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Microbial colonization is an essential process in the early life of animal hosts—a crucial phase that could help influence and determine their health status at the later stages. The establishment of bacterial community in a host has been comprehensively studied in many animal models; however, knowledge on bacterial community associated with the early life stages of Penaeus monodon (the black tiger shrimp) is still limited. Here, we examined the bacterial community structures in four life stages (nauplius, zoea, mysis and postlarva) of two black tiger shrimp families using 16S rRNA amplicon sequencing by a next-generation sequencing. Although the bacterial profiles exhibited different patterns in each developmental stage, Bacteroidetes, Proteobacteria, Actinobacteria and Planctomycetes were identified as common bacterial phyla associated with shrimp. Interestingly, the bacterial diversity became relatively stable once shrimp developed to postlarvae (5-day-old and 15-day-old postlarval stages), suggesting an establishment of the bacterial community in matured shrimp. To our knowledge, this is the first report on bacteria establishment and assembly in early developmental stages of P. monodon. Our findings showed that the bacterial compositions could be shaped by different host developmental stages where the interplay of various host-associated factors, such as physiology, immune status and required diets, could have a strong influence.

To understand the host-gut microbial relationship, we used multidisciplinary platforms, metagenome, transcriptome, and metabolome analyses, to determine shrimp and intestinal microbial interactions upon 0, 6, 12, 24, and 48 h exposure to 107 CFU/mL of Vibrio harveyi, a shrimp pathogen. The bacterial communities in the intestine of shrimp were disrupted during exposure to V. harveyi. The abundance of Vibrio ASVs in the Harveyi and Vulnificus clades was significantly increased after exposure to the pathogen (6, 12, and 24 h) and decreased later after 48 h. Conversely, Pseudoalteromonas was found in lower abundance (6, 12, and 24 h) but significantly increased after 48 h of the bacterial challenge. Gene expression analysis revealed that genes belonging to several immune-related pathways, including the Toll pathway, the immune deficiency (IMD) pathway, and pattern recognition proteins (PRPs), were significantly upregulated. Early responses in the first 6 h after exposure were the genes involved in phagocytosis, pattern recognition proteins (PRPs), and signal transduction (spätzle and ankyrin). Late responses (12–48 h) were genes related to proteinases and proteinase inhibitors (PPIs), antimicrobial peptides (AMPs), and oxidative stress. Genes related to lipid metabolisms such as fatty acid metabolism and choline metabolism were also upregulated. Metabolomics analysis also showed an increase in phospholipids, including phosphocholine groups, in the intestine of shrimp exposed to the pathogen. Taken together, the gene expression and metabolomics analyses suggest the importance of lipid metabolism in the defense mechanism against bacterial invasion. Moreover, our metabolomic analysis showed a decrease in tryptophan and indole-3-acrylic acid, metabolites related to intestinal immune homeostasis, after bacterial infection. Our observations suggest that pathogenic Vibrio disrupted biological processes in the shrimp intestine, resulting in a decrease in indole-3-acrylic acid and its derivatives which in turn compromised intestinal immunity. In addition, shrimp responded to the bacterial invasion by activating metabolites related to eicosanoid and phospholipid biosynthesis. Our findings on the interactions between shrimp and intestinal microbiota will be an indispensable gateway to systems biology to better understand the function of shrimp gut microbiota and its influence on shrimp innate immunity.

