Type III secretion system confers enhanced virulence in clinical non-O1/non-O139 Vibrio cholerae.

Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.

An Improved Genome-Scale Metabolic Model of Arthrospira platensis C1 (iAK888) and Its Application in Glycogen Overproduction.

Glycogen-enriched biomass of Arthrospira platensis has increasingly gained attention as a source for bioethanol production. To study the metabolic capabilities of glycogen production in A. platensis C1, a genome-scale metabolic model (GEM) could be a useful tool for predicting cellular behavior and suggesting strategies for glycogen overproduction. New experimentally validated GEM of A. platensis C1 namely iAK888, which has improved metabolic coverage and functionality was employed in this research. The iAK888 is a fully functional compartmentalized GEM consisting of 888 genes, 1,096 reactions, and 994 metabolites. This model was demonstrated to reasonably predict growth and glycogen fluxes under different growth conditions. In addition, iAK888 was further employed to predict the effect of deficiencies of NO3-, PO43-, or SO42- on the growth and glycogen production in A. platensis C1. The simulation results showed that these nutrient limitations led to a decrease in growth flux and an increase in glycogen flux. The experiment of A. platensis C1 confirmed the enhancement of glycogen fluxes after the cells being transferred from normal Zarrouk's medium to either NO3-, PO43-, or SO42--free Zarrouk's media. Therefore, iAK888 could be served as a predictive model for glycogen overproduction and a valuable multidisciplinary tool for further studies of this important academic and industrial organism.

Exploring Components of the CO2-Concentrating Mechanism in Alkaliphilic Cyanobacteria Through Genome-Based Analysis.

In cyanobacteria, the CO2-concentrating mechanism (CCM) is a vital biological process that provides effective photosynthetic CO2 fixation by elevating the CO2 level near the active site of Rubisco. This process enables the adaptation of cyanobacteria to various habitats, particularly in CO2-limited environments. Although CCM of freshwater and marine cyanobacteria are well studied, there is limited information on the CCM of cyanobacteria living under alkaline environments. Here, we aimed to explore the molecular components of CCM in 12 alkaliphilic cyanobacteria through genome-based analysis. These cyanobacteria included 6 moderate alkaliphiles; Pleurocapsa sp. PCC 7327, Synechococcus spp., Cyanobacterium spp., Spirulina subsalsa PCC 9445, and 6 strong alkaliphiles (i.e. Arthrospira spp.). The results showed that both groups belong to ß-cyanobacteria based on ß-carboxysome shell proteins with form 1B of Rubisco. They also contained standard genes, ccmKLMNO cluster, which is essential for ß-carboxysome formation. Most strains did not have the high-affinity Na+/HCO3- symporter SbtA and the medium-affinity ATP-dependent HCO3- transporter BCT1. Specifically, all strong alkaliphiles appeared to lack BCT1. Beside the transport systems, carboxysomal ß-CA, CcaA, was absent in all alkaliphiles, except for three moderate alkaliphiles: Pleurocapsa sp. PCC 7327, Cyanobacteriumstranieri PCC 7202, and Spirulina subsalsa PCC 9445. Furthermore, comparative analysis of the CCM components among freshwater, marine, and alkaliphilic ß-cyanobacteria revealed that the basic molecular components of the CCM in the alkaliphilic cyanobacteria seemed to share more degrees of similarity with freshwater than marine cyanobacteria. These findings provide a relationship between the CCM components of cyanobacteria and their habitats.

Transcriptomic Analysis of Male Black Tiger Shrimp (Penaeus monodon) After Polychaete Feeding to Enhance Testicular Maturation.

