biochemical responses.We measured the physiological responses of juvenile T.obscurus exposed to nitrite concentrations(0,10,20,50,and 100 mg L-1)at different salinity levels(0,10,and 20 ppt)for 96 h.We also used the integrated biomarker response(IBR)to assess ecological risk.Salinity increased the survival rates of juvenile T.obscurus exposed to nitrite.Changes in key hematological parameters,antioxidant system,malondialdehyde,Na+/K+-ATPase,and HSP70 indicated that nitrite induced considerable damage to juveniles;salinity mitigated the harmful effects.This finding reflected similar changing trends in both antioxidants and their gene expressions among different tissues.We applied an overall index,IBR,that increased under high-nitrite condition but recovered to the normal levels under salinity treatment.Analysis of the selected detection indices and IBR values showed that the overall mitigating effect of salinity on nitrite toxicity seemed to be at sub-cellular level and associated with complicated physiological responses.5.Microstructure and ultrastructure observation of adult T.obscurus under different salinity.The adult T.obscurus were placed in freshwater and saltwater(30 ppt),after 7 days,gill,intestine and kidney were dissected for section-staining and transmission electron microscopyic observation.The results showed that,in saltwater,both the number of chloride cells and the cell volume in gill was increased,the number of mitochondria also increased,and the tubular network were developed.In intestine,the goblet cells and serosas changed,the serosas were longer.In kidney,the renal corpuscle were atrophied and the number of mitochondria in the proximal convoluted tubules were decreased.The three organs were important in regulating osmotic pressure and maintaining osmotic balance,Adaptive adjustments under the salinity change were observed in the present study..These findings can be used for the further study of T.obscurus's osmoregulatory mechanism foundation and enrich the basic biological data of euryhalinity fish.6.Revealing osmotic pressure regulating mechanism of T.obscurus based on transcriptom technologyWith the help of high-throughput Illumina platform,the differentially expressed genes of T.obscurus's gill,kidney and intestine at freshwater and saltwater were sequenced.The molecular mechanism of osmotic pressure regulation of T.obscurus was analyzed from the overall level.With the increase of salinity,in terms of material transport,the ATP binding box transporter and cAMP signaling pathway in the gill and kidney were significantly reduced,in addition,the transmembrane conductance regulation factor of water channel protein and cystic fibrosis and the combined transport protein of Na+/Cl-in the intestinal were significantly increased.In terms of neuroendocrine hormone regulation,Growth hormone,prolactin,insulin-like growth factor-1 and cortisol regulat body composition more transporters,stimulate chloride cell proliferation and differentiation,so as to change capacity for membrane transport between ion and water molecules.In terms of energy metabolism and material metabolism,adenosine activated protein kinase signaling pathway and thyroid hormone signaling pathway were significantly up-regulated in the gill,and the TCA cycle pathway was also significantly increased in the kidney.This study provides the basis for the future study of the important pathways in the osmotic pressure regulation mechanism of T.obscurus.