Eyestalk ablation is commonly practiced in crustacean to induce ovarian maturation in captivity. The molecular mechanism of the ablation has not been well understood, preventing a search for alternative measures to induce ovarian maturation in aquaculture. This is the first study to employ cDNA microarray to examine effects of eyestalk ablation at the transcriptomic level and pathway mapping analysis to identify potentially affected biological pathways in the black tiger shrimp (Penaeus monodon). Microarray analysis comparing between gene expression levels of ovaries from eyestalk-intact and eyestalk-ablated brooders revealed 682 differentially expressed transcripts. Based on Hierarchical clustering of gene expression patterns, Gene Ontology annotation, and relevant functions of these differentially expressed genes, several gene groups were further examined by pathway mapping analysis. Reverse-transcriptase quantitative PCR analysis for some representative transcripts confirmed microarray data. Known reproductive genes involved in vitellogenesis were dramatically increased during the ablation. Besides these transcripts expected to be induced by the ablation, transcripts whose functions involved in electron transfer mechanism, immune responses and calcium signal transduction were significantly altered following the ablation. Pathway mapping analysis revealed that the activation of gonadotropin-releasing hormone signaling, calcium signaling, and progesterone-mediated oocyte maturation pathways were putatively crucial to ovarian maturation induced by the ablation. These findings shed light on several possible molecular mechanisms of the eyestalk ablation effect and allow more focused investigation for an ultimate goal of finding alternative methods to replace the undesirable practice of the eyestalk ablation in the future.

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Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, β-actin, and EF1-α) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-α and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-α were for ovaries from wild-caught broodstock and domesticated groups. EF1-α and β-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-α and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-α and β-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-α, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-α is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon.

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BackgroundPoor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation.ResultsIn this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon.ConclusionsWithout a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.

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BackgroundPoor reproductive maturation in captive male broodstock of the black tiger shrimp (Penaeus monodon) is one of the serious problems to the farming industries. Without genome sequence, EST libraries of P. monodon were previously constructed to identify transcripts with important biological functions. In this study, a new version of cDNA microarray, UniShrimpChip, was constructed from the Peneaus monodon EST libraries of 12 tissues, containing 5,568 non-redundant cDNA clones from 10,536 unique cDNA in the P. monodon EST database. UniShrimpChip was used to study testicular development by comparing gene expression levels of wild brooders from the West and East coasts of Thailand and domesticated brooders with different ages (10-, 14-, 18-month-old).ResultsThe overall gene expression patterns from the microarray experiments revealed distinct transcriptomic patterns between the wild and domesticated groups. Moreover, differentially expressed genes from the microarray comparisons were identified, and the expression patterns of eight selected transcripts were subsequently confirmed by reverse-transcriptase quantitative PCR (RT-qPCR). Among these, expression levels of six subunits (CSN2, 4, 5, 6, 7a, and 8) of the COP9 signalosome (CSN) gene family in wild and different ages of domesticated brooders were examined by RT-qPCR. Among the six subunits, CSN5 and CSN6 were most highly expressed in wild brooders and least expressed in the 18-month-old domesticated group; therefore, their full-length cDNA sequences were characterized.ConclusionsThis study is the first report to employ cDNA microarray to study testicular development in the black tiger shrimp. We show that there are obvious differences between the wild and domesticated shrimp at the transcriptomic level. Furthermore, our study is the first to investigate the feasibility that the CSN gene family might have involved in reproduction and development of this economically important species.