Signature selection forces and evolutionary divergence of immune-survival genes compared between two important shrimp species.

In recent years, shrimp aquaculture industry had grown significantly to become the major source of global shrimp production. Despite that, shrimp aquaculture production was impeded by various shrimp diseases over the past decades. Interestingly, different shrimp species demonstrated variable levels of immune strength and survival (immune-survival) ability towards different diseases, especially the much stronger immune-survival ability shown by the ancient shrimp species, Macrobrachium rosenbergii compared to other shrimp species. In this study, two important shrimp species, M. rosenbergii and Penaeus monodon (disease tolerant strain) (uninfected control and VpAHPND-infected) were compared to uncover the potential underlying genetic factors. The shrimp species were sampled, followed by RNA extraction and cDNA conversion. Five important immune-survival genes (C-type Lectin, HMGB, STAT, ALF3, and ATPase 8/6) were selected for PCR, sequencing, and subsequent genetics analysis. The overall genetic analyses conducted, including Analysis of Molecular Variance (AMOVA) and population differentiation, showed significant genetic differentiation (p<0.05) between different genes of M. rosenbergii and P. monodon. There was greater genetic divergence identified between HMGB subgroups of P. monodon (uninfected control and VpAHPND-infected) compared to other genes. Besides that, based on neutrality tests conducted, purifying selection was determined to be the main evolutionary driving force of M. rosenbergii and P. monodon with stronger purifying selection exhibited in M. rosenbergii genes. Potential balancing selection was identified for VpAHPND-infected HMGB subgroup whereas directional selection was detected for HMGB (both species) and ATPase 8/6 (only P. monodon) genes. The divergence times between M. rosenbergii and P. monodon genes were estimated through Bayesian molecular clock analysis, which were 438.6 mya (C-type Lectin), 1885.4 mya (HMGB), 432.6 mya (STAT), 448.1 mya (ALF3), and 426.4 mya (ATPase 8/6) respectively. In conclusion, important selection forces and evolutionary divergence information of immune-survival genes between M. rosenbergii and P. monodon were successfully identified.

Biochemical indexes and gut microbiota testing as diagnostic methods for Penaeus monodon health and physiological changes during AHPND infection with food safety concerns.

Severe shrimp disease outbreaks have a destructive impact on shrimp aquaculture and its associated downstream food processing industries. Thus, it is essential to develop proper methods for shrimp disease control, which emphasizes the importance of food safety. In this study, we performed biochemical tests and gut microbiome analysis using uninfected control and Vp AHPND-infected Penaeus monodon samples. Biochemical tests were performed to assess the phenoloxidase (PO) activity, respiratory Burst (RB) activity, nitrite concentration, superoxide dismutase (SOD) activity, total hemocyte count (THC), and total protein concentrations. Overall, upregulations were detected in these biochemical tests, which showed the activation of the immune response in P. monodon during acute hepatopancreatic necrosis disease (AHPND) infection, especially at 6 hpi and 12 hpi. Besides that, shrimp gut samples were collected and pooled (n = 3), followed by DNA extraction, PCR amplification targeting the V3/V4 16S ribosomal RNA (rRNA) region, next-generation sequencing (NGS), and bioinformatics analysis. Proteobacteria was the most abundant phylum in both samples. The Rhodobacteraceae family and Maritimibacter genus were proposed to be vital forshrimp health maintenance. Vp AHPND bacterial colonization and secondary Vibrio infections were postulated to have occurred based on the higher abundances of Vibrionaceae family and Vibrio genus in the Vp AHPND-infected sample. Firmicutes phylum together with Photobacterium and Aliiroseovarius genera were inferred to be pathogenic or related factors of AHPND infections. In conclusion, physiology (immune response activation) and gut microbiome changes of disease tolerant P. monodon during AHPND infection were identified. Both biochemical tests and 16S rRNA analysis are proposed as a combined strategy for shrimp health diagnosis for ensuring shrimp health maintenance, disease control, and food safety.

Differential STAT gene expressions of Penaeus monodon and Macrobrachium rosenbergii in response to white spot syndrome virus (WSSV) and bacterial infections: Additional insight into genetic variations and transcriptomic highlights.

