| The olive flounder Paralichthys olivaceus is an important mariculture fish in China,and the research and application of its breeding technology have always been concerned.Chromosome manipulation techniques including artificial induction and breeding of gynogenesis and triploids can be used for mono-sex culture,sex control,or rapid purification of traits.Combined with conventional breeding methods,the techniques could create a new strain and have been important ways of modern genetic breeding.In recent years,Chromosome manipulation techniques have been widely used in many fish species,but mainly in freshwater fish.The related research and application in marine fish are lack and not systematic.In this study,growth and body shape characteristics of the artificially induced meio-/mito-gynogenetic and triploid flounder stocks were clarified through long-term observation and comparison of their morphological traits.By observation under microscope and electron microscope,sex hormone level detection,and transcriptome analysis,the differences of the gonadal development of the triploid and diploid flounder were compared at histocytological,endocrinological,and molecular levels,which showed sterility of the triploid flounder.The researches would provide basic data and references for the application of gynogenesis and triploid of the flounder and other fish in genetic breeding.The main results are as follows:1.The growth and morphological characteristics of the meio-gynogenesis(MEG)and meio-gynogenesis(MIG)flounder stocks induced in 2016 and 2017 at 3,6,9,12,18,and 24 months post hatching(mph)were measured and analyzed,respectively.The results showed that the growth trends of both the MEG and MIG stocks were similar to the control stocks,and the inflection point of growth curve was at 12 mph.They all presented fast growth from 12 to 18 mph(P<0.05).Overall,the MEG fish showed a similar growth with the control diploids,while the MIG fish had a better growth performance to some extent although their growth was quite different.The difference of the specific growth rate in body weight(SGR_w)among different stocks indicated their allometric growth.In terms of the morphological characteristics,different stocks showed different shapes.Compared with the control stock,the body shape of the MEG stock is thinner and longer,while that of the MIG stock is mellower.The frame data indicated that the cumulative contributive proportions of two main principal components extracted with principal component analysis were 79.75%and78.88%in the MIG and MEG stocks at 12 mph,respectively.And the shape dispersion of the MIG stock was higher,which indicated that MIG induction and cultivation are more suitable for screening individuals with specific traits.Therefore,these results revealed that both the MEG and MIG ones have normal or even some advantages on their growth characteristics,which could be used for the flounder breeding.At the same time,gonadal development of the MEG and MIG flounder was studied,and the results showed that although a small part of their ovaries were underdeveloped,most of the ovaries and almost all of the testes of MEG developed normally,thought the proportion of MIG males is low,but there are also individuals who can produce mature sperm.All of them can produce normal eggs or normal sperm.The mature gynogenetic flounder could be used for artificial propagation to obtain the fertile offspring.2.The artificially induced triploid and diploid control flounder stocks,after ploidy identification,were respectively cultured in different culture tanks under similar culture condition.At 6 mph,the triploid and diploid fish were respectively labeled with different fluorescence markers and then mixed for culturing in equal proportion in the same tank.The growth and morphological characteristics of them at3,6,9,12,18,and 24 mph were studied.The results showed that the diploid and triploid flounder of 3–24 mph presented the similar growth patterns both before or during mixed-culture,and there was no significant difference in various growth indexes.In the meantime,observation of the external morphology and histological sections of the gonads at 18,24,and 36 mph of the diploid and triploid flounder were performed.Generally,development of the triploid gonads was severely blocked.In appearance,the gonads were obvious atrophy and not full.Histologically,the gonadal development was retarded and lagged behind the diploid.When the ovaries of the diploid reached stagesⅣ-Ⅴ,the triploid ones were still at stagesⅠ-Ⅱ.And when the testes of the diploid arrived at stageⅤ,the triploid ones were at stageⅣ.According to observation under transmission electron microscope,autophagy and apoptosis occurred,and a large number of apoptotic bodies appeared in both the male and female gonads of the triploid,which may lead to the inhibition of the triploid germ cell development.Although the 36 mph triploid flounder could produce sperm,very few sperm moved under microscope,and the motility was almost not detected.Ploidy test presented that the triploid sperm appeared haploid,haploid–diplid,and triploid,even 4-5 aneuploidy.Further observation by using scanning electron microscope was performed and the results showed that,compared with the diploid sperm,morphology of the sperm in the triploid group was almost abnormal,mainly showing different sizes of the head,deletion of the mitochondrial,and short tail.The levels of the sex hormones estradiol and testosterone in the blood and gonads of the diploid and triploid flounder at 24 mph were detected.The results presented that the levels in the gonads of the triploid females were lower than those of the diploid ones,while the levels of E2 in the triploid testes were higher than those in the diploid ones(P<0.05).The differential expression of genes of the flounder diploid and triploid gonads at 18 and 24 mph was also compared with transcriptome analysis.Transcriptome data showed that the m RNA expression levels of the triploids and diploids are quiet different.Many genes related to spermatogenesis in the triploid testis,such as mns1,dnah1 and dnah5,were down-regulated;and in the triploid ovary,genes related to testicular development,such as amh,amhr2 and wnt4,were up-regulated,which also provided molecular evidence with the testicular-like gonads observed in the histology of the triploid ovary.As for the gonads of the diploid and triploid at 18 and 24 mph,the enriched pathways were concerned with metabolism and cell function,which are related to cell proliferation,differentiation,and migration.These results give the further evidences to explain why the gonads of the flounder triploid are developmental retardation,low fertility,and even infertility. |
PDF Full Text Request