Project description:Myxozoans are widely distributed aquatic obligate endoparasites that were recently recognized as belonging within the phylum Cnidaria. They have complex life cycles with waterborne transmission stages: resistant, infectious spores that are unique to myxozoans. However, little is known about the processes that give rise to these transmission stages. To understand the molecular underpinnings of spore formation, we conducted proteomics on Ceratonova shasta, a highly pathogenic myxozoan that causes severe mortalities in wild and hatchery-reared salmonid fishes. We compared proteomic profiles between developmental stages from inside the fish host, and the mature myxospore, which is released into the water where it drifts passively, ready to infect the next host. We found that C. shasta contains 2123 proteins; representing the first proteomic catalog of a myxozoan myxospore. Analysis of proteins differentially expressed between developing and mature spore stages uncovered processes that are active during spore formation. Our data highlight dynamic changes in the actin cytoskeleton, which provides myxozoan developmental stages with mobility through lamellipodia and filopodia, whereas in the mature myxospore the actin network supports F-actin stabilization that reinforces the transmission stage. These findings provide molecular insight into the myxozoan life cycle stages and, particularly, into the process of sporogenesis.
Project description:Motility factors are fundamental for parasite invasion, migration, proliferation and immune evasion and thus can influence parasitic disease pathogenesis and virulence. Salmonid enteronecrosis is caused by a myxozoan (Phylum Cnidarian) parasite, Ceratonova shasta. Three parasite genotypes (0, I, II) occur, with varying degrees of virulence in its host, making it a good model for examining the role of motility in virulence. We compare C. shasta cell motility between genotypes and describe how the cellular protrusions interact with the host. We support these observations with motility gene expression analyses. C. shasta stages can move by single or combined used of filopodia, lamellipodia and blebs, with different behaviors such as static adhesion, crawling or blebbing, some previously unobserved in myxozoans. C. shasta stages showed high flexibility of switching between different morphotypes, suggesting a high capacity to adapt to their microenvironment. Exposure to fibronectin showed that C. shasta stages have extraordinary adhesive affinities to glycoprotein components of the extracellular matrix (ECM). When comparing C. shasta genotypes 0 (low virulence, no mortality) and IIR (high virulence, high mortality) infections in rainbow trout, major differences were observed with regard to their migration to the target organ, gene expression patterns and proliferation rate in the host. IIR is characterized by rapid multiplication and fast amoeboid bleb-based migration to the gut, where adhesion (mediated by integrin-? and talin), ECM disruption and virulent systemic dispersion of the parasite causes massive pathology. Genotype 0 is characterized by low proliferation rates, slow directional and early adhesive migration and localized, non-destructive development in the gut. We conclude that parasite adhesion drives virulence in C. shasta and that effectors, such as integrins, reveal themselves as attractive therapeutic targets in a group of parasites for which no effective treatments are known.
Project description:Infectious hematopoietic necrosis virus (IHNV) can cause widespread death of rainbow trout (Oncorhynchus mykiss), understanding the molecular mechanisms that occur in the rainbow trout in response to IHNV infection will be useful to decrease IHN-related morbidity and mortality in trout aquaculture. However, the molecular mechanisms of rainbow trout in response to IHNV are very limited. This study performed analysis of mRNAs and miRNAs based on RNA-seq technology on the intestine of rainbow trout infected with IHNV and control. There were 80 differentially expressed miRNAs that regulated 3355 target mRNAs, which overlapped with differentially expressed mRNAs obtained from RNA-seq. The expression patterns of DEGs and miRNAs differentially expressed were validated by qRT-PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DEGs were mainly enriched in immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and IL-17 signaling pathway. These findings improve our understanding of the molecular mechanisms of IHNV infection. The study analyzed the immune regulatory target gene pairs and signal pathways of rainbow trout intestine against IHNV infection at the transcriptional level, and provided basic data for the study of rainbow trout against IHNV immune regulatory.
Project description:Infectious hematopoietic necrosis virus (IHNV) can cause widespread death of rainbow trout (Oncorhynchus mykiss), understanding the molecular mechanisms that occur in the rainbow trout in response to IHNV infection will be useful to decrease IHN-related morbidity and mortality in trout aquaculture. However, the molecular mechanisms of rainbow trout in response to IHNV are very limited. This study performed analysis of mRNAs and miRNAs based on RNA-seq technology on the intestine of rainbow trout infected with IHNV and control. There were 80 differentially expressed miRNAs that regulated 3355 target mRNAs, which overlapped with differentially expressed mRNAs obtained from RNA-seq. The expression patterns of DEGs and miRNAs differentially expressed were validated by qRT-PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DEGs were mainly enriched in immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and IL-17 signaling pathway. These findings improve our understanding of the molecular mechanisms of IHNV infection. The study analyzed the immune regulatory target gene pairs and signal pathways of rainbow trout intestine against IHNV infection at the transcriptional level, and provided basic data for the study of rainbow trout against IHNV immune regulatory.
Project description:Hypoxia negatively affects the behavior, growth, reproduction, and survival of fish, causing serious economic losses to aquaculture. Rainbow trout (Oncorhynchus mykiss), an important economic fish worldwide, belongs to a hypoxia-sensitive fish species. However, the regulatory mechanisms of miRNAs under hypoxia stress response of rainbow trout remains unclear. In this study, rainbow trout were subjected to hypoxia stress (DO: 3.5 mg/L) for 3 h (H3h_L), 12 h (H12h_L), 24 h (H24h_L) and 3 h reoxygenation (R3h_L) to systemically evaluate the changes of miRNA expression profiles in liver, and the functions of sha-miR-92a_L+2R+4 were investigated. We found 17, 144, 57 and 55 differentially expressed (DE) miRNAs in H3h_L vs. control (N_L), H12h_L vs. N_L, H24h_L vs. N_L and R3h_L vs. N_L comparisons, respectively. Enrichment analysis revealed that the targets of these DE miRNAs were significantly enriched in HIF signaling pathway, VEGF signaling pathway, FoxO signaling pathway and glycolysis/gluconeogenesis. Through miRNA-mRNA nteraction and weighted gene co-expression network analysis (WGCNA), five key DE miRNAs (sha-miR-92a_L+2R+4, ssa-miR-128-3p, ssa-miR-101b-3p_R+1, ola-miR-199a-5p_R+2 and tni-miR-199_1ss18CG) were identified, which can target at least two hypoxia-responsive genes, such as vegfaa, ho, glut1a and junb. Functional analysis found that sha-miR-92a_L+2R+4 directly regulated vegfaa expression by targeting its 3′-UTR, overexpression of sha-miR-92a_L+2R+4 significantly decreased vegfaa expression in rainbow trout liver cells, while opposite results were obtained after transfection of sha-miR-92a_L+2R+4 inhibitor. Furthermore, overexpression of sha-miR-92a_L+2R+4 promoted rainbow trout liver cell proliferation and inhibited apoptosis. These results deepen our understanding of the crucial roles of miRNAs under hypoxia stress in rainbow trout, and provide valuable information for further studying the regulatory mechanisms of key hypoxia-responsive miRNAs and breeding hypoxia-tolerant rainbow trout species.