| The oriental river prawn, Macrobrachium nipponense (Crustaccea; Decapoda;Palaemonidae), is an important commercial prawn species with an annual aquaculture production of 205,010 tons and an annual commercial benefits of over 10 billion. The males grow faster and gain more weight at harvest time compared to that of female,thus the all-male population has dramatic application prospect and commercial benefits. The formation of animal sex depends on two processes,including sex-determination and sex-differentiation. Therefore, the realization of sex-determination and sex-differentiation mechanisms is the foundation of sex control and all-male seed population techniques. The androgenic gland is a specific gland in crustacean species, producing hormones, which play crucial roles in sexual differentiation to maleness,including the development of the testes and male sexual characteristics. The histology slice analysis of mature androgenic gland has been performed in many crustacean species, including M. nipponense. Studies on gene expression in androgenic gland were mainly focused on IAG (insulin-like androgenic gland hormone), which has been analyzed in many crustacean species. However, to the best of our knowledge, studies on the developmental process of androgenic gland during the post-larvae prawn, other sex related genes expressed in androgenic gland and their expression patterns were rarely reported.In this study, we are aimed to analyze the differentiation points, developmental process and mature points of androgenic gland and sex glands by using histology slice and steroid hormone detection technique during the post-larvae prawns; the transcriptome and miRNA library were constructed by using Illumina sequencing platform, in order to scientifically find out the sex-related genes and miRNAs in androgenic gland, based on the annotation and expression level in androgenic gland; the sex-related gene functions were analyzed, combined with the histological observation, qRT-PCR analysis in different mature tissues and developmental stages and RNAi:1. Histology observation and sex hormone detection of sex gonad development during post-larval development in M. nipponenseThe differentiation points,development process and mature points of androgenic gland and sex glands during post-larval developmental stages at 30? were analyzed by using histology slice and steroid hormone detection.According to the histology slice analysis, the androgenic gland was developed at post-larvae developmental stage 10 (PL 10) in M. nipponense; the prawns formed the funicular structure with less androgenic gland cells. The androgenic gland undergone 3 developmental stages (proliferating stages, synthesis phase and secretary phase) and matured at PL 19, the androgenic gland forned complete structure. Both of the testis and ovary were developed at PL 13 in M. nipponense. At PL 13, the spermatogonias with irregular arrangement were the first time to be observed. Testis was small. Ovogoniums,differentiated from germinal epithelium, were oval or polygon. The testis undergone 4 developmental stages (spermatogonium stage, spermatocyte stage, sperm cell stage and sperm stage) and matured at PL22, the seminiferous tubule of matured testis was filled with mature spermatozoa; the ovary undergone 4 developmental stages (oogonia stage, primary ovocyte stage, secondary ovocyte stage and ovum stage) and matured at PL 19, the matured ovary was full of ovum. The volume of 17?-E2 was gradually increased from 10.26 pg/pp at PL1 to 19.77 pg/pp at PL 19, and remained stable until PL25, and then the volume gradually decreased; the volume of 17?-T was gradually increased from 33.69 pg/pp at PL1 to 69.27 pg/pp at PL22, and remained stable until PL25, and then gradually decreased.These results indicated that both of the development of androgenic gland and sex glands undergone 10 days, while the development and maturement of androgenic gland was 3 days earlier than that of sex glands. The study provides information for the screening of sex and reproduction related genes and the mechanism of sex control during the developmental process.2. The construction of androgenic gland transcriptome library of M. nipponenseThe transcriptome of androgenic gland was constructed by using Illumina Hiseq2000 sequencing platform. A total of 5 ?g total RNA was used to construct the androgenic gland transcriptome. The raw reads produced 78,408 contigs through de novo assembling,ranged from 351 to 23,217 bp. These 78,408 isosequences produced a total of 57,619 non-redundant transcripts with an average of 1,244.19 bp. Through comparison the obtained isosequences with the non-redundant protein database and nucleotide sequence in NCBI using Blastp and Blastx at an E-value of 10-5, a total of 70,702 non-redundant transcripts were annotated in Nr database, while the other 7,706 unannotated transcripts represent novel genes whose functions have not yet been identified. In addition, a total of 33,203 non-redundant transcripts, 13,817 non-redundant transcripts and 17,9868 non-redundant transcripts were highly homologous with the genes in GO, COG and KEGG database.Based on the functional annotation of non-redundant transcripts, a total of 47 sex-determination and sex-differentiation gene families were identified from the androgenic gland transcriptome of M. nipponense, including IAG, sex-lethal and transformer-2.Through performing the comparative transcriptome analysis with vasa deferentia, ovaries and