To mitigate disease outbreak, an alternative approach through enhancing shrimp immunity was explored. Mannan oligosaccharides (MOS) have been previously reported to enhance shrimp immune system. Here, coprameal samples were digested with mannanase to yield MOS, namely, mannanase-hydrolyzed coprameal (MCM) and feasibility of MCM as shrimp immunostimulant in grow-out ponds was determined. Pacific white shrimp (Litopenaeus vannamei) were fed with the commercial diet containing 1% MCM as the MCM-supplemented group and compared to the non-MCM control diet. There was no significant difference in survival rates between the MCM-supplemented and the control groups throughout the 4-month-period of the trial (p > 0.05). Gene expression analysis in shrimp intestines revealed that the transcript levels of antimicrobial peptides (anti-lipopolysaccharide factor isoform 1 (alf1), penaeidin (pen3a) and crustin (crus)) and lysozymes (lyz) were not significantly different in the MCM-supplemented group. Meanwhile, C-type lectin and toll-like receptor transcript levels, whose gene products play roles as pattern recognition proteins, were significantly higher in a group fed with MCM for 2- and 4-month periods than those of the control group (p < 0.05). The increased transcript levels of C-type lectin and toll-like receptor provide evidence for potential implementation of MCM as feed supplement to modulate shrimp immune system.

To salvage marine ecosystems from fishery overexploitation, sustainable and efficient aquaculture must be emphasized. The knowledge obtained from available genome sequence of marine organisms has accelerated marine aquaculture in many cases. The black tiger shrimp (Penaeus monodon) is one of the most prominent cultured penaeid shrimps (Crustacean) with an average annual global production of half a million tons in the last decade. However, its currently available genome assemblies lack the contiguity and completeness required for accurate genome annotation due to the highly repetitive nature of the genome and technical difficulty in extracting high‐quality, high‐molecular weight DNA. Here, we report the first chromosome‐level whole‐genome assembly of P. monodon. The combination of long‐read Pacific Biosciences (PacBio) and long‐range Chicago and Hi‐C technologies enabled a successful assembly of this first high‐quality genome sequence. The final assembly covered 2.39 Gb (92.3% of the estimated genome size) and contained 44 pseudomolecules, corresponding to the haploid chromosome number. Repetitive elements occupied a substantial portion of the assembly (62.5%), the highest of the figures reported among crustacean species. The availability of this high‐quality genome assembly enabled the identification of genes associated with rapid growth in the black tiger shrimp through the comparison of hepatopancreas transcriptome of slow‐growing and fast‐growing shrimps. The results highlighted several growth‐associated genes. Our high‐quality genome assembly provides an invaluable resource for genetic improvement and breeding penaeid shrimp in aquaculture. The availability of P. monodon genome enables analyses of ecological impact, environment adaptation and evolution, as well as the role of the genome to protect the ecological resources by promoting sustainable shrimp farming.

Understanding the correlation between shrimp growth and their intestinal bacteria would be necessary to optimize animal’s growth performance. Here, we compared the bacterial profiles along with the shrimp’s gene expression responses and metabolites in the intestines between the Top and the Bottom weight groups. Black tiger shrimp (Penaeus monodon) were collected from the same population and rearing environments. The two weight groups, the Top-weight group with an average weight of 36.82 ± 0.41 g and the Bottom-weight group with an average weight of 17.80 ± 11.81 g, were selected. Intestines were aseptically collected and subjected to microbiota, transcriptomic and metabolomic profile analyses. The weighted-principal coordinates analysis (PCoA) based on UniFrac distances showed similar bacterial profiles between the two groups, suggesting similar relative composition of the overall bacterial community structures. This observed similarity was likely due to the fact that shrimp were from the same genetic background and reared under the same habitat and diets. On the other hand, the unweighted-distance matrix revealed that the bacterial profiles associated in intestines of the Top-weight group were clustered distinctly from those of the Bottom-weight shrimp, suggesting that some unique non-dominant bacterial genera were found associated with either group. The key bacterial members associated to the Top-weight shrimp were mostly from Firmicutes (Brevibacillus and Fusibacter) and Bacteroidetes (Spongiimonas), both of which were found in significantly higher abundance than those of the Bottom-weight shrimp. Transcriptomic profile of shrimp intestines found significant upregulation of genes mostly involved in nutrient metabolisms and energy storage in the Top-weight shrimp. In addition to significantly expressed metabolic-related genes, the Bottom-weight shrimp also showed significant upregulation of stress and immune-related genes, suggesting that these pathways might contribute to different degrees of shrimp growth performance. A non-targeted metabolome analysis from shrimp intestines revealed different metabolic responsive patterns, in which the Top-weight shrimp contained significantly higher levels of short chain fatty acids, lipids and organic compounds than the Bottom-weight shrimp. The identified metabolites included those that were known to be produced by intestinal bacteria such as butyric acid, 4-indolecarbaldehyde and L-3-phenyllactic acid as well as those produced by shrimp such as acyl-carnitines and lysophosphatidylcholine. The functions of these metabolites were related to nutrient absorption and metabolisms. Our findings provide the first report utilizing multi-omics integration approach to investigate microbiota, metabolic and transcriptomics profiles of the host shrimp and their potential roles and relationship to shrimp growth performance.

