| Anemonefish is not only an important member of the coral reef ecosystem,but also one of the most successfully bred marine ornamental fish.As a group-living fish with a size-based dominance hierarchical social system,and in order to adapt to complex aquatic habitats and produce more offspring,anemonefish has evolved a variety of ecological adaptation strategies.Anemonefish is mutualistic symbiotic with sea anemones,which provide shelter and spawning grounds.Previous studies have shown that the thick mucus layer on the surface of anemonefish could protect fish from being stung by sea anemones,which is the basis of symbiosis.However,whether the mucus on the surface of anemonefish has other physiological and ecological functions remains to be studied.In addition,anemonefish is unique,socially controlled,protandrous sex changer with a monogamous mating system.In general,anemonefish forms a social unit that consists of a monogamous breeding pair and 0-4 non-breeders.The monogamy protandrous is the optimal reproductive mechanism in the natural environment,and the existence of undifferentiated individuals in social unit provides a timely supplement for the loss of broodstock.However,the molecular basis for regulating this reproductive pattern had not been comprehensively analyzed.Therefore,the present study took yellow tail clownfish(Amphiprion clarkii)as the research object to further explore its ecological adaptation mechanism from the perspectives of surface mucus immunity and sex development regulation.Detailed results and main conclusions are listed as follows:1.Analysis of immunological activity of skin mucus derived from A.clarkiiIn this study,the activity of several immune-related enzymes(lysozyme,protease,anti-protease,cathepsin B,alkaline phosphatase and peroxidase)of A.clarkii skin mucus of were analysed.The results showed that the levels of lysozyme,protease,antiprotease and alkaline phosphatase were significantly lower than that of most other marine fish reported.A.clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria,particularly against Vibrio parahaemolyticus and Aeromonas hydrophila.The skin mucus was able to cause bacteria deformation and aggregation,cell membrane rupture and leakage of contents.The bactericidal activity functioned within a short time in a distinct time-and dose-dependent manner,while showing good acid-base tolerance and thermal stability.Meanwhile,A.clarkii skin mucus showed an apparent antiparasitic activity against Cryptocaryon irritans theronts with a distinct dose-and time-dependent relationship.After treated with skin mucus,the theronts quickly stopped their swimming and cilia movement,cells became rounded,cilia shed,small bubbles formed on the surface,cell nucleolus enlarged,cytoskeleton deformed,cell membranes ruptured and cell content leaked out.Therefore,we speculated that there may be some new cationic antibacterial peptide in the mucus of A.clarkii to help it resist the threat of bacteria and parasites,which is crucial for fish to adapt to the social status competition and living environment in the group mode.2.Gonadal development and its relationship with sex steroid hormones in A.clarkiiThe gonads of A.clarkii were bi-lobed and asymmetrical,and the germinal epithelium contained the cell types from all stages of spermatogenesis or oogenesis.The gonads of male A.clarkii can be ovo-testis.The male gonads can be divided into testicular region and the ovarian region.There was no basement membrane can be found between testicular and ovarian region.In testicular region,the scattered primary oocytes can be found embedded inside the testicular tissue,and oocytes in male and female gonads have no morphological difference.There was no special membrane structure or connective tissue can be found between oocytes and spermatids or sperms.In ovarian region,a large number of oocytes in perinucleolus stage or earlier stages can be found,with lobules structures which further develop into testis tissue can be seen between the oocytes.The gonads of female A.clarkii were ovarian structures without the presence of any testicular tissue or sperm cells.The gonadal structure of A.clarkii determined that the direction of sex development can only be from male to female,while female individuals can no longer be reversed into male.The expression level of T was the highest in the undifferentiated stage and increased significantly with the development and maturity of gonads in male stage,while there was also a small amount of expression,but it did not change with the development of the gonads in the female stage.The results indicated that T not only promotes the male gonad development and matureness of A.clarkii,but also promotes the competitive behavior of juvenile fish and the ruling behavior and defensive behavior of female individuals.The expression trend of 11-KT in the undifferentiated and male stages were similar to that of T,so its role may be the same as T during this period.However,unlike T,11-KT was hardly expressed in the female stage,indicating that it has no effect on female behavior and gonadal development.The expression patterns of E2 and DHP were similar to those of most other teleost fish.The expression levels of E2 increased significantly with the development