Circulating Phylotypes of White Spot Syndrome Virus in Bangladesh and Their Virulence

White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the south-east (Cox’s Bazar) and south-west (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal–Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira) and over the study periods (2017–2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71 ± 0.76 ppt, 3.7 ± 0.1 ppm, 34.11 ± 0.38 °C and 8.23 ± 0.38, respectively, in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at the 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 × 109 copies of WSSV per mL resulted in a median LT50 value of 73 h and 75 h, respectively. The in vivo trial showed higher mean Log WSSV copies (6.47 ± 2.07 per mg tissue) in BD1-challenged shrimp PL compared to BD2 (4.75 ± 0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 h and 62 h of challenge, respectively, with mean Log WSSV copies of 12.06 ± 0.48 and 9.95 ± 0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs), penaeidin and lysozyme expression were lower in the BD1-challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.

Penaeus monodon Interferon Regulatory Factor (PmIRF) Activates IFNs and Antimicrobial Peptide Expression via a STING-Dependent DNA Sensing Pathway

Interferon regulatory factors (IRFs) are transcription factors found in both vertebrates and invertebrates that were recently identified and found to play an important role in antiviral immunity in black tiger shrimp Penaeus monodon. In this study, we investigated the mechanism by which P. monodon IRF (PmIRF) regulates the immune-related genes downstream of the cytosolic DNA sensing pathway. Depletion of PmIRF by double-stranded RNA-mediated gene silencing significantly reduced the mRNA expression levels of the IFN-like factors PmVago1, PmVago4, and PmVago5 and antilipopolysaccharide factor 6 (ALFPm6) in shrimp. In human embryonic kidney (HEK293T) cells transfected with PmIRF or co-transfected with DEAD-box polypeptide (PmDDX41) and simulator of IFN genes (PmSTING) expression plasmids, the promoter activity of IFN-β, nuclear factor (NF-κB), and ALFPm6 was synergistically enhanced following stimulation with the nucleic acid mimics deoxyadenylic–deoxythymidylic acid sodium salt [poly(dA:dT)] and high molecular weight (HMW) polyinosinic–polycytidylic acid [poly(I:C)]. Both nucleic acid mimics also significantly induced PmSTING, PmIRF, and ALFPm6 gene expression. Co-immunoprecipitation experiments showed that PmIRF interacted with PmSTING in cells stimulated with poly(dA:dT). PmSTING, PmIRF, and PmDDX41 were localized in the cytoplasm of unstimulated HEK293T cells and PmIRF and PmDDX41 were translocated to the nucleus upon stimulation with the nucleic acid mimics while PmSTING remained in the cytoplasm. These results indicate that PmIRF transduces the pathogen signal via the PmDDX41–PmSTING DNA sensing pathway to induce downstream production of interferon-like molecules and antimicrobial peptides.

Metagenomic Analysis of Bacterial Communities and Antibiotic Resistance Genes in Penaeus monodon Biofloc-Based Aquaculture Environments

Biofloc technology (BFT) is one of the most promising technologies in global aquaculture for the purpose of improving water quality, waste treatment, and disease prevention in intensive aquaculture systems. However, characterization of the microbial species and antibiotic resistance potentially present in biofloc-based aquaculture environments is needed. In this study, we used high-throughput sequencing technology to comprehensively compare the bacterial communities in mariculture ponds of Penaeus monodon (P. monodon), by testing of water, biofloc, and intestine of P. monodon. Operational taxonomic units (OTUs) cluster analysis showed that the nine samples tested divided into 45 phyla and 457 genera. Proteobacteria was the dominant bacteria in water, biofloc and prawn intestine. In biofloc and intestine, the Ruegeria (2.23–6.31%) genus represented the largest proportion of bacteria, with Marivita (14.01–20.94%) the largest group in water. Microbial functional annotation revealed that in all the samples, genes encoding metabolism were predominant. The antibiotic resistance gene annotation showed the highest absolute abundance of patB, adeF, OXA-243, and Brucella_suis_mprF from Proteobacteria. PatB (11.33–15.01%), adeF (15.79–18.16%), OXA-243 (35.65%), and Brucella_suis_mprF (10.03%) showed the highest absolute abundance of antibiotic resistance genes in water, biofloc, and intestines, respectively. These findings may greatly increase our understanding of the characteristics of the microbiota of shrimp biofloc-based aquaculture systems and the complex interactions among shrimp, ambient microflora, and environmental variables. It provides a reference basis for policy on breeding, environmental safety, and maintaining food safety in the production of P. monodon.

The effect of deproteinization methods on the properties of glucosamine hydrochloride from shells of white leg shrimp (Litopenaeus vannamei) and black tiger shrimp (Penaeus monodon)

ABSTRACT: The effect of methods to remove protein content on the properties of glucosamine hydrochloride from the shells of white leg shrimp (Litopenaeus vannamei) and black tiger shrimp (Penaeus monodon) was investigated. Chitin from shrimp shells was obtained by demineralization in 6% HCl for 12h, deproteinization by two different methods (first group soaked in 8% NaOH for 36h and second group treated in Alcalase enzyme at the concentration of 0.2% for 36h). Two group samples were converted to glucosamine hydrochloride by soaking in 36.76% HCl solution for 5h at 85 °C. The results of fourier transform infrared spectroscopy (FTIR), solubility and recovery yield analysis showed that deproteinization methods did not significantly affect the properties of glucosamine hydrochloride. However, glucosamine hydrochloride from white leg shrimp shells contained higher recovery yield and solubility than black tiger shrimp shells.