Shrimp microbiome and immune development in the early life stages.

With its contribution to nutrition, development, and disease resistance, gut microbiome has been recognized as a crucial component of the animal's health and well-being. Microbiome in the gastrointestinal tract constantly interacts with the host animal's immune systems as part of the normal function of the intestines. Interactions between the microbiome and the immune system are complex and dynamic, with the microbiome shaping immune development and function. In contrast, the immune system modulates the composition and activity of the microbiome. In shrimp, as with all other aquatic animals, the interaction between the microbiome and the animals occurs at the early developmental stages. This early interaction is likely essential to the development of immune responses of the animal as well as many key physiological developments that further contribute to the health of shrimp. This review provides background knowledge on the early developmental stage of shrimp and its microbiome, examines the interaction between the microbiome and the immune system in the early life stage of shrimp, and discusses potential pitfalls and challenges associated with microbiome research. Understanding the interaction between the microbiome and shrimp immune system at this crucial developmental stage could have the potential to aid in the establishment of a healthy microbiome, improve shrimp survival, and provide ways to shape the microbiome with feed supplements or other strategies.

Isolation and Characterization of Mannanase-Producing Bacteria for Potential Synbiotic Application in Shrimp Farming.

Prebiotics such as mannan-oligosaccharides (MOS) are a promising approach to improve performance and disease resistance in shrimp. To improve prebiotic utilization, we investigated the potential probiotics and their feasibility of synbiotic use in vitro. Two bacterial isolates, Man26 and Man122, were isolated from shrimp intestines and screened for mannanase, the enzyme for mannan digestion. The crude mannanase from both isolates showed optimal activities at pH 8 with optimum temperatures at 60 °C and 50 °C, respectively. The enzymes remained stable at pH 8−10 for 3 h (>70% relative activity). The thermostability range of Man26 was 20−40 °C for 20 min (>50%), while that of Man122 was 20−60 °C for 30 min (>50%). The Vmax of Man122 against locust bean gum substrate was 41.15 ± 12.33 U·mg−1, six times higher than that of Man26. The Km of Man26 and Man122 were 18.92 ± 4.36 mg·mL−1 and 34.53 ± 14.46 mg·mL−1, respectively. With the addition of crude enzymes, reducing sugars of copra meal, palm kernel cake, and soybean meal were significantly increased (p < 0.05), as well as protein release. The results suggest that Man26 and Man122 could potentially be used in animal feeds and synbiotically with copra meal to improve absorption and utilization of feedstuffs.

Complete Genome Sequences of Mannanase-Producing Bacillus and Niallia Strains Isolated from the Intestine of the Black Tiger Shrimp (Penaeus monodon).

Here, we report the complete genome sequences of mannanase-producing bacteria, namely, Niallia sp. strain Man26 and Bacillus subtilis strain Man122, isolated from the intestine of Penaeus monodon, the black tiger shrimp. Mannanases are used in various industries, such as food, animal feed, and biorefinery, to hydrolyze mannan to oligomers and mannose.

Transcriptomic analysis of the black tiger shrimp (Penaeus monodon) reveals insights into immune development in their early life stages.

With the rapid growth in the global demand, the shrimp industry needs integrated approaches for sustainable production. A high-quality shrimp larva is one of the crucial key requirements to maximize shrimp production. Survival and growth rates during larval development are often criteria to evaluate larval quality, however many aspects of gene regulation during shrimp larval development have not yet been identified. To further our understanding of biological processes in their early life, transcriptomic analysis of larval developmental stages (nauplius, zoea, mysis, and postlarva) were determined in the black tiger shrimp, Penaeus monodon using next-generation RNA sequencing. Gene clustering and gene enrichment analyses revealed that most of the transcripts were mainly related to metabolic processes, cell and growth development, and immune system. Interestingly, Spätzle and Toll receptors were found in nauplius stage, providing evidence that Toll pathway was a baseline immune system established in early larval stages. Genes encoding pathogen pattern-recognition proteins (LGBP, PL5-2 and c-type lectin), prophenoloxidase system (PPAE2, PPAF2 and serpin), antimicrobial peptides (crustin and antiviral protein), blood clotting system (hemolymph clottable protein) and heat shock protein (HSP70) were expressed as they developed further, suggesting that these immune defense mechanisms were established in later larval stages.

A chromosome-level assembly of the black tiger shrimp (Penaeus monodon) genome facilitates the identification of growth-associated genes.

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.

Comparison of the Effects of Microbial Inoculants on Fermentation Quality and Microbiota in Napier Grass (Pennisetum purpureum) and Corn (Zea mays L.) Silage.

