Artificial Seedling Techniques Of Pseudopleuronectes Yokohamae And Its Related Genes Expression Analysis During Early Developmental Stage

Factory aquaculture of fish has developed a mature Large-scale aquaculture industry which had rised suddenly in marine aquaculture in China.However,the factory aquaculture of Pseudopleuronectes yokohamae has not researched and popularized until now.P.Yokohamae is one of the most economically important aquaculture species in China.However,the present abundance of P.yokohamae is sharply declining,its artificial breeding techniques urgently need to breakthrough.In this study,basically reproductive biology of P.yokohamae including fertility and sperm activity duration were researched according to its biological characteristics during early developmental stage.In this study,Pseudopleuronectes yokohamae was studied by the techniques of breeding stocks rearing,artificial dry fertilization,and so on.Artificial incubation was produced through hatching barrel and bolting cloth net.We also observed and recorded embryonic development since fertilized egg stage using NIKON-100 microscope.Furthermore,the effects of starvation on growth,morphology and behavior were studied.At the same time,the effect of light intensity on feeding was also detected.In addition,the mRNA expression of key genes(Shh and Hox)in early larval development stage were analyzed.The major obtained results were as follows:1.In the present study,reproductive biology of 32 healthy P.yokohamae captured in Lvshun sea area was researched.P.yokohamae is a kind of single spawning fish.The egg diameter of mature eggs is distributed in 0.80~0.95 mm,and the absolute fecundity F is 0.5~1.5 million eggs.The relative fecundity FL is 10~50 thousand eggs per centimeter,and the relative fecundity Fw is 1~5 thousand eggs per gram.All the preserved sperm refrigerated at 4 ? and 0 ? were inactive after 8 and 11 days later,respectively.2.Our results showed that hatching rate were 36.99% and 93.38% by hatching barrel and bolting cloth net.Meanwhile,survival rate were 41.33% and 82.49%,respectively.3.We found that egg surface of P.yokohamae showed floral designs by observation and record of hatching process.It takes about 216 h from fertilization to larval hatching at the water temperature 11 ?.The embryonic development at different stages is shown in Appendix plate I.4.The effects of starvation on larval morphology,growth and behavior of P.Yokohamae was studied.The results showed that the larvae began to feed after 4 days post hatch.The first feeding rate of starved larvae reached the highest value 100% at 6 days post hatch.At 8 days post hatch,starved larvae reached the point of no return(PNR),and the first feeding rate of starved larvae was 46.78%.The results showed that starvation plays a negative role in the process of growth and development compared with normal larvae,and the hunger tolerance of P.yokohamae larvae is weak.5.The effect of different light intensity on the P.yokohamae larvae feeding was studied.There were obvious feeding peaks at 10:00~12:00 a.m.and 16:00~18:00 p.m.in the natural light group and the artificial continuous illumination group.In total,the feeding rate of P.Yokohamae in the artificial continuous illumination group was higher than that in the natural light group at night.The suitable light intensity of P.yokohamae larvae feeding was 100~600 lx.Before benthic life,the larvae feeding ability was the best when the light intensity was 400 lx.After benthic life,the optimum light intensity for feeding ability gradually shifted from 400 to 100 lx.The feeding efficiency reached the peak level at the first 10 min and decreased with the extension of time.Our results showed that excessive light intensity plays an positive role in the process of growth and development of P.Yokohamae.6.The results of the mRNA expression of key genes(Shh and Hox)in early larval development stage showed that the expression levels of Shh and Hox-d4 in the first 6 days were significantly higher than those in the latter days.Furthermore,Shh gene continuously expressed in the whole stage of larval development,while Hox-d4 gene intermittently expressed.