Diseases have remained the major issue for shrimp aquaculture industry for decades by which different shrimp species demonstrated alternative disease resistance or tolerance. However, there had been insufficient studies on the underlying host mechanisms of such phenomenon. Hence, in this study, the main objective involves gaining a deeper understanding into the functional importance of shrimp STAT gene from the aspects of expression, sequence, structure, and associated genes. STAT gene was selected primarily because of its vital signalling roles in stress, endocrine, and immune response. The differential gene expressions of Macrobrachium rosenbergii STAT (MrST) and Penaeus monodon STAT (PmST) under White Spot Syndrome Virus (WSSV) and Vibrio parahaemolyticus/VpAHPND infections were identified through qPCR analysis. Notably, during both pathogenic infections, MrST demonstrated significant gene expression down-regulations (during either early or later post-infection time points) whereas PmST showed only significant gene expression up-regulations. Important sequence conservation or divergence was highlighted through STAT sequence comparison especially amino acid alterations at 614 aa [K (Lysine) to E (Glutamic Acid)] and 629 aa [F (Phenylalanine) to V (Valine)] from PmST (AY327491.1) to PmST (disease tolerant strain). There were significant differences observed between in silico characterized structures of MrST and PmST proteins. Important functional differentially expressed genes (DEGs) in the aspects of stress, endocrine, immune, signalling, and structural were uncovered through comparative transcriptomic analysis. The DEGs associated with STAT functioning were identified including inositol 1,4,5-trisphosphate receptor, hsp90, caspase, ATP binding cassette transmembrane transporter, C-type Lectin, HMGB, ALF1, ALF3, superoxide dismutase, glutathione peroxidase, catalase, and TBK1. The main findings of this study are STAT differential gene expression patterns, sequence divergence, structural differences, and associated functional DEGs. These findings can be further utilized for shrimp health or host response diagnostic studies. STAT gene can also be proposed as a suitable candidate for future studies of shrimp innate immune enhancement.

Potential muscle activity disturbance in Penaeus monodon during Acute Hepatopancreatic Necrosis Disease (AHPND) infection: Inference through gene expression, calcium concentration, and MicroRNA.

Global shrimp aquaculture farmers have suffered major economic losses due to disease outbreaks. A notable shrimp disease is Acute Hepatopancreatic Necrosis Disease (AHPND), which is caused by a new strain of Vibrio parahaemolyticus bacteria (VpAHPND) that mainly inhabits the shrimp gut and damages the hepatopancreas. Fewer studies have investigated whether this disease will affect shrimp muscle functioning or cause any muscle damage. We challenged Penaeus monodon shrimp with VpAHPND bacteria using an immersion method. Expression of Dystrophin gene, an important regulatory gene for maintenance of muscle integrity, was quantified from muscle samples using qRT-PCR. Additional verification was conducted by determining calcium concentration and bta-miR-4286 and dre-miR-107b miRNAs expression. P. monodon dystrophin gene demonstrated the highest expression level during AHPND infection when muscle calcium concentration was detected at its lowest level at 6 h post-infection (hpi). The highest muscle calcium concentration, determined at 36 hpi, was supported by higher bta-miR-4286 miRNA expression and lower dre-miR-107b miRNA expression in VpAHPND-infected samples compared to uninfected samples at the same time point. We deduced an interactive relationship between dystrophin gene expression, calcium concentration, and miRNA expression in P. monodon muscle tissues triggered by the invading VpAHPND bacterium.

Differential transcriptome analysis of the disease tolerant Madagascar-Malaysia crossbred black tiger shrimp, Penaeus monodon hepatopancreas in response to acute hepatopancreatic necrosis disease (AHPND) infection: inference on immune gene response and interaction.