Shrimp is an important food source consumed worldwide. An intensive aquaculture system with overuse of feed in combination with detrimental effects from climate change are serious problems leading to mass mortality of cultured shrimp. Biofloc technology is an approach to managing water quality and controlling the disease to counter the negative side of intensive culture system, however, most of the biofloc applications are naturally formed, which could be inconsistent. In this study, we employed an established optimal ratio of microbial consortium called “ex-situ biofloc (BF)” to be used as a feed supplement in shrimp cultured in a zero-water discharged system at low salinity conditions. Three feeding groups (100%commercial pellet (C), 95%C+BF, 90%C+BF) of shrimp were cultured for six weeks. The effect of an ex-situ biofloc supplement with commercial pellet reduction showed that levels of ammonium, nitrite, nitrate and phosphate were significantly decreased in water culture. Shrimp fed with ex-situ biofloc supplement with commercial pellet reduction exhibited significantly increased shrimp weight and survival, and significantly expressed growth-related genes involving lipolysis and energy metabolism higher than those fed with 100% commercial pellet. Nutritional analysis indicated a significant increase of docosahexaenoic acid (DHA) and eicosenoic acid (C20:1) concentrations in the ex-situ biofloc supplemented shrimp. This finding revealed the potential of ex-situ biofloc to manage water quality, improve shrimp growth performance and enhance shrimp nutritional value under intensive culture at low salinity conditions. The beneficial effects of the ex-situ biofloc in shrimp culture system make it a promising alternative strategy to mitigate climate change effects leading to the sustainable production of high-quality shrimp in the future.

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.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

The delay in ovarian maturation in farmed black tiger shrimp Penaeus monodon has resulted in the widespread practice of feeding broodstock with the polychaetes Perinereis nuntia and their unilateral eyestalk ablation. Although this practice alters fatty acid content in shrimp ovaries and hepatopancreas, its effects on fatty acid regulatory genes have yet to be systematically examined. Here, microarray analysis was performed on hepatopancreas and ovary cDNA collected from P. monodon at different ovarian maturation stages, revealing that 72 and 58 genes in fatty acid regulatory pathways were differentially expressed in hepatopancreas and ovaries respectively. Quantitative real-time PCR analysis revealed that ovarian maturation was associated with higher expression levels of acetyl-CoA acetyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase 3 and long-chain fatty acid transport protein 4 in hepatopancreas, whereas the expression levels of 15 fatty acid regulatory genes were increased in shrimp ovaries. To distinguish the effects of different treatments, transcriptional changes were examined in P. monodon with stage 1 ovaries before polychaete feeding, after one-month of polychaete feeding and after eyestalk ablation. Polychaete feeding resulted in lower expression levels of enoyl-CoA hydratase and acyl-CoA synthetase medium-chain family member 4, while the expression level of phosphatidylinositide phosphatase SAC1 was higher in shrimp hepatopancreas and ovaries. Additionally, eyestalk ablation resulted in a higher expression level of long-chain fatty acid-CoA ligase 4 in both tissues. Together, our findings describe the dynamics of fatty acid regulatory pathways during crustacean ovarian development and provide potential target genes for alternatives to eyestalk ablation in the future.