To reveal molecular mechanism of how polychaetes enhanced reproductive maturation in the male black tiger shrimp (Penaeus monodon), transcriptomic profiles of male reproductive organs (testes and vas deferens) between polychaete-fed and commercial pellet-fed male brooders were compared using cDNA microarray. The overall profiles were distinguishingly different between the two feed groups as well as between testes and vas deferens. Additionally, six of 11 differentially expressed gene identified by the microarray (HNRPUL1 and GCP4 in testes, MAT2B, CDC16, and CSN5 in vas deferens, and SLD5 in both organs) were validated by quantitative real-time PCR (qPCR) and found to exhibit significantly higher expression levels in polychaete-fed shrimp than those in commercial pellet-fed shrimp. From microarray and qPCR results, the differentially expressed transcripts in both testes and vas deferens between different feeds belonged to DNA replication and microtubule nucleation pathways. Interestingly, while the transcripts involved in nutrient uptake and nucleotide biosynthesis were increased only in testes, those involved in protein refolding and apoptosis were increased only in vas deferens. These findings suggest that polychaetes may enhance spermatogenesis by increasing spermatogonia proliferation in testes and by regulating mature spermatozoa in vas deferens.

Cyanobacterial Biofuels: Strategies and Developments on Network and Modeling.

Cyanobacteria, the phototrophic microorganisms, have attracted much attention recently as a promising source for environmentally sustainable biofuels production. However, barriers for commercial markets of cyanobacteria-based biofuels concern the economic feasibility. Miscellaneous strategies for improving the production performance of cyanobacteria have thus been developed. Among these, the simple ad hoc strategies resulting in failure to optimize fully cell growth coupled with desired product yield are explored. With the advancement of genomics and systems biology, a new paradigm toward systems metabolic engineering has been recognized. In particular, a genome-scale metabolic network reconstruction and modeling is a crucial systems-based tool for whole-cell-wide investigation and prediction. In this review, the cyanobacterial genome-scale metabolic models, which offer a system-level understanding of cyanobacterial metabolism, are described. The main process of metabolic network reconstruction and modeling of cyanobacteria are summarized. Strategies and developments on genome-scale network and modeling through the systems metabolic engineering approach are advanced and employed for efficient cyanobacterial-based biofuels production.

Genome-scale metabolic modeling of Mucor circinelloides and comparative analysis with other oleaginous species.

We present a novel genome-scale metabolic model iWV1213 of Mucor circinelloides, which is an oleaginous fungus for industrial applications. The model contains 1213 genes, 1413 metabolites and 1326 metabolic reactions across different compartments. We demonstrate that iWV1213 is able to accurately predict the growth rates of M. circinelloides on various nutrient sources and culture conditions using Flux Balance Analysis and Phenotypic Phase Plane analysis. Comparative analysis of three oleaginous genome-scale models, including M. circinelloides (iWV1213), Mortierella alpina (iCY1106) and Yarrowia lipolytica (iYL619_PCP) revealed that iWV1213 possesses a higher number of genes involved in carbohydrate, amino acid, and lipid metabolisms that might contribute to its versatility in nutrient utilization. Moreover, the identification of unique and common active reactions among the Zygomycetes oleaginous models using Flux Variability Analysis unveiled a set of gene/enzyme candidates as metabolic engineering targets for cellular improvement. Thus, iWV1213 offers a powerful metabolic engineering tool for multi-level omics analysis, enabling strain optimization as a cell factory platform of lipid-based production.

Bacterial dynamics in intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure.