of female gonads,reaching the highest in mature female stage,and there was no significant change during male development process;DHP levels showed two peaks at mature stages of both male and female phases,indicating that E2 has the effect of promoting yolk accumulation,while DHP play a role in the final maturaion of oocytes and sperm cells.The above results indicated that the expression of sex steroid hormone is not only related to gonad development,but also closely related to social group structure,social level and social behavior.3.Transcriptome analysis of gonads at different stages of development in A.clarkiiIn this study,we used RNA-seq technology to investigate transcriptomes of gonads at different developmental stages of A.clarkii.It was found that the gene expression patterns of undifferentiated-phased gonads and male-phased gonads were relatively similar,and the difference were relatively large with that of female-phased gonads,which was consistent with its protandrous development pattern.Differentially expressed genes were mainly involved in biological processes such as metabolism and development,and have various functions such as catalysis,protein binding and transcriptional regulation.Among the differentially expressed genes,we screened out 19 genes that were annotated to participate in the process of sex differentiation and gonadal development,and the expression patterns of multiple genes were significantly different from those of other fish,suggesting that they may perform different physiological function in A.clarkii.Sox8 gene was up-regulated in male stage,which may relate to the development of male gonads;Foxl2,Cypl9a1a,wnt4a,NR5A1a,gnb2,gnb4,gng2,Pgr,lipeb and hsd3b7 genes were up-regulated in female stage,possibly involved in the development of female gonads through regulation of steroidogenesisrelated pathways;Raraa was also up-regulated in the female stage,possibly induced the development of female gonads by binding retinoic acid molecules;Col4a1 and Col6a1 genes were also up-regulated in female stage,possibly related to the maintenance and development of ovarian tissue;Ar and NR0B1 genes were upregulated only in female stage and may be involved in the regulation of female social behavior;the expression profile of skib gene was down-regulated in male stage and significantly up-regulated in female stage.It was speculated that skib gene may induce the development of male and female gonads by regulating the bmp 15 activity;both Amh and tdrp genes were down-regulated and may be involved in the early development of gonadal tissues in A.clarkii.4.Cloning and expression patterns analysis of sex-related gene inA.clarkiiIn this study,we cloned the cDNA sequences of Vasa,Dmrtl,Foxl2 and Foxl3 genes,and investigated the tissue distributions and gonad expression patterns of these four key sex-regulated genes in A.clarkii.The results showed that the Vasa was specifically only expressed in gonadal tissues,and the highest expression level was found in undifferentiated gonads.Vasa signals can be detected in early germ cells,including spermatogonia,primary and secondary spermatocytes,oogonia,oocytes and previtellogenic oocytes.Our results indicated that Vasa do not regulate the development of gonads,and its main function may be invo lved in the regulation of the development and differentiation of early germ cells.Dmrt1 gene was only highly expressed in gonadal tissues,and the highest expression level was observed at mature female-phased gonad,with little or no expression in other tissues.Dmrt1 gene expressed in the spermatogonia,primary and secondary spermatocytes,Sertoli cells,Perinucleolus stage and earlier oocytes in male-phased gonad of A.clarkii,which means that Dmrt1 gene may have important function in the development and maintain of testis.However,Dmrt1 signals can also be detected in both the Cortical Alveoli stage and earlier oocytes in female-phased gonad,which is different from other teleost,suggesting that Dmrt1 gene may also promote the development of A.clarkii female gonads.Foxl2 and Foxl3 were paralogous genes with high expression levels in both gills and gonads.The common feature in gills between Foxl2 and Foxl3 may indicate that they retain the role from their common ancestor in regulating somatic metabolism.Foxl2 gene was expressed only in female gonads,and the expression level of mature femalephased gonad was higher than that of immature female-phased gonad.Foxl2 signals can only be detected in granulosa cells,which is similar to most other teleost,suggesting that Foxl2 may play a role in the differentiation of granulosa cells and the maintenance of ovarian function.In teleost,the study of Foxl3 gene was still limited.In this study,Foxl3 gene was the most highly expressed in immature female-phased gonads,followed by undifferentiated-phased gonads,and there was no significant difference between the other three phases.Foxl3 signals can be detected in spermatogonia,Perinucleolus stage and earlier oocytes.Therefore,we speculated that the Foxl3 gene may be involved in the early differentiation of fish germline stem cells and induce the sexual reversal differentiation of germline stem cells through some feedback mechanism. |
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