Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp.
ABSTRACT: Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.
Project description:Comparisons of gene expression profiles in hepatopancreas and ovary of domesticated broodstock at different ovarian maturation stages were made through a cDNA microarray in the black tiger shrimp (Penaeus monodon). Differentially expressed genes were identified through the microarray analysis, and the microarray results were confirmed by real-time PCR. Selected genes were further characterized. Overall design: A cDNA microarray was constructed from the EST libraries of P. monodon, consisting of 6826 features. RNA samples were extracted from both hepatopancreas and ovaries of pooled mature broodstock (11 month-old, pooled from n=10 ), individual broodstocks from Surath Thani Provice in Thailand. The RNA from ovaries and hepatopancreases of shrimp at week 0 (before the experiment began) were labeled with Cy3 dye as a reference and those of ovaries and hepatopancreas after treatment (4 weeks of feeding with polychaetes and unilateral eyestalk ablation) were labeled with Cy5 dye.
Project description:Eyestalk ablation is the most common method to induce ovarian maturation in decapod crustacean aquaculture, but it jeopardizes broodstock survival and larvae production. It is important to understand the molecular basis underlying the maturation triggered by ablation and thereby develop an alternative measure for maturation manipulation. In this study, we investigate alterations of ovarian proteome and miRNA profile after ablation in a commercially important marine crab Portunus trituberculatus. Quantitative proteomic analysis using iTRAQ reveals that 163 proteins are differentially expressed following ablation, and modulation of methyl farnesoate metabolism and activation of calcium signaling may play important roles in the ovarian maturation induced by ablation. miRNA expression profiling identifies 31 miRNAs that show statistically significant changes. Integration of miRNA and proteome expression data with miRNA target prediction algorithms generates a potential regulatory network consisting of 26 miRNAs and 30 proteins linked by 71 possible functional associations. The miRNA-protein network analysis suggests that miRNAs are involved in promoting ovarian maturation by controlling expression of proteins related to methyl farnesoate synthesis, calcium signals, and energy metabolism. Experimental validation and temporal expression analysis indicate multiple miRNAs can act synergistically to regulate expression of Farnesoic acid O-methyltransferase and Calmodulin. Our findings provide new insights for elucidating the mechanisms underlying eyestalk ablation-induced ovarian maturation and could be useful for devising an alternative technique for manipulating reproduction in P. trituberculatus and other decapods.
Project description:The study aimed to determine effect of polychaetes as a shrimp feed on male reproductive maturation at transcriptional level through a cDNA microarray in the black tiger shrimp (Penaeus monodon). Thus, the experiment was to compare transcriptomic profiles of two different parts of reproductive organs, namely testes (TT) and vas deferens (VD), of domesticated 17-month-old between two different feeds, namely commercial pellet and polychaetes after feeding for one month. Differentially expressed genes were identified through the microarray analysis, and the microarray results were confirmed by real-time PCR. Selected genes were further characterized. Overall design: A cDNA microarray was constructed from the EST libraries of P. monodon, consisting of 5,973 features. RNA samples were extracted from TT and VD of before feeding (week 0; w0) and after feeding with commercial pellet (C) and polychaetes (P) for one month (week 4; w4) from Shrimp Genetic Improvement Center , Thailand. The RNA from week 0 were labeled with Cy3 dye as a reference and those from week 4were labeled with Cy5 dye.
Project description:Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)-E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA-E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA-GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon.
Project description:BACKGROUND: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. RESULTS: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. CONCLUSIONS: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.
Project description: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, ?-actin, and EF1-?) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-? and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-? were for ovaries from wild-caught broodstock and domesticated groups. EF1-? and ?-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-? and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-? and ?-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-?, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-? is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon.
Project description:Chk1 is a cell-cycle regulator. Chk1 has been identified in organisms ranging from yeast to humans, but few researchers have studied Chk1 in shrimps. We cloned Chk1 from the black tiger shrimp (Penaeus monodon). The full-length cDNA sequence of PmChk1 had 3,334 base pairs (bp), with an open reading frame of 1,455 bp. The complete genomic sequence of PmChk1 (11,081 bp) contained 10 exons separated by nine introns. qRT-PCR showed that PmChk1 was highly expressed in the ovaries and gills of P. monodon. The lowest PmChk1 expression was noted in stage III of ovarian development in P. monodon. PmChk1 expression decreased significantly after injection of 5-hydroxytryptamine and eyestalk ablation in P. monodon ovaries. RNA interference experiments were undertaken to examine the expression of PmChk1, PmCDC2, and PmCyclin B. PmChk1 knockdown in the ovaries and hepatopancreas by dsRNA-Chk1 was successful. The localization and level of PmChk1 expression in the hepatopancreas was studied using in situ hybridization, which showed that data were in accordance with those of qRT-PCR. The Gonadosomatic Index of P. monodon after dsRNA-Chk1 injection was significantly higher than that after injection of dsRNA-GFP or phosphate-buffered saline. These data suggest that PmChk1 may have important roles in the ovarian maturation of P. monodon.
