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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.

The use of probiotics in aquaculture is a practical alternative to promote animal health and disease prevention. Meanwhile, this practice can also reduce the use of prophylactic antibiotics. The purpose of this study was to identify candidate probiotics that could control pathogen populations in host's gastrointestinal (GI) tract and stimulate host immunity in shrimp aquaculture. Bacillus aryabhattai TBRC8450, a bacterial strain isolated from the environment in a shrimp farm, has an antimicrobial activity against many pathogenic strains of Vibrio harveyi and V. parahaemolyticus. Supplementation of B. aryabhattai to Pacific white shrimp (Litopenaeus vannamei) not only decreased the abundance of Vibrio populations, but also shifted the bacterial community in the shrimp GI tract. We found that supplementation of B. aryabhattai triggered shrimp innate immunity and antioxidant activities. mRNA expression of genes encoding microbial peptides and antioxidant enzymes, including C-type lectin, penaeidin-3, heat shock protein 60, thioredoxin, and ferritin, was significantly upregulated in the hepatopancreas of shrimp fed B. aryabhattai. Furthermore, phenoloxidase activity in the hemocytes and the total antioxidant activity in the plasma were increased, indicating enhanced immune and antioxidant responses at the systemic level. In contrast, supplementation of B. aryabhattai had no effect on the total hemocyte count and superoxide dismutase activity in the plasma and hepatopancreas. Importantly, a pathogen challenge test using V. harveyi 1562 showed a significant increase in survival rates of shrimp fed B. aryabhattai compared to the control group. Our findings suggest that B. aryabhattai TBRC8450 can likely be used as a probiotic to reduce the population of V. harveyi in the shrimp GI tract and to enhance shrimp innate immunity and antioxidant capacity for vibriosis resistance in shrimp aquaculture.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

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.

Long-chain polyunsaturated fatty acids (LC-PUFAs) have been shown to promote growth and health of many larval marine animals. Producing high amounts of LC-PUFAs, thraustochytrids are potential alternatives to fish oil as feed supplement for marine larviculture. Their applications have been assessed in many larval aquatic species but not yet in larval and post-larval (PL) shrimp. Here, growth performance and health of Penaeus vannamei PL were evaluated when fed with a thraustochytrid, Aurantiochytrium limacinum BCC52274 (AL), via enrichment in instar-II Artemia. Instar-II Artemia was enriched with AL for 4 h at the ratio of 400,000 individuals: 600 mg of freeze-dried AL per 1l of 30-ppt water. The feeding experiment consisted of treatment groups fed with varied proportions (from 0 to 100%) of the number of meals offering the non-enriched and enriched instar-II Artemia. Additionally, a reference group was included, in which PL were fed only with instar-I Artemia, the stage commonly used in commercial practice. AL enrichment replenished DHA deficiency in Artemia and increased LC-PUFA content in the fed PL. Increased feeding amounts of the enriched Artemia yielded positive effects on increased biomass gain, longer average length and decreased length variation coefficient of the PL (P 0.05). These findings suggest benefits of AL on growth performance, increasing hypo-salinity tolerance and swimming strength of shrimp PL.

Bacterial community associated with the gastrointestinal (GI) tract of aquaculture animals can play important roles in health, nutrition and disease. Compared with the GI tract of aquatic vertebrates such as fish, crustacean GI tract has unique structures and surfaces in different segments that may contribute to differences in the bacterial communities. This study examined the bacterial composition and distribution in different segments along the GI tract and in digesta of wild-caught adult Penaeus monodon using Automated Ribosomal Intergenic Spacer Analysis (ARISA), real-time quantitative PCR and clone libraries of 16S rRNA genes. Thirty-nine bacterial species in four phyla including Proteobacteria (α, β, e, γ), Firmicutes, Bacteroidetes and Actinobacteria were represented in the GI tract of adult P. monodon. Proteobacteria comprised over 80% abundance of the bacterial community in most segments of the GI tract, except the middle intestine that was dominated by Firmicutes (~50% abundance). The results also showed that bacterial communities showed significant differences along the GI tract segments, particularly the hindgut (p