Forage preservation for livestock feeding is usually done by drying the plant material and storing it as hay or ensiling it into silage. During the ensiling process, the pH in the system is lowered by the activities of lactic acid-producing bacteria (LAB), inhibiting the growth of spoilage microorganisms and maintaining the quality of the ensiled product. To improve this process, inoculation of LAB could be used as starter cultures to shorten the ensiling time and control the fermentation process. Here, we compared fermentation quality and bacterial dynamics in two plant materials, whole-plant corn (Zea mays L.) and Napier grass (Pennisetum purpureum), with and without starter inoculation. The efficacy of Lactobacillus plantarum, L. brevis, and Pediococcus pentosaceus as starter cultures were also compared in the ensiling system. In whole-plant corn, pH decreased significantly, while lactic acid content increased significantly on Day 3 in both the non-inoculated and LAB-inoculated groups. Prior to ensiling, the predominant LAB bacteria were Weissella, Enterococcus, and Lactococcus, which shifted to Lactobacillus during ensiling of whole-plant corn in both the non-inoculated and LAB inoculated groups. Interestingly, the epiphytic LAB associated with Napier grass were much lower than those of whole-plant corn before ensiling. Consequently, the fermentation quality of Napier grass was improved by the addition of LAB inoculants, especially L. plantarum and a combination of all three selected LAB strains showed better fermentation quality than the non-inoculated control. Therefore, the different abundance and diversity of epiphytic LAB in plant raw materials could be one of the most important factors determining whether LAB starter cultures would be necessary for silage fermentation.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

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.

Bacterial analysis in the early developmental stages of the black tiger shrimp (Penaeus monodon).

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.

Characterization and function of a tachylectin 5-like immune molecule in Penaeus monodon.

Tachylectin5A and its homolog, tachylectin5B both contain a fibrinogen-related domain (FReD) and have been studied in horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5-like genes in shrimp, Penaeus monodon, designated as Penlectin5-1 (PL5-1) and Penlectin5-2 (PL5-2), which both contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites and they are both structurally similar to horseshoe crab tachylectin/carcinolectin5. The PL5-1and PL5-2 transcript were expressed in various shrimp tissues in normal shrimp, and their expression was upregulated in tissues such as hemocytes and hindgut following challenge with pathogenic Vibrio harveyi. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. They have hemagglutination activity on human erythrocyte and bacterial agglutination activity to both Gram negative and Gram positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the GlcNAc-MurNAc cell wall of the peptidoglycan since the binding could be inhibited by GlcNAc or GalNAC. The presence of PL5-2 protein in both circulating hemolymph and intestine, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body, and probably to control and maintain the normal flora in the intestine.

Involvement of a tachylectin-like gene and its protein in pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in the shrimp, Penaeus monodon.

A shrimp disease, the so-called acute hepatopancreatic necrosis disease (AHPND) is caused by a specific strain of Vibrio parahaemolyticus (VP) and it has resulted in significant losses to the global shrimp farming industry. In our previous study, three of tachylectin-like genes were cloned and characterized from the intestine of Penaeus monodon, designated as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). These three genes all contain fibrinogen-related domain (FReD). The expression level of PL5-1, PL5-2 and PL5-3 was elevated in the stomach after oral administration with AHPND-causing V. parahaemolyticus 3HP (VP3HP). A polyclonal antibody to PL5-2 was successfully produced in a rabbit using the purified recombinant PL5-2 as an immunogen, and this because only the predominant protein PL5-2 could be successfully purified from shrimp plasma by affinity chromatography using a N-Acetyl-d-glucosamine column allowed us to perform functional studies of this lectin. The native purified PL5-2 protein had binding and agglutination activities towards VP3HP. To further understand the functions and the involvements of this lectin in response to AHPND in shrimp, RNAi-mediated knockdown of PL5-1, PL5-2 or PL5-3 was performed prior to an oral administration of VP3HP. As a result, Penlectin5-silencing in shrimp challenged with VP3HP showed higher mortality and resulted in more severe histopathological changes in the hepatopancreas with typical signs of AHPND. These results therefore suggest a role for crustacean fibrinogen-related proteins (FRePs) in innate immune response during the development of AHPND, and maybe also during other infections.

Cloning and characterization of a melanization inhibition protein (PmMIP) of the black tiger shrimp, Penaeus monodon.

Melanization is an important component of the innate immune responses in invertebrates and it is essential for defense against invading microorganism. Melanin formation, which is a result of activation of the so called prophenoloxidase activating system, needs to be controlled due to the dangerous effects of quinones and melanin which are produced during the process of melanization. Here, a cDNA for a melanization inhibition protein (MIP), named PmMIP, was identified from the black tiger shrimp, Penaeus monodon by RT-PCR using degenerated oligonucleotide primers and RACE-PCR. The complete sequence significantly matched MIP of the freshwater crayfish Pacifastacus leniusculus (PlMIP). PmMIP contains an N-terminal signal peptide and a fibrinogen related domain (FReD). RT-PCR was applied to examine the expression profiles of PmMIP in various tissues of juvenile P. monodon. PmMIP was expressed in all examined tissues except hemocytes and at very low levels in hepatopancreas and ovaries. The expression of this gene was very low during the larval stages and hardly present in egg and at the nauplius stage. A time-course expression analysis of PmMIP upon Vibrio harveyi challenge at protein levels in plasma was determined. The result shows that MIP protein in plasma was induced at 6 h and disappeared at 12 and 24 h and then the protein reappeared at 48 and 72 h post injection. These results suggest that upon bacterial infection the PmMIP protein is first released from tissues into hemolymph and then degraded to allow melanization to occur for fighting against bacteria.