Project description:Comparisons of gene expression profiles between ovaries of before (day 0) and after eyestalk ablation (days 1, 4 and 7) of domesticated 14-month-old black tiger shrimp (Penaeus monodon) were made using a cDNA microarray. Differentially expressed genes were identified through the microarray analysis, and the microarray results were confirmed by real-time PCR. Selected genes were further characterized. A cDNA microarray consisting of 5,568 features was constructed from EST libraries of P. monodon. RNA samples were extracted from the ovaries of before (day 0) and after eyestalk ablation at days 1, 4 and 7 from shrimp from the Shrimp Genetic Improvement Center, Thailand. The RNA was converted into cDNA by indirect aminoallyl-cDNA labelling method (LabelStar Array, Qiagen). aa-cDNA from day 0 samples were coupled with Cy3 dye as a reference, and those from days 1, 4 and 7 were coupled with Cy5 dye.
Project description:Background: Aquaculture of the black tiger prawn Penaeus monodon remains severely constrained by an almost total dependence on wild-caught broodstock. Reliance on wild-caught broodstock stems, for the most part, from reduced reproductive potential of captive-reared females. Reproductive performance of captive-reared females is usually characterised by longer latency period, lower egg production, egg hatch rates and post-larval survivorship compared with their wild-caught counterparts. Improved understanding of the cellular and associated molecular events occurring during peneaid ovarian maturation could therefore be fundamental to improving reproductive success of captive-reared animals. Methodology/Principle Findings: In support of other studies, our histological analyses of developing oocytes revealed differences between wild-caught and captive-reared P. monodon, including reduced lipid accumulation in oocytes of captive-reared animals. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Custom oligonucleotide microarrays were constructed and screened with transcripts derived from the ovary, cephalothorax and eyestalk from animals of all ovarian maturation stages. Ovarian maturation-related differential expression patterns were observed for 111 transcripts, with 53 transcripts displaying differential expression between wild-caught and captive-reared animals. Notably transcripts encoding vitellogenin - the major egg yolk protein precursor, and a lipid storage droplet protein (which we named pmLSD) which is involved in lipid accumulation, were found to be more highly expressed in wild-caught animals. pmLSD transcripts localise to pre-vitellogenic oocytes of wild-caught animals and the pmLSD protein is exclusively localised to the surface of lipid droplets of oocytes at vitellogenic and cortical rod stages. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Target preparation and microarray hybridisation. Ovarian RNA samples from nine wild-caught animals representing six ovarian maturation stages (P, 2, 24, V, R, E) were used in microarray hybridisations. Similarly, RNA samples from three captive-reared animals representing four maturation stages (P, 24, V, E) were used in microarray hybridisations. For wild-caught animals, samples from each ovarian maturation stage were pooled into groups of four and five, enabling two hybridisations. For captive-reared animals, samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage. Importantly, as the four stages for captive-reared animals were (1) pre-ablation pre-vitellogenic, (2) post-ablation pre-vitellogenic, (3) post-ablation vitellogenic, (4) post-ablation vitellogenic with cortical rods, this arrangement allowed for 2 samples of captive-reared pre-vitellogenic and 2 samples of captive-reared vitellogenic, thereby enabling t-tests between samples, while also allowing analysis across the whole 4 stages via cluster analysis. All hybridisations were single channel hybridisations conducted using equal amounts of RNA pooled from each individual.
Project description:With the worldwide demand for tropical penaeid prawn increasing in recent decades, more research on shrimp culture methods is needed to enhance efficiency and profitability for shrimp farmers. The objective of this study was to develop a technique to boost the productivity, feed efficiency, and profitability of the giant tiger prawn (Penaeus monodon). To accomplish this, a novel culture setup was established in which two benthic organisms, a filamentous green alga (Chaetomorpha sp.) and a microsnail (Stenothyra sp.), were propagated together with P. monodon post-larvae during an early culture stage and then offered to shrimp as supplementary live feeds in intensive aquaculture ponds. For the experiment, shrimp post-larvae (density: approximately 33 individuals m-2) were cultured in outdoor concrete ponds (9 × 9 × 1.2 m) under either control (fed only artificial feed, n = 3) or experimental (fed artificial feed and benthic organisms, n = 3) conditions until they reached marketable size (15 weeks). Apparent green algae consumption was 6.81 kg (8.4% green alga to total feed consumption), whereas microsnail consumption was 1.96 kg (2.4% microsnail to total feed consumption). Compared with the control group of giant tiger prawn, the experimental group showed significantly higher productivity (total number of shrimp produced: 118%; total shrimp production: 133%), feed efficiency (feed conversion ratio of artificial shrimp feed: 89%), and profitability (shrimp sales: 139%; balance between shrimp sales and costs: 146%), while labor and financial costs were kept minimal. These results can be explained by the enhanced growth of shrimp at the early stages of culture. The techniques developed in this study will help to advance the efficiency of intensive aquaculture operations for giant tiger prawn and also improve profitability for shrimp farmers.