| The species in the genus Babylonia(Mollusca,Gastropoda,Neogastropoda,Babyloniidae)are considered to be the most promising economic marine gastropods in the present century,because of their wide distribution along most parts of Asian coastlines,their delicate flavor and their high market reception.Babylonia areolata and B.lutosa are two common species in China,which are good study materials for not only scientific researches on the early development and metamorphosis of gastropods but also artificial breeding technology of the genus Babylonia.In recent years,omics technologies have brought us new approaches to analysis lots of scientific problems with broader and more comprehensive perspectives.Therefore,it is of great significance to combine several omics technologies to investigate the early development in Babylonia and to illuminate deeper mechanisms that remain under cover.In this study,we collected five varieties of Babylonia as research objects,including B.areolata from Hainan,Thailand,the reciprocal hybrid offspring of the two varieties above and B.lutosa.Optical microscope and transmission electron microscope were used to study the ultrastructure of sperms in five varieties.Principal component analysis was conducted to reveal the morphological difference in the measuring data.The results showed that the morphological difference could be used as a basis for classification of the two species of B.areolata and B.lutosa.The reciprocal hybrid offspring of the two B.areolata varieties from Hainan and Thailand had similar ultrastructure of sperms with their parents,though showed preference to female or male parent in the size of detailed structures.This study aimed at comparing the ultrastructure of sperms in five Babyloniavarieties and revealing the difference between varieties.We hope this study could provide fundamental data and theoretical basis for both genetic studies and aquacultural researches of the genus Babylonia.In this study,the Illumina HiSeq sequencing technology was used to conduct transcriptome analysis of the samples from three developmental stages ofB.areolata,including the middle stage and late stage of veliger(MV and LV)and young snail stage(YS).After obtaining the sequence raw data,we used several bioinformatics tools to conduct bioinformatics analysis.Assembly of the clean reads was conducted using the software Trinity.Gene annotation was conducted based on seven databases,including Nr,Nt,Pfam,KOG/COG,Swiss-Prot,GO and KEGG.We used the software RSEM and DESeq to conduct the analysis of gene expression level andgene differential expression.GO and KEGG enrichment analysis were conducted based on GO database and KEGG database.The results showed that 495,795 transcripts and 424,075 unigenes were generated,and 142,593 unigenes were annotated in at least one database.In the result of GO enrichment analysis,the number of genes involved in various metabolic processes were the most abundant,indicating that metabolic processes were active and vigorous during the early development stages of B.areolata.In the result of KEGG enrichment analysis,the number of genes involved in ribosome pathway and lysosome pathway were the most abundant,indicating that protein synthesis and immune response were also active during the studied stages,in order to adapt the rapid development of the larvae and defense the attack from pathogens.In this study,we also used Q-exactive to conduct LC-MS/MS proteomics analysis of the three developmental stages of B.areolata as in the transcriptome analysis.The data was analyzed using used a label-freeproteomics approach,including Maxquant and Perseus algorithm.The differentially expressed proteins were annotated and enriched in GO database and KEGG database,in order to better analyze protein expression during the three stages.We identified 5,583 proteins in total,among which 1,419 proteins showed significant differential expression.In the result of GO enrichment analysis,the number of proteins involved in metabolic' process and cellular process were the most abundant.Those proteins mostly acted with functions such as binding,catalytic activity and transporter activity.In the result of KEGG enrichment analysis,the number of proteins involved in ribosome pathway,carbon metabolism pathway and lysosome pathway were the most abundant,indicating that protein synthesis and immune response were active during three stages,which was consistent with the results of transcriptome.In this study,we used HILICUHPLC-Q-TOF MS technology to conduct metabolomics analysis of four groups ofB.areolata larvae in different nourishment states,including the newly hatched larvae with vitellus capsule(DX2),the larvae that finished absorbing the yolk reserves and just started feeding(DX3),stayed fed for 24 h(DX4)or unfed 24 h(DX5).After obtaining the primary mass spectrometry and secondary mass spectrum data,we usedXCMS to extract peaks and identify metabolites.Quality control experiments showed that the instrument analysis system had good stability and the experimental data was steady and reliable,which indicated that the difference in metabolic spectrums could reflect the biological differences among samples.In the results of principal component analysis,the metabolic spectrums of the larvae in different nourishment states showed disparity from each other.We screened out thedifferentially produced metabolites and conducted clustering analysis and KEGG metabolic pathway analysis of the metabolites.The results showed that 5,793 differentially produced metabolites were identified in the positive ion mode,among which 74 metabolites were described in detail;5,621 differentially produced metabolites were identified in the negative ion mode,among which 62 metabolites were described in detail.In the result of KEGG metabolic pathway analysis,the differentially produced metabolites were mainly from the metabolic pathway,biosynthesis of secondary metabolites,biosynthesis of antibiotics and biosynthesis of amino acids.In this study,we also used proteomic technology to study the growth and survival situation three groups ofB.areolata larvae after exposing to low pH sea water,including veliger larvae before attaching(E1)and its control(C1),veliger larvae after attaching(E2)and its control(C2)and carnivorousjuvenile snail(E3)its control(C3).We identified 720 proteins using two-dimension liquid chromatography tandem mass spectrum,among which 245 proteins can be identified quantitatively.The result showed that the differential proteins between E2 and C2 were 148,and the number between E1 and Cl and E3 and C3 was 57 and 26 respectively.In the result of subcellular localization,the up-regulated proteins in E1 were mainly from cytoplasm,ribosome,myosin complexes and mitochondria;the down-regulated proteins in E1 were mainly from cell nucleus,ribosome and cytoskeleton in cytoplasm.The up-regulated proteins in E2 were mainly from cytoplasm,ribosome,mitochondria,membrane structure and endoplasmic reticulum;the down-regulated proteins in E2 were mainly from cytoplasm,cell nucleus,ribosome and cytoskeleton in cytoplasm.The up-regulated proteins in E3 were mainly from cytoplasm and mitochondria;the down-regulated proteins in E3 were mainly from cytoplasm,cell nucleus,ribosome and extracellular space.This is the first study on the early developmental stages of B.areolata combining three omics technology,including transcriptomics,proteomics and metabonomics,which could fill up the gap of the relevant data resources to a large extent.We hope that this study could provide reference data to related researches of other marine gastropods by comprehensively analyzing the gene and protein expression level and metabolites composition of B.areolata from the omics perspective. |
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