| The anchovy Coilia nasus,order Clupeiformes,family Engraulidae,is a rare and valuable migratory fish in the Yangtze River,famous for its fresh-tasting,tender meat.Developments in the aquaculture of C.nasus have resulted in increasing demand for seed and fry,and solutions to overcome problems associated with large-scale seed propagation and breeding of C.nasus are urgently required.However,asynchronous spawning and strong stress responses in C.nasus populations have severely restricted further developments in large-scale artificial breeding.In this study,we investigated the phenomenon of population asynchronous ovarian development and the spawning pattern of C.nasus cultivated in ponds.We used an integrated genomic,transcriptomic,and metabolomics approach to screen important signal transduction pathways and key regulatory genes involved in C.nasus ovarian development to elucidate the mechanism responsible for asynchronous spawning.We also investigated the regulation of ovarian development by exogenous hormones during spawning-induction.The results of this study will help to establish a method for synchronizing ovarian development in C.nasus populations,thus laying the foundations for its large-scale artificial propagation.1.Pond Cultivation and Natural Spawning Pattern of C.nasus Broodstock.We assessed the water temperature,main hydrochemical indices,bait-feeding strategy,and natural spawning patterns of C.nasus broodstock after pond culture,under artificial rearing conditions.The annual water temperature in the breeding ponds ranged from 5?-34?,and the C.nasus broodstock consumed food all year round.Live bait,including dace fry and Caridina nilotica gracilipes,was used as enrichment feed during the rearing,overwintering,and ripening stages.The maturation factor of the female broodstock reached 16.5%after enrichment culture;they were well developed and the overall survival rate was 97.6%.The phenomenon of asynchronous spawning was evident,with spawning lasting from late April to late July,with a peak from May to June.Spawning was concentrated around 20:00-20:30,and the fertilization rate was 80.0%-92.0%.Spawning of C.nasus was closely related to water temperature,with an optimal water temperature for spawning of 20.0?-28.0?.In this study,the C.nasus broodstock were all cultured under the same conditions in terms of nutrients(abundant food source)and the water environment,but their gonadal development was still asynchronous,as described previously,suggesting that asynchronous spawning in C.nasus is mainly caused by genetic rather than environmental factors.2.Whole Genome Sequencing,Assembly,and Annotation of C.nasus.The whole genome of C.nasus was sequenced using a classic shotgun sequencing strategy and Hiseq 2500 sequencing platform,followed by de novo genome assembly.C.nasus gene structure(including gene locus,open reading frames(ORFs),initiators,terminators,introns,exon regions,variable cleavage sites,and protein-coding sequences)was determined and functional information(protein domains and functions,and metabolic pathways)was annotated,and their evolutionary characteristics were analyzed.Sequencing of the genomic library of C.nasus generated a total of 277.92 Gb of sequence data,and 181.74 Gb of high-quality sequence data after filtration.Under 17-mer conditions,the estimated genome size of C.nasus was 714 Mb,with high genome heterozygosity of 1.04%.The total number of assembled contigs was 84,691,and the N50 was 19 kb.The number of contigs longer than 2 kb was 59,201,and the longest contig was about 230 kb.The number of assembled scaffolds was 1,159,and the N50 was about 1.8 Mb.The total assembly length of the scaffolds was about 786 Mb,which was 1.1 times as large as the estimated genome size.The GC content of the C.nasus genome was mainly between 18.6%and 79.8%,and the maximum peak value was at 44.2%,consistent with a normal distribution,demonstrating that the sequencing process was not subject to external contamination.The average sequencing depth of the C.nasus genome was 258x;the maximum depth of base coverage was 300x,and the percentage with a depth of coverage<10× was<5%.The total length of repetitive sequences in the C.nasus genome was about 245.66 Mb,accounting for 31.24%of the genome.GLEAN integration indicated 20,353 coding genes,based on sequence alignment with transcriptome sequences of C.nasus.A total of 18,547 C.nasus genes with defined functions were identified by five annotation platforms,including InterPro,GO,KEGG,Swissprot,and TrEMBL,accounting for 91.13%of the total genes of C.nasus.Only 8.87%of the total genes could not be matched to corresponding functions in currently known databases,indicating that these genes were unique to C.nasus.C.nasus belongs to the Clupeocephala,and is closely related to Electrophorus electricus(order Gymnotiformes)and Danio rerio(order Cypriniformes),while Clupea harengus,which is in the same order(Clupeiformes),differentiated at an earlier time with C.nasus.Internal differentiation within the order Clupeiformes may thus be the main reason for the controversial relationship between Clupeiformes and Otophysi,which do not form a separate monophyletic group.3.Analysis of Population-asynchronous