| Pigmentation processes occur from invertebrates to mammals.Body wall pigmentation in animals serves many functions,in addition to achieving rare and beautiful coloration.Body color and coloration patterns are important phenotypic traits associated with the survival and reproductive activities in many organisms,including camouflage,thermoregulation,mating selection,social interactions,desiccation resistance,salinity adaptation,and immunity.Furthermore,Pigmentation processes provide a traceable and relevant trait for understanding key issues in evolutionary biology such as adaptation,speciation and the maintenance of balanced polymorphisms.Echinoderms have been a rich source of pigment and pigmentation research for over 125 years.These animals are enormously diverse in color with rich differences even within a species.In addition,they are also basal branching deuterostomes whose sister taxa include chordates.Therefore,many of the genes involved in development,in regeneration,and in pigment formation are more closely related to mammals.Here,we consider pigment formation in Apostichopus japonicus.The sea cucumber Apostichopus japonicus,which has nutritive and medical properties,is considered the most valuable commercial species in many parts of Asia.Compared with the green morph,the purple and white morph are rare and have great appeal to consumers.However,little is currently known about the molecular and genetic mechanism of body color formation in A.japonicus,even in echinoderm.In this study,we used Illumina sequencing,IBT sequencing,LC-QTOF-MS,ChIPseq,real time PCR,in-situ hybridization,immunohistochemistry,genetic breeding,et al.to reveal the molecular and genetic mechanism of body color formation in A.japonicus.Main results are as follows: 1.Comparative study on the pigment type,pigment content and melanocytes of A.japonicusIn this study,we conducted research on pigment composition and melanin content among white,light-green,dark-green,and purple morphs of sea cucumber,A.japonicus.The results of the comparison experiments of pigment types and contents showed that there were fewer types of pigments in white sea cucumber than in the other color morphs;the biological color pigments melanin,astaxanthin,β-carotene,and lutein were detected in light-green,dark-green,and purple sea cucumbers;melanin played the most important role in the formation of body color;the concentration of guanine which belonged to structural color is highest in all color morphs.Transmission electron microscopy analyses revealed that white sea cucumbers had the fewest epidermal melanocytes in the body wall,and their melanocytes contained fewer melanosomes as well as non-pigmented premelanosomes.Sea cucumbers with deeper body colors contained more melanin granules.In the body wall of dark-green and purple sea cucumbers,melanin granules were secreted out of the cell.2.Growth,histology and ultrastructure differences in three pigmentation stages of green,white and purple morphs of A.japonicusIn this study,the growth,histology and ultrastructure of two newly discovered white and purple color morphs were investigated and compared with the common green morph,in the same breeding environment.At 50 days old,the growth and survival rates and the extent of pigmentation were the highest in purple,then green color morphs,and were lowest in white morphs.The white morph had fewer,and less developed,epidermal melanocytes compared with the green and purple color morphs.The results suggested that growth and survival rates were highest in purple sea cucumber and lowest in white sea cucumber.Epidermis thickness and melanin content may be the newfound factors contributed to the differences of growth and survival rate among three color morphs.3.Transcriptome analysis of three sea cucumber color morphs in three pigmentation stagesIn this study,Illumina sequencing was performed to analyze the gene expression dynamic trend of white,green and purple sea cucumbers in three pigmentation stages,and to screen the differential expressed genes related to albinism,pigmentation and body color polymorphism.These results may provide base for systematically analyzing the molecular regulation mechanism of body color formation and albinism of sea cucumbers.Surprisingly,the major enzyme responsible for melanin synthesis and converting tyrosine to DOPA,tyrosinase—a copper containing phenol oxidase—is not detectable yet,neither in the transcriptome data obtained in this study nor the genome data of sea cucumbers.Perhaps this enzyme activity is accomplished by various different genes or is highly variable in sequence in certain taxa.There is also the possibility of exogenous intake of melanin.4.IBT-based quantitative proteomics analysis of purple sea cucumber in three pigmentation stagesIn this study,isobaric tags(IBT)were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber.We analyzed the proteomes of purple sea cucumber in early pigmentation stage(Pa),mid pigmentation stage(Pb)and late pigmentation stage(Pc),resulting in the identification of 5580 proteins,including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb.GO and KEGG analyses revealed possible differentially expressed proteins,including “melanogenesis”,“melanosome”,“melanoma”,“pigment-biosynthetic process”,“Epidermis development”,“Ras-signaling pathway”,“Wntsignaling pathway”,involved in pigment synthesis and regulation in purple sea cucumbers.The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers.Furthermore,these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma,albinism,UV damage,and other diseases in sea cucumbers.5.Metabolome analysis of three sea cucumber color morphs in mid-pigmentation stageIn this study,UHPLC-QTOF/MS technique was performed to analyze the metabolome of white sea cucumber,green sea cucumber and purple sea cucumber in the mid-pigmentation stage.A total of 2633 metabolites were identified.And the differential metabolites potentially related to pigmentation and body color formation were screened out,which lays a foundation for elucidating the related mechanisms of the metabolic process and body color formation mechanism of sea cucumbers.6.Differences of MITF gene in white,green and purple sea cucumbersThe MITF gene is one of the most critical genes in melanocyte development and melanin synthesis pathways.In this study,we conducted in situ hybridization,immunohistochemistry,ChIPseq,real time PCR,Western Blot,and polymorphism analysis experiments.Among them,real time PCR experiment showed that the expression level of MITF mRNA was the highest in purple sea cucumber and the lowest in white sea cucumber;Western Blot experiment showed that the expression level of MITF protein was the highest in purple sea cucumber and the lowest in white sea cucumber;immunohistochemistry experiment showed that the MITF protein was mainly expressed in the epidermis.The in situ hybridization experiment showed that the MITF gene was mainly expressed in the epidermis.Gene polymorphism experiment showed that there were 20 SNPs in exon 1-9,two of which were non-synonymous mutations,and the rest were synonymous mutations.The ChIP-seq experiment screened out DNA sequences that might be bound by MITF.This study comprehensively explained the expression of MITF gene in different color morphs,and screened out the SNP related to body color,which will be applied in the breeding.7.Self-crossing and Hybridization family construction of sea cucumbersIn this study,nine families were constructed using a complete double-column hybrid design.The results showed that,the body color of wild white sea cucumber is not stable and requires continuous breeding.Purple sea cucumber is an ideal material for studying the color inheritance of sea cucumber.There may be hybridization advantages in the growth of sea cucumber,and the superior combinations can be obtained through different hybrid families.The genetic mechanism of sea cucumber body color is affected by maternal traits. |
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