RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari.

RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

Expression profiling and cellular localization of myxozoan minicollagens during nematocyst formation and sporogenesis.

In free-living cnidarians, minicollagens are major structural components in the biogenesis of nematocysts. Recent sequence mining and proteomic analysis demonstrate that minicollagens are also expressed by myxozoans, a group of evolutionarily ancient cnidarian endoparasites. Nonetheless, the presence and abundance of nematocyst-associated genes/proteins in nematocyst morphogenesis have never been studied in Myxozoa. Here, we report the gene expression profiles of three myxozoan minicollagens, ncol-1, ncol-3, and the recently identified noncanonical ncol-5, during the intrapiscine development of Myxidium lieberkuehni, the myxozoan parasite of the northern pike, Esox lucius. Moreover, we localized the myxozoan-specific minicollagen Ncol-5 in the developing myxosporean stages by Western blotting, immunofluorescence, and immunogold electron microscopy. We found that expression of minicollagens was spatiotemporally restricted to developing nematocysts within the myxospores during sporogenesis. Intriguingly, Ncol-5 is localized in the walls of nematocysts and predominantly in nematocyst tubules. Overall, we demonstrate that despite being significantly reduced in morphology, myxozoans retain structural components associated with nematocyst development in free-living cnidarians. Furthermore, our findings have practical implications for future functional and comparative studies as minicollagens are useful markers of the developmental phase of myxozoan parasites.

The myxozoan minicollagen gene repertoire was not simplified by the parasitic lifestyle: computational identification of a novel myxozoan minicollagen gene.

BACKGROUND: Lineage-specific gene expansions represent one of the driving forces in the evolutionary dynamics of unique phylum traits. Myxozoa, a cnidarian subphylum of obligate parasites, are evolutionarily altered and highly reduced organisms with a simple body plan including cnidarian-specific organelles and polar capsules (a type of nematocyst). Minicollagens, a group of structural proteins, are prominent constituents of nematocysts linking Myxozoa and Cnidaria. Despite recent advances in the identification of minicollagens in Myxozoa, the evolutionary history and diversity of minicollagens in Myxozoa and Cnidaria remain elusive. RESULTS: We generated new transcriptomes of two myxozoan species using a novel pipeline for filtering of closely related contaminant species in RNA-seq data. Mining of our transcriptomes and published omics data confirmed the existence of myxozoan Ncol-4, reported only once previously, and revealed a novel noncanonical minicollagen, Ncol-5, which is exclusive to Myxozoa. Phylogenetic analyses support a close relationship between myxozoan Ncol-1-3 with minicollagens of Polypodium hydriforme, but suggest independent evolution in the case of the myxozoan minicollagens Ncol-4 and Ncol-5. Additional genome- and transcriptome-wide searches of cnidarian minicollagens expanded the dataset to better clarify the evolutionary trajectories of minicollagen. CONCLUSIONS: The development of a new approach for the handling of next-generation data contaminated by closely related species represents a useful tool for future applications beyond the field of myxozoan research. This data processing pipeline allowed us to expand the dataset and study the evolution and diversity of minicollagen genes in Myxozoa and Cnidaria. We identified a novel type of minicollagen in Myxozoa (Ncol-5). We suggest that the large number of minicollagen paralogs in some cnidarians is a result of several recent large gene multiplication events. We revealed close juxtaposition of minicollagens Ncol-1 and Ncol-4 in myxozoan genomes, suggesting their common evolutionary history. The unique gene structure of myxozoan Ncol-5 suggests a specific function in the myxozoan polar capsule or tubule. Despite the fact that myxozoans possess only one type of nematocyst, their gene repertoire is similar to those of other cnidarians.

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians.

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.