Here, we developed a 9-plex bead-based array as a tool to evaluate molecular effects on transcription levels of immune-related genes in the black tiger shrimp (Penaeus monodon). The bead array technology allows simultaneous detection of multiple target genes in a single sample, reducing time, labor and cost. The oligonucleotide probes were designed to target eight immune-related genes that involve in antimicrobial activity, melanization, pathogen pattern recognition proteins, lysozyme and one housekeeping gene as an internal control. The nine probes were coupled to carboxylated-magnetic bead sets. The 9-plex PCR primers were designed and optimized for conditions to allow multiplex detection. The specificity of the assay was validated and the sensitivity was determined to be 103 copies/μL for all target genes. The 9-plex immune gene expression assay was applied to determine transcript levels in gills of P. monodon under exposure to a shrimp pathogen, Vibrio harveyi, and gene expression patterns were consistent to patterns observed under a traditional realtime PCR method. While realtime PCR method gave a better sensitivity but limited multiplexity, our 9-plex immune gene expression assay was able to simultaneously measure expression of multiple target genes, providing useful alternative assay in the need of higher-throughput gene expression analysis such as evaluation of immune stimulatory effects in different feed additives under various dosages and time points in shrimp.

This study investigated the effects of dietary mannooligosaccharides (MOS) from copra meal as a dietary feed additive on growth performance, disease resistance, and immune enhancement of the Pacific white shrimp Litopenaeus vannamei . Experimental shrimp were fed with diets supplemented with 2, 3 and 4 g MOS per kg of pellet feed for 6 weeks prior to evaluation of their growth performance, pathogen resistance to Vibrio harveyi and innate immune responses. No significant differences in shrimp average weight and feed conversion ratios were found among all diet groups ( p > 0.05). While phagocytic indices and respiratory burst activities were not significantly different either, bactericidal activities were higher in the MOS-supplemented diet groups than the control group. Furthermore, when all diet groups were challenged with V. harveyi , the groups fed with MOS-supplemented diets showed significantly higher survival rates than the control group on day 3 ( p

Sand polychaetes (Perinereis nuntia) have been used as a live feed to boost reproductive maturation before mating in hatchery farms in Thailand. However, no study has evaluated the effect of polychaetes on performance and physiology of the sperm of the domesticated black tiger shrimp (Penaues monodon). Therefore, this study aims to study the effects of sand polychaetes on growth, survival, reproductive performance and sperm physiology and morphology of domesticated male broodstock P. monodon. After feeding with either polychaetes or commercial pellets for four weeks, growth and survival rates of polychaete-fed group were significantly higher than those of pellet-fed group. Spermatophore weight and total sperm counts of the polychaete-fed group were significantly higher than those of the pellet-fed group at Weeks 3 and 4, whereas % abnormal sperm cells and % acrosome reaction of polychaete-fed group were significantly lower and higher than those of pellet-fed group only at Week 4, respectively. Physiology of spermatophores in the polychaete-fed group was white and opaque while that of the pellet-fed group was pigmented. Morphological changes of sperm revealed less abnormal sperms (misshaped half, misshaped head, and misshaped tails) in the polychaete-fed group. Nutritional analysis revealed that polychaetes have significantly higher total protein, fat contents and essential fatty acids (arachidonic and eicosapentaenoic acids) but significantly lower fiber content. Moreover, histological analysis of hepatopancreas revealed more vacuoles, which are storages for glycogen and lipids in the polychaete-fed group than in the pellet-fed group. Therefore, this study provides an evidence that polychaetes provide benefits to growth, survival and sperm performance for boosting reproductive maturation in male domesticated broodstock Penaeus monodon.

