Differential responses of hepatopancreas transcriptome between fast and slow growth in giant freshwater prawns (Macrobrachium rosenbergii) fed a plant-based diet.

Efficient utilisation of plant-based diets in the giant freshwater prawn, Marcrobrachium rosenbergii, varies according to individual, suggesting that it might be associated with differences in physiological and metabolic responses. Therefore, we aimed to investigate the individual differences in the growth response of shrimp fed to a soybean-based diet (SBM). Two hundred shrimp were fed SBM for 90 days, and specific growth rate (SGR) was determined individually. Fast- and slow-growing shrimp (F-shrimp vs. S-shrimp), with the highest and lowest 5% SGRs, respectively, were sampled to determine haemolymph chemistry and carcass composition. The hepatopancreas of these shrimps were used for transcriptome analysis through RNA sequencing (RNA-Seq). The results showed no significant differences in haemolymph chemistry parameters. In terms of carcass proximate composition, F-shrimp exhibited higher protein composition than did S-shrimp, suggesting that F-shrimp have higher protein anabolism. Using RNA-seq and real-time reverse transcription polymerase chain reaction (qRT-PCR), the expression levels of several genes encoding physiologic and metabolic enzymes were found to be upregulated in F-shrimp compared to in S-shrimp, suggesting that these enzymes/proteins mediated the efficient use of SBM-based diets for growth promotion in shrimp. Various DEGs associated with the immune system were observed, indicating a difference in immune processes between F- and S-shrimp. The expression of several housekeeping genes was found to be upregulated in S-shrimp. Collectively, the upregulated expression of several enzymes associated with physiological and/or metabolic processes and increased protein anabolism may be attributed to the efficient use of SBM for maximal growth in shrimp.

Evaluation of Bacillus spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (Litopenaeus vannamei) Farms.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75-95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80-95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly.

Combining segments 9 and 10 in DNA and recombinant protein vaccines conferred superior protection against tilapia lake virus in hybrid red tilapia (oreochromis sp.) compared to single segment vaccines.

Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 105 TCID50 per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.

Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp.

Acute hepatopancreatic necrosis disease (AHPND), also called early mortality syndrome (EMS), is a recently emergent shrimp bacterial disease that has resulted in substantial economic losses since 2009. AHPND is known to be caused by strains of Vibrio parahaemolyticus that contain a unique virulence plasmid, but the pathology of the disease is still unclear. In this study, we show that AHPND-causing strains of V. parahaemolyticus secrete the plasmid-encoded binary toxin PirAB(vp) into the culture medium. We further determined that, after shrimp were challenged with AHPND-causing bacteria, the bacteria initially colonized the stomach, where they started to produce PirAB(vp) toxin. At the same early time point (6 hpi), PirB(vp) toxin, but not PirA(vp) toxin, was detected in the hepatopancreas, and the characteristic histopathological signs of AHPND, including sloughing of the epithelial cells of the hepatopancreatic tubules, were also seen. Although some previous studies have found that both components of the binary PirAB(vp) toxin are necessary to induce a toxic effect, our present results are consistent with other studies which have suggested that PirB(vp) alone may be sufficient to cause cellular damage. At later time points, the bacteria and PirA(vp) and PirB(vp) toxins were all detected in the hepatopancreas. We also show that Raman spectroscopy "Whole organism fingerprints" were unable to distinguish between AHPND-causing and non-AHPND causing strains. Lastly, by using minimum inhibitory concentrations, we found that both virulent and non-virulent V. parahaemolyticus strains were resistant to several antibiotics, suggesting that the use of antibiotics in shrimp culture should be more strictly regulated.

Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

Draft Genome Sequences of Six Strains of Vibrio parahaemolyticus Isolated from Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease Shrimp in Thailand.

Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus.

BACKGROUND: Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome. RESULTS: Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences. CONCLUSIONS: Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.