The intestinal microbiota play important roles in health of their host, contributing to maintaining the balance and resilience against pathogen. To investigate effects of pathogen to intestinal microbiota, the bacterial dynamics upon a shrimp pathogen, Vibrio harveyi, exposures were determined in two economically important shrimp species; the black tiger shrimp (BT) and the Pacific white shrimp (PW). Both shrimp species were reared under the same diet and environmental conditions. Shrimp survival rates after the V. harveyi exposure revealed that the PW shrimp had a higher resistance to the pathogen than the BT shrimp. The intestinal bacterial profiles were determined by denaturing gradient gel electrophoresis (DGGE) and barcoded pyrosequencing of the 16S rRNA sequences under no pathogen challenge control and under pathogenic V. harveyi challenge. The DGGE profiles showed that the presence of V. harveyi altered the intestinal bacterial patterns in comparison to the control in BT and PW intestines. This implies that bacterial balance in shrimp intestines was disrupted in the presence of V. harveyi. The barcoded pyrosequencing analysis showed the similar bacterial community structures in intestines of BT and PW shrimp under a normal condition. However, during the time course exposure to V. harveyi, the relative abundance of bacteria belong to Vibrio genus was higher in the BT intestines at 12h after the exposure, whereas relative abundance of vibrios was more stable in PW intestines. The principle coordinates analysis based on weighted-UniFrac analysis showed that intestinal bacterial population in the BT shrimp lost their ability to restore their bacterial balance during the 72-h period of exposure to the pathogen, while the PW shrimp were able to reestablish their bacterial population to resemble those seen in the unexposed control group. This observation of bacterial disruption might correlate to different mortality rates observed between the two shrimp species. Our findings provide evidence of intestinal bacterial population altered by a presence of the pathogen in shrimp intestines and intestinal bacterial stability might provide colonization resistance against the invading pathogen in the host shrimp. Hence, intestinal microbial ecology management may potentially contribute to disease prevention in aquaculture.

Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp (Penaeus monodon).

The black tiger shrimp (Penaeus monodon) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated P. monodon broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely i) Proteobacteria (Vibrio, Photobacterium, Novosphingobium, Pseudomonas, Sphingomonas and Undibacterium), ii) Firmicutes (Fusibacter), and iii) Bacteroidetes (Cloacibacterium). The shared bacterial members in P. monodon from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of Vibrio and Photobacterium in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp.

Expression of transcripts related to intestinal ion and nutrient absorption in pregnant and lactating rats as determined by custom-designed cDNA microarray.

In pregnancy and lactation, maternal adaptation for the enhancement of intestinal ion and nutrient absorption is of paramount importance for fetal development and lactogenesis. This nutrient hyperabsorption has been reported to result from upregulation of transporter gene expression, in part, under control of lactogenic hormone prolactin (PRL). Since a number of gene families are responsible for ion and nutrient transport in the rat small intestine, we herein developed a custom-designed cDNA microarray (CalGeneArray) to determine the transcriptome responses of duodenal epithelial cells during these reproductive periods, which was subsequently validated by quantitative real-time PCR. We thus designed 277 oligonucleotide probes to detect 113 transcripts related to ion/nutrient transport, bone/calcium metabolism, paracrine regulator, and cell metabolism. Pregnancy was found to upregulate the expressions of several duodenal transporters, e.g., Trpm6, Trpm7, Glut5, and Trpv6. Pregnant rats subjected to 7-day injection of bromocriptine, an inhibitor of PRL release, showed the increased levels of some other transcripts, e.g., insulin-2 and Cyp27b1, compared to untreated pregnant rats. Bromocriptine also increased the mRNA levels of insulin-2, glucose transporter-1 (Sglt1), and Cyp27b1, while decreasing those of Fgfr2c, Atp1b2, and Cldn19 in early lactation. During late lactation, the levels of eight studied transcripts (i.e., NaPi-IIb, Cyp27b1, Cldn18, Casr, Atp1b2, Xpnpep, Pept1, and Trpm7) were altered. In conclusion, the CalGeneArray was powerful to help reveal that pregnancy and lactation modulated the expression of genes related to duodenal nutrient transport and cell metabolism. Our findings supported the physiological significance of PRL in regulating nutrient absorption during pregnancy and lactation.

Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

Development of bacteria identification array to detect lactobacilli in Thai fermented sausage.