Spawning Mechanism in C.nasus Based on Transcriptomics and Metabolomics.We investigated the mechanisms responsible for regulating asynchronous ovarian development in C.nasus using an integrated transcriptomic and metabolomic approach to identify the key signaling pathways in ovaries at development stages ?,?,and ?.We identified 565 metabolites and 47,049 unigenes.During ovarian development from stage ?to ?,55 metabolites and 830 unigenes were up-regulated and 27 metabolites and 642 unigenes were down-regulated,and 31 metabolites and 1,932 unigenes were significantly up-regulated and four metabolites and 764 unigenes were significantly down-regulated from stage ? to ?.These differentially expressed genes and metabolites were enriched.Forty-three and 50 pathways played important roles in ovarian development from stage ?to ? and from stage ? to ?,respectively.Among these above signaling pathways,39 pathways played important roles in ovarian development from stage ? to ?.Interaction analysis of the metabolites and genes in these pathways revealed three core pathways with key roles in regulating asynchronous ovarian development in C.nasus populations:"squalene and cholesterol biosynthesis","steroid hormone biosynthesis",and "arachidonic acid metabolism and prostaglandin formation".4.Cloning and Regulation of Expression of Prostaglandin E2 Gene in C.nasusProstaglandin E2(PGE2)has a variety of biological activities and participates in various physiological processes.It is synthesized by three consecutive enzymatic reactions,of which the isomerization of PGH2 to PGE2 catalyzed by prostaglandin E synthase(PTGES)is a key step.The PTGES2 gene is a key gene in the pathway of arachidonic acid metabolism and prostaglandin formation,and plays an important role in regulating asynchronous ovarian development in C.nasus.There are two orthologs of PTGES2 in C.nasus,PTGES2a and PTGES2b.The full-length PTGES2a cDNA was 1,575 bp and contained a 1,167-bp ORF that encoded 388 amino acids.The 5' and 3' untranslated regions were 269 bp and 139 bp,respectively.The full-length PTGES2a DNA was 3,222 bp,composed of seven exons and six introns.The full-length PTGES2b cDNA was 1,457 bp and contained a 729-bp ORF encoding 242 amino acids.The 5' and 3' untranslated regions were 402 bp and 326 bp,respectively.The full-length PTGES2b DNA was 1,908 bp,including six exons and five introns.Real Time Quantitative Fluorescence analysis showed that PTGES2a was highly expressed in gills and liver tissues,while PTGES2b was highly expressed in gills and testes.The expression patterns of PTGES2a and PTGES2b were similar and were significantly decreased during gonadal development in C.nasus.However,PTGES2a expression was significantly down-regulated during spawning induction in C.nasus,while expression of PTGES2b was significantly up-regulated.These results suggested that both PTGES2a and PTGES2b played important roles in early gonadal development in C.nasus,while PTGES2b was also important during the spawning process.5.Effect of LHRH-A2 Injection on Spawning Induction in C.nasus and Plasma Biochemical Indices in Female Fish.To further our understanding of the factors affecting artificial propagation of C.nasus,we investigated the effect of LHRH-A2 on spawning induction in C.nasus and plasma biochemical indices in female fish.Cultivated two-winter-old C.nasus were used in these experiments.Spawning was induced by LHRH-A2 injection.Plasma biochemical indices in female C.nasus during the spawning-induction process were determined systematically by radioimmunoassay and chemiluminescence methods.Female C.nasus were injected with 30 ?g/kg LHRH-A2 and males with half that dose.Broodstock mortality was 16.67±3.34%.The effect time in the surviving broodstock population was 21-24 h,the rate of induced spawning was 83.33±3.34%,and the fertilization rate was 75.06±6.19%.Within 21 h after injection of oxytocin,plasma levels of 17?-estradiol,prolactin,thyroxine,cortisol,total protein,and lactate initially increased and then decreased during the spawning-induction process,levels of triiodothyronine,triglycerides,and Cl-decreased continuously,while alanine aminotransferase and aspartate aminotransferase levels,osmotic pressure,and K+and Na+ increased continuously.Comparison of the above plasma indices between spawning and non-spawning fish indicated that spawning was associated with a plasma environment including high concentrations of cortisol,total protein,triglyceride,Na+,K+,and high osmotic pressure,and low concentrations of 17?-estradiol,prolactin,lactate,thyroxine,triiodothyronine,Cl-,alanine aminotransferase,and aspartate aminotransferase compared with levels of these plasma indices in the non-spawning group(P<0.05).These results indicated that the spawning-induction process affected normal liver and kidney functions,which thus inhibited the positive regulation of eggs by LHRH-A2.It has been suggested that the pituitary-thyroid-liver axis may act as a bridge between stress and reproduction in C.nasus,and the success rate of induced spawning could thus be increased by reducing stress and by simultaneous injection of appropriate doses of thyroxine. |
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