Tze Hann Ng

An Invertebrate Warburg Effect: A Shrimp Virus Achieves Successful Replication by Altering the Host Metabolome via the PI3K-Akt-mTOR Pathway

In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the viruss requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

Shrimp hemocytes release extracellular traps that kill bacteria

Extracellular traps (ETs) are formed from the DNA, histones and cytoplasmic antimicrobial proteins that are released from a range of vertebrate immune-cells in response to pathogenic stimulation. This novel defense mechanism has not been demonstrated in invertebrates. In this study, we investigated the formation of ETs in the crustacean Litopenaeus vannamei. We found that stimulation of shrimp hemocytes with phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and live Escherichia coli all led to the formation of the characteristic ET fibers made from host cell DNA. After E. coli stimulation, we found that histone proteins were co-localized with these extracellular DNA fibers. The results further showed that E. coli were trapped by these ET-like fibers and that some of the trapped bacteria were permeabilized. All of these results are characteristic of the ETs that are seen in vertebrates and we therefore conclude that shrimp are also capable of forming extracellular traps.

The DNA fibers of shrimp hemocyte extracellular traps are essential for the clearance of Escherichia coli

Extracellular traps (ETs) are a part of the vertebrate immune response that was only recently discovered. These structures are formed in response to pathogenic invasion and they act to kill the invader. Vertebrate ETs are composed of chromosomal DNA, histone proteins and other antimicrobial cytoplasmic proteins. Pathogenic stimulation was also recently shown to trigger a similar ET response in shrimp hemocytes, and in the present study, we evaluate the role of the DNA fibers in the bactericidal properties of these invertebrate ETs. When the formation of shrimp ETs was disrupted by DNase I, the ETs anti-bacterial activity was also reduced, indicating that the DNA fibers are important for ET-mediated bacterial clearance. We also found that at high bacterial densities, shrimp ETs were a more effective anti-bacterial response than phagocytosis.

Microbiome Dynamics in a Shrimp Grow-out Pond with Possible Outbreak of Acute Hepatopancreatic Necrosis Disease

Acute hepatopancreatic necrosis disease (AHPND) (formerly, early mortality syndrome) is a high-mortality-rate shrimp disease prevalent in shrimp farming areas. Although AHPND is known to be caused by pathogenic Vibrio parahaemolyticus hosting the plasmid-related PirABvp toxin gene, the effects of disturbances in microbiome have not yet been studied. We took 62 samples from a grow-out pond during an AHPND developing period from Days 23 to 37 after stocking white postlarvae shrimp and sequenced the 16S rRNA genes with Illumina sequencing technology. The microbiomes of pond seawater and shrimp stomachs underwent varied dynamic succession during the period. Despite copies of PirABvp, principal co-ordinates analysis revealed two distinctive stages of change in stomach microbiomes associated with AHPND. AHPND markedly changed the bacterial diversity in the stomachs; it decreased the Shannon index by 53.6% within approximately 7 days, shifted the microbiome with Vibrio and Candidatus Bacilloplasma as predominant populations, and altered the species-to-species connectivity and complexity of the interaction network. The AHPND-causing Vibrio species were predicted to develop a co-occurrence pattern with several resident and transit members within Candidatus Bacilloplasma and Cyanobacteria. This study’s insights into microbiome dynamics during AHPND infection can be valuable for minimising this disease in shrimp farming ponds.

Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes)

Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN−/− F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN−/− F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.

Draft Genome Sequence of Vibrio parahaemolyticus Strain M1-1, Which Causes Acute Hepatopancreatic Necrosis Disease in Shrimp in Vietnam

ABSTRACT We report here the genome sequence of Vibrio parahaemolyticus strain M1-1, which causes a mild form of shrimp acute hepatopancreatic necrosis disease (AHPND). Compared to other virulent strains, the M1-1 genome appeared to express several additional genes, while some genes were missing. These instabilities may be related to the reduced virulence of M1-1.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND‐associated plasmid pVA1 encoding a virulent toxin (Pirvp) that damages the shrimps hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimps stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND‐induced changes in the stomach of AHPND‐causing V. parahaemolyticus (5HP)‐infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene‐to‐gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP‐infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.