To improve the quality and safety of food products, there is a need in the food industry for a reliable method for simultaneously monitoring multiple bacterial strains. Microarray technology is a high-throughput screening approach that can provide an alternative for bacteria detection. A total of 164 bacteria-specific probes were designed from 16S rRNA gene sequences to target 12 bacteria species, including lactic acid bacteria and selected food pathogens. After fabrication onto aminosilane-coated slides, hybridization conditions of the array were optimized for high specificity and signal intensities. The array was applied to detect 12 bacteria individually and was specific to all (Lactobacillus plantarum group, L. fermentum, L. brevis, L. delbrueckii, L. casei, L. sakei, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes) except L. animalis. Multiplex detection using mixed bacteria populations was evaluated and accurate detection was obtained. The feasibility of using the array to detect the target bacteria in food was evaluated with Thai fermented sausages (Nham). Meat samples were collected on days 2, 3 and 7 after natural fermentation, L. plantarum-inoculated fermentation and L. brevis-inoculated fermentation before applying to the array. The naturally-fermented Nham contained L. sakei, L. delbrueckii, L. plantarum and L. fermentum. The L. plantarum-inoculated Nham showed a similar lactic acid bacteria population but the positive signal level for L. plantarum was higher than with natural fermentation. The L. brevis-inoculated Nham contained L. brevis, L. plantarum, L. delbrueckii and L. fermentum. The array was used to monitor bacteria population dynamics during the fermentation process. The naturally-fermented and L. brevis-inoculated samples showed lower positive signal levels of L. plantarum on day 2, but signals gradually increased on days 3 and 7 of the fermentation. In contrast, the L. plantarum-started fermentation showed a higher positive signal level on day 2 than the natural and L. brevis-inoculated samples, and the positive signal level remained high on days 3 and 7. The bacteria identification array was proven to be useful as an alternative method to detect and monitor target bacteria populations during food fermentation.

Systematic identification of Lactobacillus plantarum auxotrophs for fermented Nham using genome-scale metabolic model.

In this study, a systematic strategy is presented, which identifies auxotrophic starters for the popular Thai fermented sausage product, called Nham, using a genome-scale metabolic model. A published genome-scale model of Lactobacillus plantarum WCFS1 is adopted for studying the L. plantarum BCC9546 characteristics cultured on Simulated Nham Broth. Single gene deletion analysis is performed to determine the genes essential for cell growth. Strains lacking such essential genes are considered potential auxotrophic mutants. Then, metabolite supplement analysis is introduced to determine a list of metabolites supplements for each mutant required to restore its growth. Herein, 9 potential auxotrophs are proposed for use in Nham fermentation, along with their metabolite supplements. Simulation studies showed that the secreted fluxes of organic acids, as well as amino-derived flavor compounds of these auxotrophs, are similar to those of the wild-type, indicating that Nham fermented by these auxotrophs would have similar tastes and flavors as Nham fermented by the wild-type. These proposed auxotrophs and corresponding nutritional supplements will be useful for the design of auxotroph starter culture utilized for Nham production in the laboratory. The systematic strategy presented here will facilitate the analysis and development of auxotroph starters used in the food industry.

iAK692: a genome-scale metabolic model of Spirulina platensis C1.