testes transcriptomes, a total of 40 candidate sex-related genes were found out, which may be involved in the sex-determination and sex-differentiation mechanism in M.nipponense, including 24 specific expressed genes with high expression level in androgenic gland and 16 generally expressed genes which have similar expression pattern (dramatically high expression level in androgenic gland and rare expression in vasa deferentia, ovaries and testes) with IAG. A total of 12,437 SSRs and 65,535 high-confident SNPs were identified in this EST database.3.' The construction of androgenic gland miRNA transcriptome library of M.nipponenseThe androgenic gland miRNA library of M. nipponense was constructed using high-throughput Illumina Solexa analysis. A total of 22,549,954 high-quality reads were obtained, through eliminating the low quality raw reads. The total number of reads with the length of 18-32 nt was 17,574,958. The majority reads (43.96%) were from 20 to 23 nt in length.Through annotation in miRBase database, a total of 1,077 miRNAs, ranged from 1 to 1,766,725. The miRNAs with number of reads, greater than 60,000, include miR-100,bantam, miR-184, miR-315, let-7, miR-315, miR-8-5p, miR-10, miR-8-3p, and miR-12. A total of 8,248, 76,011, and 78,307 target genes were predicted, using the EST and SRA sequences in NCBI, and in the androgenic gland transcriptome of M. nipponense. A total of 48 candidate' sex-related miRNAs were found out, according to the annotation of the target genes.4. Cloning, tissues and temporal-spatial expression, and function analysis of important sex-related genes in M. nipponenseAccording to the gene annotation, seuqence with high homologous with Foxl2 in fish and mammal was chosen from androgenic gland transcriptome of M. nipponense for cloning and gene function analysis, in order to carry out the Foxl2 function in M.nipponense. In addition,Sst and Sti with high expression level in androgenic gland of M.nipponense were chosen for cloning and qRT-PCR analysis, in order to analyze whether they are sex-related genes in M. nipponense.4.1 Cloning, qRT-PCR and gene function analysis of Foxl2 in M. nipponenseThe function of Foxl2 in M. nipponense (Mn-Foxl2) were analyzed by using RACE,qRT-PCR and RNAi techniques.The full-length of Mn-Foxl2 cDNA sequence was 2,097 bp with an open reading frame of 1,740 bp, encoding 579 amino acids. The identity of Mn-Foxl2 amino acid sequence with the Foxl2 amino acid sequences in other fish and mammal species reached to 60%-75%, whereas the coverage was only 20%. The amino acid sequence of Mn-Foxl2 was extremely longer than that of fish and mammal species with a highly-conserved sequence between 245 and 400 amino acids inMn-Foxl2.According to the qRT-PCR analysis in different matured tissues,Mn-Foxl2 was dramatically expressed in testis and androgenic gland. The expression of Mn-Foxl2 in male prawns was extremely higher than that in female prawn in the same tissues,including hearts, intestines, eyestalks, and gills, indicating Mn-Foxl2 may be involved in the male sexual differentiation and development in M. nipponense. The Mn-Foxl2 expressions during different developmental stages indicate that the high expression was observed at PL 10, which is consistent with the testis differentiation point. The above results pridicted that Mn-Foxl2 was involved in the -sex-differentiation and male development in M.nipponense. RNAi resulted in the significant decrease of Mn-Foxl2expression in testes and ovaries,whereas the GSI difference was not observed between the injected and control group. However, the Mn-Foxl2 function needs further research.4.2 Cloning and qRT-PCR analysis of Sst and Sti in M. nipponenseThe function of Sst and Sti in M. nipponense (Mn-sst andMn-sti) were analyzed by using RACE and qRT-PCR techniques. The full length cDNA sequence of Mn-Sst and Mn-Sti was 977 and 1,926 bp, respectively. The cDNA sequences of these two genes were the same from 1 bp to 823 bp, and the differences were from 824 bp. Both of these genes contain an 852 bp open reading frame, encoding 283 amino acid protein. The identity between Mn-Sst and Mn-Sti reached to 95.82%, and the main difference of amino acid sequence was at C-terminal between 269 aa and 283 aa. According to the polygenetic tree analysis, Mn-Sst and Mn-Sti have the closest relationship with slow type of tropomyosin in other crustacean species.Both of the Mn-Sst and Mn-Sti were significantly expressed in androgenic gland, and showed significant difference with that in other tissues. This indicates that these two genes may play essential roles in androgenic gland. The qRT-PCR results during the different developmental stages implies these two genes have other functions except the sex differentiation and development in M. nipponense.This study carried out the differentiation points, developmental process and mature point of androgenic gland and sex glands for the first time by using histological observation and steroid hormone detection techniques,and scientifically find out the candidate sex-related genes and miRNAs in androgenic gland for the first time by the construction of androgenic gland transcriptome and miRNA transcriptome of M. nipponense, providing basic information for the sex-determination and sex-differentiation related studies in M.nipponense, further may advance the studies of sex-determination mechanism in M.nipponense and even the whole crustacean species. |
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