| Strongylocentyotus intermedius is one of the most economically valuable sea urchin species in northern China,and its breeding has made some progress.However,in the breeding process,there are still many problems in the selection of diets and the development of artificial diets.The requirements of microalgae in the larvae period were strict,and the culture of microalgae is subject to environmental conditions.In the creeping stage,the dominant species of the benthic diatoms are not stable,so those were needed to be re-collected and cultured every year.In the growing period,sea urchins mostly feed on fresh large algae,but kelp and wakame are more expensive,and the supply was subject to seasonal restrictions.Therefore,the selection of the optimum diet at each stage and the development of artificial diet are an important issue in the process of sea urchin breeding.This experiment is aimed at the characteristics of diets in three different stages of life history of sea urchin.Contrast experiments were carried out on sea urchins in the larvae,creeping and growing stages using different microalgae,benthic diatoms and large algae.The effects of different diets on sea urchin growth,fatty acid composition and expression of MYP gene and related fatty acid transforming genes were investigated.The main results are as follows:1.Setting up mechanical flow an significantly increase the growth rate of microalgae cells compared with artificial stirring when Chaetoceros muelleri and Nitzschia closteriumf.minutis-sima are cultured.12W(5000 L/H)compared with 3W and 6W had better growth effects in tank of 200L.The growth density can reach 1.4 million/cell/mL in 6 days,while the control group is still less than 1 million/cell/mL.The study found that there is a significant correlation between microalgae cell density and medium salinity and pH.The density of microalgae cells can be roughly predicted by pH and salinity.2.The larvae fed with C.muelleri and mixed microalgae had the fastest development rate and had better stomach length and width than the larvae fed with D.tetriolecta and I.galbana.Both of them are the most suitable diets for the larvae period in this study.The high EPA/DHA,EPA/ARA ratio and high n-3 PUFA and n-6 PUFA content in the microalgae of this experiment can promote the growth and development of sea urchin larvae.In addition,the accumulation of n-6 and n-3 LC-PUFA and higher ARA/EPA in larvae can improve the status of larvae.The results also indicate that the larvae can be synthesized from α-linolenic acid(18:3n-3)and linoleic acid(18:2n-6)or assimilated from the fatty acid of the diet and accumulate long-chain polyunsaturated fatty acids(LC-PUFA).)3.In this study,eight kinds of benthic diatoms were isolated,purified and identified from mixed diatoms collected from the sea area.Respectively Achnanthes biasolettiana(Kutz.)Grun、Amphora exigua Gregory、Cocconeis scutellum var.parva Grunow、Amphiprora hyalina、Diploneis puella(Schumarm)Cleve、Navicula sp、Nitzschia sp a and Nitzschia sp b.The effects of sea urchin metamorphosis and growth on sea urchin during the creeping period were tested using three of them and collecting mixed diatoms.The effects of four kinds of diatoms on the induction of sea urchin metamorphosis:Amphiprora hyalina>Nitzschia sp a>Achnanthes biasolettiana(Kutz.)Grun>mixture,but there were no significant differences among the four experimental groups,and the metamorphosis rates were all above 70%;On the growth side,the sea urchin fed with Achnanthes biasolettiana(Kutz.)Grun had significantly higher growth rate than the other three kinds of diatom diets,so it could be considered that the Achnanthes biasolettiana(Kutz.)Grun is the best diet in this experiment.It can be seen that the high content of n-3 PUFA and EPA/ARA in the diet can promote the growth of sea urchin during the creeping stage.At the same time,the accumulation of n-6 and n-3 LC-PUFA in sea urchin and the higher ARA/EPA can improve the state of sea urchin.In addition,the accumulation of n-6 and n-3 LC-PUFA and higher ARA/EPA in larvae can improve the status of larvae.The results also indicate that the larvae can be synthesized from α-linolenic acid(18:3n-3)and linoleic acid(18:2n-6)or assimilated from the fatty acid of the diets and accumulate long-chain polyunsaturated fatty acids(LC-PUFA).)4.The experiment found that sea urchins fed with L.japonica and U.pinnatifida in the three ages of sea urchin during the growing period had significant better performance in test diameter,test height,body weight,specific growth rate,gonadal index and gonad color than fed with U.pertusa.However,there is no difference between sea urchins fed with L.japonica and U.pinnatifida,and it can be considered that the best diets in this experiment is Laminaria japonica and U.pinnatifida.Different diets had no significant effect on SOD,T-AOC,CAT,MDA and digestive enzymes of sea urchin in most time.The high content of ARA,EPA and n-3PUFA in the diet can promote the growth of sea urchin,and the accumulation of C18.2N6,C18.3N6 and ARAn-6 PUFA and Total PUFA in the larvae contribute to the growth of sea urchin.Sea urchins can also be synthesized by the synthesis of α-linolenic acid(18:3n-3)and linoleic acid(18:2n-6)or from the fatty acid of the diet and accumulate long-chain polyunsaturated fatty acids(LC-PUFA).5.During the larvae period:the expression level of MYP gene in the larvae fed with C.muelleri and mixed algae was significantly higher than that of D.tetriolecta and I.galbana.In addition,the trend of MYP gene expression in different stages was 6-arm stage>8-arm stage>4-arm stage>Fertilized egg>prism stage.In creeping period,Achnanthes biasolettiana(Kutz.)Grun>Nitzschia sp a>mixture>Amphiprora hyalina.In the intestinal tract of three ages of sea urchin,the MYP expression of the U.pinnatifida group and the L.japonica group was significantly higher than that of the U.pertusa group;the MYP gene of the sea urchin gonads fed with U.pinnatifida in the gonads was significantly higher than that of the L.japonica group.In the U.pertusa group,the expression level first increased and then decreased.In addition to the development of the gonads,the expression of the MYP gene in other stages is closely related to the growth state.The expression of MYP is present throughout the life history of S.intermedius and is expressed in both female and male individuals.6、In terms of the larvae period and the growing period,it can be considered that△Fad6 is the rate-limiting enzyme for the synthesis of long-chain unsaturated fatty acids in the larvae and staging phases of S.intermedius.Although benthic diatoms lack essential fatty acids,larvae can still normalize into C20 and longer C chain unsaturated fatty acids,possibly converting 18:2n-6 and 18:3n-3 into fatty acids and converting them to 18:3n.In addition to the basic pathways of-6 and 18:4n-3,echinoderms have other synthetic pathways.The expression patterns of Elovl4 and Elovl5 in the larvae and pupa sea urchins were basically the same as those in Fad,indicating that low DHA,EPA and n-3 PUFA in the diet promoted the expression of △6Fad and also promoted Elovl4.Elovl5,a synergistic effect between desaturase and elongase.It can be seen from the growing period that a diet rich in C18 unsaturated fatty acids(C18:1n-9/C18:2n-6/C18:3n-3)can promote the expression of △Fad6,Elovl4,Elovl5 genes,and is rich in HUFA.Diet can inhibit its gene expression. |