The black tiger shrimp (Penaeus monodon) is an aquatic animal with considerable economic importance. Poor reproductive maturation in captivity impedes sustainable aquaculture production of this species. This study aims to provide transcriptomic information on reproductive organs using 454 pyrosequencing technology. The transcriptome analysis of ovaries and testes revealed 41,136 transcripts with 20,192 contigs. We found novel sets of transcripts completing several important reproductive pathways such as gonadotropin-releasing hormone (GnRH) signaling and progesterone-mediated oocyte maturation. In addition, we found transcripts encoding for receptors crucial for initiation of the maturation process, such as GnRH receptor (GnRHR), voltage-dependent calcium channel L type alpha-1C (CACNA1C) and epidermal growth factor receptor (EGFR). Moreover, we found a putative novel vigillin encoding for an estrogen–induced polysome-associated protein, which has not been reported in penaeid shrimp. These results suggest that the regulatory mechanism of the pathways important to reproductive maturation might be similar to those in the vertebrate. The obtained data will consequently accelerate the study of reproductive biology of this important species to ensure a sustainable shrimp farming industry.

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.

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.

In all previous studies, to study shrimp immune response, bacteria were directly injected into the shrimp body and as a consequence the initial step of a natural interaction was omitted. In this study we have instead used an immersion technique, which is a more natural way of establishing an infection, to study immune responses in black tiger shrimp (Penaeus monodon). Normally, Vibrio harveyi (Vh) is highly pathogenic to post-larval shrimp, but not to juveniles which usually resist an infection. In post-larvae, Vh causes a massive destruction of the digestive system, especially in the hepatopancreas and in the anterior gut. We have therefore investigated changes in transcription levels of fifteen immune-related genes and morphological changes in juvenile shrimp following an immersion of shrimp in Vh suspension. We found that a pathogenic bacterium, Vh, has the capacity to induce a local expression of some immune-related genes in shrimp after such a bacterial immersion. Our results show that in the juvenile gut small changes in expression of the antimicrobial peptide (AMP) genes such as antilipopolysaccharide factor isoform 3, crustin and penaeidin were observed. However some other genes were more strongly induced in their expression compared to the AMP genes. C-type lectin, Tachylectin 5a1 and mucin-like peritrophic membrane were increased in their expression and the C-type lectin was affected most in its expression. Several other examined genes did not change their expression levels. By performing histology studies it was found that Vh infection induced a strong perturbation of the midgut epithelium in some regions. As a consequence, the epithelial cells and basement membrane of the infected site were completely damaged and necrotic and massive hemocyte infiltration occurred underneath the affected tissue to combat the infection.

The depletion of three banned nitroimidazole drugs – dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) – was investigated in black tiger shrimp (Penaeus monodon) following in-water medication. The highest concentrations of residues were measured immediately after the 24-h immersion (d0). At this time, MNZ and MNZ-OH residues were measured in shrimp tissue samples at concentrations ranging from 361 to 4189 and from 0.28 to 6.6 μg kg−1, respectively. DMZ and its metabolites HMMNI ranged in concentration between 31 509 and 37 780 and between 15.0 and 31.9 μg kg−1, respectively. RNZ and HMMNI concentrations ranged from 14 530 to 24 206 and from 25.0 to 55 μg kg−1, respectively. MNZ, DMZ and RNZ were the more persistent marker residues and can be detected for at least 8 days post-treatment. MNZ-OH was only detectable on d0 following treatment with MNZ. HMMNI residues were only detectable up to d1 (0.97–3.2 μg kg−1) or d2 (1.2–4.5 μg kg−1) following DMZ and RNZ treatment, respectively. The parent drugs MNZ, DMZ and RNZ were still measureable on d8 at 0.12–1.0, 40.5–55 and 8.8–18.7 μg kg−1, respectively. The study also investigated the stability of nitroimidazole residues under various cooking procedures (frying, grilling, boiling, and boiling followed by microwaving). The experiments were carried out in shrimp muscle tissue containing both high and low concentrations of these residues. Different cooking procedures showed the impact on nitroimidazole residue concentration in shrimp tissue. Residue concentration depleted significantly, but partially, by boiling and/or microwaving, but the compounds were largely resistant to conventional grilling or frying. Cooking cannot therefore be considered as a safeguard against harmful nitroimidazole residues in 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, 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.

