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.
Methods: We have employed high-throughput RNA-Seq technology to uncover the transcriptome changes of P. monodon hepatopancreas when challenged with VpAHPND. The shrimps were challenged with VpAHPND 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 VpAHPND 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 VpAHPND, 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 VpAHPND 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.