BACKGROUND: Spirulina (Arthrospira) platensis is a well-known filamentous cyanobacterium used in the production of many industrial products, including high value compounds, healthy food supplements, animal feeds, pharmaceuticals and cosmetics, for example. It has been increasingly studied around the world for scientific purposes, especially for its genome, biology, physiology, and also for the analysis of its small-scale metabolic network. However, the overall description of the metabolic and biotechnological capabilities of S. platensis requires the development of a whole cellular metabolism model. Recently, the S. platensis C1 (Arthrospira sp. PCC9438) genome sequence has become available, allowing systems-level studies of this commercial cyanobacterium. RESULTS: In this work, we present the genome-scale metabolic network analysis of S. platensis C1, iAK692, its topological properties, and its metabolic capabilities and functions. The network was reconstructed from the S. platensis C1 annotated genomic sequence using Pathway Tools software to generate a preliminary network. Then, manual curation was performed based on a collective knowledge base and a combination of genomic, biochemical, and physiological information. The genome-scale metabolic model consists of 692 genes, 837 metabolites, and 875 reactions. We validated iAK692 by conducting fermentation experiments and simulating the model under autotrophic, heterotrophic, and mixotrophic growth conditions using COBRA toolbox. The model predictions under these growth conditions were consistent with the experimental results. The iAK692 model was further used to predict the unique active reactions and essential genes for each growth condition. Additionally, the metabolic states of iAK692 during autotrophic and mixotrophic growths were described by phenotypic phase plane (PhPP) analysis. CONCLUSIONS: This study proposes the first genome-scale model of S. platensis C1, iAK692, which is a predictive metabolic platform for a global understanding of physiological behaviors and metabolic engineering. This platform could accelerate the integrative analysis of various "-omics" data, leading to strain improvement towards a diverse range of desired industrial products from Spirulina.

Draft genome sequence of Arthrospira platensis C1 (PCC9438).

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Bacterial community associated with the intestinal tract of P. monodon in commercial farms.

The potentially important roles of intestinal bacteria on immune response, disease resistance, and nutrition for the black tiger shrimp Penaeus monodon have been increasingly investigated. However, so far, little is known about the intestinal bacterial community of the shrimp in the commercial aquaculture settings. In this study, the intestinal bacterial communities of juvenile P. monodon (70 individuals) from eight commercial farms in Thailand were examined using 16S rDNA PCR-DGGE, and seven 16S rDNA clone libraries from representative DGGE profiles were constructed. Bacteria in the γ-Proteobacteria class were the only common bacteria group found in the intestinal tracts of shrimp from all farms. The dominant bacterial genera in the intestinal population of each shrimp varied among different farms, and these genera were Vibrio, Photobacterium, Aeromonas, or Propionigenium (phylum Fusobacteria). Other commonly found genera included Actinomyces, Anaerobaculum, Halospirulina, Pseudomonas, Mycoplasma, and Shewanella. Twelve phyla of bacteria including Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria, Tenericutes, Deinococcus-Thermus, Planctomycetes, Spirochaetes, Synergistetes, Thermotogae, and Verrucomicrobia were represented in the sequences. Additionally, strictly anaerobic bacteria such as Propionigenium and Fusibacter were found. These intestinal bacterial communities varied significantly among different commercial farms and were distinct from their rearing water. The results provide descriptive structures of the intestinal bacterial communities of P. monodon in commercial farms, which can further be applied to areas of research on the immunity, disease resistance, and nutrition of shrimp to improve aquaculture of the black tiger shrimp.

Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp.

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, ß-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 ß-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 ß-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.

Systems biology and metabolic engineering of Arthrospira cell factories.

Arthrospira are attractive candidates to serve as cell factories for production of many valuable compounds useful for food, feed, fuel and pharmaceutical industries. In connection with the development of sustainable bioprocessing, it is a challenge to design and develop efficient Arthrospira cell factories which can certify effective conversion from the raw materials (i.e. CO2 and sun light) into desired products. With the current availability of the genome sequences and metabolic models of Arthrospira, the development of Arthrospira factories can now be accelerated by means of systems biology and the metabolic engineering approach. Here, we review recent research involving the use of Arthrospira cell factories for industrial applications, as well as the exploitation of systems biology and the metabolic engineering approach for studying Arthrospira. The current status of genomics and proteomics through the development of the genome-scale metabolic model of Arthrospira, as well as the use of mathematical modeling to simulate the phenotypes resulting from the different metabolic engineering strategies are discussed. At the end, the perspective and future direction on Arthrospira cell factories for industrial biotechnology are presented.

Insights into eyestalk ablation mechanism to induce ovarian maturation in the black tiger shrimp.

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.