Background Poor 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). Results The 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. Conclusions This 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.

A sudden increase in temperature results in heat shock stress of the cultured shrimp. To cope with the stress, shrimp has to overcome by triggering a response known as heat shock response. To understand the heat shock response in the black tiger shrimp (Penaeus monodon), we examined expression patterns and distribution of three heat shock protein (hsp) genes in P. monodon juveniles. The expression levels of hsp21, hsp70 and hsp90 were determined by quantitative real-time PCR in nine tissues (gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocyte) under untreated and heat shock conditions. Under untreated condition, all three hsp genes were differentially expressed in all examined tissues where the hsp70 transcript showed the highest basal level. Under heat shock condition, only hsp90 was inducible in all nine tissues when comparing to its untreated level. The time-course induction experiment in gill and hepatopancreas revealed that the transcriptional levels of hsp21, hsp70 and hsp90 were inducible under the heat shock condition and in time-dependent manner. To determine the response of the hsp genes upon bacterial exposure, we further determined transcript levels of the hsp genes in gill of P. monodon after Vibrio harveyi injection. The expression levels of hsp70 and hsp90 were significantly increased after a 3-h exposure to V. harveyi where the hsp21 transcript was induced later after a 24-h exposure. This evidence suggests for putative roles and involvement of the hsp genes as a part of immunity response against V. harveyi in P. 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.

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.

Disease outbreak has continued to be a major challenge for shrimp aquaculture industry. In recent years, manipulation of gut microbiota has been shown to improve the health of host shrimp and disease resistance. Therefore, understanding how various factors can shape the gut microbiota of shrimp is crucial for future utilization of gut microbiota as tools for disease controls. The salinity of the rearing environment can potentially affect shrimp gut microbiota as well as the host-microbe interactions. The effects of salinity adaptation on intestinal microbiota and transcriptomes of juvenile black tiger shrimp Penaeus monodon were examined in this experiment. Intestinal microbiota and transcriptomic profiles from juvenile black tiger shrimp of the same cohort adapted to three different salinity levels were examined using 16S rDNA amplicon sequencing and RNA sequencing, respectively. Shrimp were acclimatized from the original 20 ppt salinity to either 10 ppt or 30 ppt over a period of 10 days, and intestinal samples were collected on Day 0, Day 10, and Day 20 after reaching the target salinity. Across all salinity levels, the dominant phyla composition in the intestine of shrimp was relatively similar with Proteobacteria (83.4%), Bacteroidetes (8.1%), Planctomycetes (3.2%), Verrucomicrobia (2.5%), and Firmicutes (1.5%). The most abundant genus was Vibrio. While it can be found at all salinity levels, a relative abundance of Vibrio was lower at 10 ppt than at higher salinity. A shift toward the higher relative abundance of Vibrio ASVs belonging to the Harveyi clade was observed at higher salinity. On the other hand, the relative abundance of Shewanella was higher at 10 ppt than at higher salinity. Other dominant genera in the intestines not affected by the change in salinity were Pseudoaltermonas and Tenacibaculum. The shift in the rearing water microbiota with different salinity levels was also observed. The most abundant intestinal transcripts responding to different salinity levels based on GO classification were intracellular anatomical structure (cellular component), protein binding (molecular function), and organic substance metabolic process (biological function). Genes involved with stress and immune responses were differentially expressed, and correlations between potentially pathogenic Vibrio and genes relating to innate immunity and peritrophic matrix were observed. The results confirmed the influence of rearing water salinity on both the intestinal microbiota and transcriptomes of shrimp. Efforts to shape the microbiota of aquatic animals should take into consideration the rearing salinity to ensure its efficacy. Using multi-omic platform allowed for better understanding of the microbiota and the host-microbe interaction, which will help support the sustainability of aquaculture production.