Previous work has shown that non-retroviral endogenous viral elements (EVE) are common in crustaceans, including penaeid shrimp. So far, they have been reported for infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). For the latter, it was shown that shrimp sperm were positive for an EVE of WSSV called EVE366, suggesting that it was heritable, since shrimp sperm (non-motile) do not contain mitochondria. However, to prove this hypothesis that EVE366 was heritable and located in chromosomal DNA, it was necessary to carry out mating tests to show that EVE366 could be detected in parental shrimp and distributed in their offspring in a Mendelian fashion. To do this, we analyzed two shrimp crosses using polyacrylamide gels with a multiple-allele, microsatellite marker Pmo11 as a quality control for single allele detection. In both crosses, all of the shrimp (parents and siblings) were positive for 2 Pmo11 alleles as expected. In Cross 1, the female was PCR-positive for EVE366 while the male was negative, and in Cross 2, both the female and male were PCR-positive for EVE366. Individual analysis of the offspring of Cross 1 revealed a distribution of 1:1 for EVE366, indicating that the EVE366-positive female parent was heterozygous for EVE366. In the second cross, the distribution of EVE366 in the offspring was 3:1, indicating that both PCR-positive parents were heterozygous for EVE366. These results supported the hypothesis that EVE366 was present in shrimp chromosomal DNA and was heritable in a Mendelian fashion. This work provides a model to screen for heritable EVE in shrimp and shows that selection of one parent heterozygous for an EVE and the other negative for it can result in approximately half of the siblings positive and half negative for that EVE as expected. Dividing the siblings of such a cross into an EVE positive group and an EVE negative group followed by challenge with the originating lethal virus should reveal whether or not possession of that specific EVE results in any significant protection against disease caused by the homologous virus.

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research.

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.

A science content course in food chemistry was offered as a 4-day summer workshop from 1999 to 2001 to 4th grade school teachers in the Seattle School District. The objectives of the workshop were to increase the teachers' knowledge of food science, to perform simple experiments that could be used in the 4th grade classroom, and to help the teachers become more familiar with the scientific method. The workshop is described, and challenges associated with running the workshop from the instructors' perspective are discussed. Protocol for hands-on experiments used in the workshop, discussion questions/answers, and comments about each experiment are included in Appendices. The use of hands-on experiments that were easily adaptable to the 4th grade classroom and the comfortable atmosphere for discussion appear to have contributed the most to a successful workshop.

This study was conducted to determine the effects of increasing dietary lipid concentrations on fillet characteristics of post-juvenile rainbow trout. A feeding trial was conducted with fish meal based diets containing 10, 15, 20, 25, or 30% lipid for 24 wk. Menhaden oil was the lipid ingredient. Weight gain was significantly greater in fish fed the 30% lipid diet than in fish fed either 10%, 15% or 20% lipid diets. There were no significant differences in visceral somatic index. Fillet lipid concentration of fish fed the 30% lipid diet (9.2-g lipid/ 100-g fillet) was significantly higher (P < 0.05) than fish fed either the 10% or 15% lipid diets (5.8- and 6.9-g lipid/100-g fillet, respectively). In samples stored for 3 d at 5 C or S wk at -20 C, sensory panelists reported that the cooked fillets from fish fed the 30% lipid diet were “more fishy” than fish fed the 15% lipid diet, and preferred the cooked fillets from the 15% lipid treatment over the 30% lipid treatment. Triangle tests and fillet colorimetry showed no significant differences between fillets from fish fed the 15% and 30% lipid diets at any sampling time point. No significant differences in fillet concentrations of thiobarbituric reactive substances were observed among dietary treatments stored at either 5 C or -20 C. These results suggest that two main effects of feeding a 30% lipid diet (with fish oil as the lipid source) are a higher lipid concentration in the fillet and a “fishier” aroma compared to fillets from fish fed a 15% lipid diet.

The recent trend for rainbow trout feeds to be higher than 15% lipid may improve feed conversion ratios (FCRs) and increase the lipid concentration of the fish. However, one disadvantage of the higher lipid concentration in fish is the potential for increased rates of lipid oxidation, which could affect fish health and contribute to off-flavors in fillets. The objective of this experiment was to determine the effects of adequate and high concentrations of dietary lipid and the antioxidant vitamin E on fish performance, fish health, and fillet product quality of rainbow trout. A 10-week feeding trial was conducted with diets containing either 15 or 30 g lipid/100 g feed, and either 300 (e) or 1500 (E) mg dl-α-tocopheryl acetate/kg feed (15e, 15E, 30e, and 30E). Fish fed 30% lipid diets had significantly higher final body weight, specific growth rate (SGR), and protein efficiency ratio (PER) than fish fed 15% lipid diets. All health parameters of fish measured in the experiment were within normal ranges of healthy fish although several parameters were affected by increasing dietary lipid concentration. Lipid concentration in fish body (whole body, fillets, and viscera) reflected dietary lipid concentration. Increasing the lipid concentration in the feed from 15% to 30% increased the fillet lipid concentration from 8.4 to 9.6 g lipid/100 g fillet. Vitamin E concentrations in fillets reflected dietary vitamin E concentration and ranged from 8.3 mg/kg (30e) to 49.1 mg/kg (15E) (P

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.