Abstract Background The oriental river prawn, Macrobrachium nipponense, is an economically and nutritionally important species of the Palaemonidae family of decapod crustaceans. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag EST dataset for M. Methodology and Principal Findings Total RNA was isolated from eyestalk, gill, heart, ovary, testis, hepatopancreas, muscle, and embryos at the cleavage, gastrula, nauplius and zoea stages. Using pyrosequencing technology, we generated a total of , high quality reads
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Abstract Background The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China, even in whole of Asia. The androgenic gland produces hormones that play crucial roles in sexual differentiation to maleness. This study is the first de novo M. We generated 70,, high quality reads after eliminating adapter sequences and filtering out low-quality reads. A total of 78, isosequences were obtained by clustering and assembly of the clean reads, producing 57, non-redundant transcripts with an average length of In total 70, isosequences were matched to the Nr database, additional analyses were performed by GO 33, , KEGG 17, , and COG analyses 13, , identifying the potential genes and their functions.
A total of 47 sex-determination related gene families were identified from the M. Furthermore, a total of 40 candidate novel genes were found, that may contribute to sex-determination based on their extremely high expression levels in the androgenic compared to other sex glands,. Conclusion Our study provides new sequence information for M. More importantly, this study dramatically improves understanding of sex-determination mechanisms, and advances sex-determination research in all crustacean species.
The huge number of potential SSR and SNP markers isolated from the transcriptome may shed the lights on research in many fields, including the evolution and molecular ecology of Macrobrachium species.
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Introduction The oriental river prawn, Macrobrachium nipponense Crustacea; Decapoda; Palaemonidae , is an important commercial prawn species, that is widely distributed in freshwater and low-salinity estuarine regions in China and other Asian countries  —  with an aquaculture production of , tons annually for aquaculture only . As known, within many other Macrobrachium species, the males grow faster and gain more weight at harvest time than females.
Facing stiff market competition, Macrobrachium producers require improvement in fish production and performance traits to obtain more profit. Thus, culture of all-male populations would be necessary for economic purpose. Therefore, the long-term goals of the M. Some cDNA libraries and transcriptome-level datasets have been generated and serving as a basis for functional genomics approaches aimed at improving the aquaculture performance of this species  — .
However, little information on sex-determination and sex-differentiation related genes in the androgenic gland of M. The androgenic gland is found in most crustacean species, it produces hormones that play crucial roles in sexual differentiation to maleness, including the development of the testes and male sexual characteristics .
Androgenic gland hormone AGH and its relative producers are recognized as important factors when the sex-differentiation and determination mechanisms are concerned, their functions have been widely studied in many crustaceans  — . In Macrobrachium rosenbergii, the males undergoes sex reversal to the females after androgenic gland was ablated from males.
Considering the fact that ablation or implantation of the androgenic gland at a certain stage of development can result in sex reversal to male or female  ,  —  , the expression pattern of androgenic gland hormone genes in crustaceans have received much attention in recent years  — . Previous studies have focused on morphology and anatomy on the M. However, no information to date was reported on genetic underpinnings of the androgenic gland in M.
More novel genes, potentially involved in the sex-determination mechanism, are necessary to be identified from the androgenic gland.
The transcriptome is the total set of transcripts, mRNA, and non-coding RNA, in one or a population of cells during a specific developmental stage or in response to a particular physiological condition using high-throughput technology .
Transcriptome analysis provides the foundation for gene structure and function research and determines when genes are expressed and how they are regulated. The development of next-generation sequencing NGS technologies allows the acquisition of more sequence data per run at a substantially lower cost than in long-read technologies .
To date, there are only 81, expressed sequence tags ESTs from M. In this study, we generate the first M. The EST sequences generated were assembled and annotated with putative functions where possible, and database mining was performed to identify sex-determination and differentiation related genes. A variety of markers potentially useful for genomic population studies including simple sequence repeats SSRs located within coding regions and single nucleotide polymorphisms SNPs detected amongst deep coverage sequence region reads are also reported.
Results and Discussion Transcriptome Analysis To the best of our knowledge, this is the first comprehensive study of the M. In the present study, the transcriptome profile was used to study the sex-determination and differentiation genes in the androgenic gland using next-generation sequencing technologies.
Interestingly, the results revealed that there were genes highly expressed in the androgenic gland, which may be potentially involved in the regulatory mechanism of sex-determination in the oriental river prawn. The reads produced by the Illumina Hiseq were used for clustering and de novo assembly. The reads yielded a total of approximately After eliminating adapter sequences and filtering out the low-quality reads the number of bases in each read was less than 25 bp before de novo assembly by the SeqPreq program, Illumina Hiseq sequencing yielded a total of 70,, Table S1 , Table S2 high-quality transcriptome reads with a total size of 6,,, bp.
Therefore, the raw data were assembled de novo using the Trinity program resulting in 78, contigs, ranging from to 23, bp Table 1. Most of the contigs These 78, isosequences yielded a total of 57, non-redundant transcripts with an average of 1, In prawn, the earliest cDNA libraries were constructed in , based on hemocytes and hepatopancreas from Litopenaeus vannamei and L.
Three cDNA libraries based on the material from the testes, ovaries, and milti-tissues were constructed in previous studies on M. However, only limited numbers of genes were obtained from these three cDNA libraries.
Compared with those studies, a great number of genes were generated in the current study, taking advantage of Illumina Hiseq NGS which can sequence in higher throughput and provide more candidate genes. Besides, the average length of each isosequence is much longer than the previous studies after clustering and de novo assembly in current study, which can promote further studies on these isosequences, including RT- PCR and western-blot.
The 57, non-redundant transcripts in this study provide a transcriptome database for future analyses of sex-determination and sex- differentiation related genes in androgenic gland tissue. Therefore, this transcriptome dataset accelerates the understanding toward the sex-determination mechanisms in M.
Received May 16; Accepted Sep Abstract The structural change of the oviduct of freshwater shrimp Macrobrachium nipponense during spawning was examined by electron microscopy. The oviduct wall structural characteristics seem to be influenced significantly by the spawning process. Before the parturition and ovulation, two types of epithelial cells types I and II are found in the epithelium.