BACKGROUND: Penaeus monodon is the second most widely cultured marine shrimp species in the global shrimp aquaculture industry. However, the growth of P. monodon production has been constantly impaired by disease outbreaks. Recently, there is a lethal bacterial infection, known as acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus AHPND strain (Vp AHPND), which led to mass mortalities in P. monodon. Unfortunately, there is still insufficient knowledge about the underlying immune response of P. monodon upon AHPND infection. The present study aims to provide an insight into the antibacterial immune response elicited by P. monodon hepatopancreas towards AHPND infection. METHODS: We have employed high-throughput RNA-Seq technology to uncover the transcriptome changes of P. monodon hepatopancreas when challenged with Vp AHPND. The shrimps were challenged with Vp AHPND through immersion method with dissected hepatopancreas samples for the control group (APm-CTL) and treatment group at 3 (APm-T3), 6 (APm-T6), and 24 (APm-T24) hours post-AHPND infection sent for RNA-Seq. The transcriptome de novo assembly and Unigene expression determination were conducted using Trinity, Tgicl, Bowtie2, and RSEM software. The differentially expressed transcripts were functionally annotated mainly through COG, GO, and KEGG databases. RESULTS: The sequencing reads generated were filtered to obtain 312.77 Mb clean reads and assembled into 48662 Unigenes. Based on the DEGs pattern identified, it is inferred that the PAMPs carried by Vp AHPND or associated toxins are capable of activating PRRs, which leads to subsequent pathway activation, transcriptional modification, and antibacterial responses (Phagocytosis, AMPs, proPO system). DAMPs are released in response to cell stress or damage to further activate the sequential immune responses. The comprehensive interactions between Vp AHPND, chitin, GbpA, mucin, chitinase, and chitin deacetylase were postulated to be involved in bacterial colonization or antibacterial response. CONCLUSIONS: The outcomes of this research correlate the different stages of P. monodon immune response to different time points of AHPND infection. This finding supports the development of biomarkers for the detection of early stages of Vp AHPND colonization in P. monodon through host immune expression changes. The potential genes to be utilized as biomarkers include but not limited to C-type lectin, HMGB1, IMD, ALF, serine proteinase, and DSCAM.

Dystrophin gene expression and intracellular calcium changes in the giant freshwater prawn, Macrobrachium rosenbergii, in response to white spot symptom disease infection.

BACKGROUND: Dystrophin, an essential protein functional in the maintenance of muscle structural integrity is known to be responsible for muscle deterioration during white spot syndrome virus (WSSV) infection among prawn species. Previous studies have shown the upregulation of dystrophin protein in Macrobrachium rosenbergii (the giant freshwater prawn) upon white spot syndrome virus (WSSV) infection. The literature has also suggested the important role of calcium ion alterations in causing such muscle diseases. Thus, the interest of this study lies within the linkage between dystrophin functioning, intracellular calcium and white spot syndrome virus (WSSV) infection condition. METHODS: In this study, the dystrophin gene from M. rosenbergii (MrDys) was first characterised followed by the characterization of dystrophin gene from a closely related shrimp species, Penaeus monodon (PmDys). Dystrophin sequences from different phyla were then used for evolutionary comparison through BLAST analysis, conserved domain analysis and phylogenetic analysis. The changes in mRNA expression levels of dystrophin and the alteration of intracellular calcium concentrations in WSSV infected muscle cells were then studied. RESULTS: A 1246 base pair long dystrophin sequence was identified in the giant freshwater prawn, Macrobrachium rosenbergii (MrDys) followed by 1082 base pair long dystrophin sequence in P. monodon (PmDys). Four conserved domains were identified from the thirteen dystrophin sequences compared which were classified into 5 different phyla. From the phylogenetic analysis, aside from PmDys, the characterised MrDys was shown to be most similar to the invertebrate phylum of Nematoda. In addition, an initial down-regulation of dystrophin gene expression followed by eventual up-regulation, together with an increase in intracellular calcium concentration [Ca2+] i were shown upon WSSV experimental infection. DISCUSSION: Both the functionality of the dystrophin protein and the intracellular calcium concentration were affected by WSSV infection which resulted in progressive muscle degeneration. An increased understanding of the role of dystrophin-calcium in MrDys and the interactions between these two components is necessary to prevent or reduce occurrences of muscle degeneration caused by WSSV infection, thereby reducing economic losses in the prawn farming industry from such disease.