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

© 2021 by the authors.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.This research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, grant number LTAUSA17201; by the European Commission under the H2020 Programme—ParaFishControl, grant number 634429; by the Czech Science Foundation, grant number 19-28399X (to A. S. Holzer, G. Alama-Bermejo, and J. Kyslík) and 21-16565S and by the Czech Academy of Sciences and Hungarian Academy of Sciences, grant number MTA 19-07. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein

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

Acknowledgments: We thank to Baveesh Pudhuvai (BC CAS, Budweis, Czech Republic) for help in the PCR verification of Buddenbrockia stefin. We also thank Ivan Fiala (BC CAS, Budweis, Czech Republic) for providing suggestions to improve the manuscript and for sharing M. lieberkuehni and N. pickii transcriptomic data. We are grateful to Hanna Hartikainen (ETH Zurich, Switzerland) for sharing T. bryosalmonae genome data. Funding: This research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, grant number LTAUSA17201; by the European Commission under the H2020 Programme— ParaFishControl, grant number 634429; by the Czech Science Foundation, grant number 19-28399X (to A. S. Holzer, G. Alama-Bermejo, and J. Kyslík) and 21-16565S and by the Czech Academy of Sciences and Hungarian Academy of Sciences, grant number MTA 19-07. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein.Peer reviewedPublisher PD

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

Phylogeny of Myxozoa based on cnidarian specific genes

Current myxozoan phylogeny relies mostly on single 18S rDNA or concatenated analysis of a limited number of 18S + 28S rDNA and exceptionally EF-2 gene sequences. Although these widely-used markers support particular phylogenetic relationships, they are not sufficient to reconstruct the pattern of some early branching nodes, important for the understanding of the main phylogenetic trends of Myxozoa. The present study describes new potential phylogenetic markers nematogalectin genes, which are unique cnidarian-specific genes. Phylogenetic reconstruction based on nematogalectins showed congruency with 18S rDNA based phylogenies and concatenated analysis of 18S rDNA and nematogalectins revealed better support of early branching nodes in the myxozoan tree. Recently published and newly obtained data of these cnidarian-specific genes represent important insights into the phylogeny of Myxozoa

New phylogenetic markers for phylogenetic reconstruction of Myxozoa

Phylogenetic placement of the Myxozoa within the Cnidaria is still not clearly understood, although there are many supports of close relationships between Myxozoa and Medusozoa. This thesis addresses the issue of the phylogenetic position of Myxozoa based on mitochondrial genes (rns and NADH 1). Determination of the position and order of these genes in the mitochodrial genome together with results of phylogenetic analysis might be the key for deciphering of myxozoan phylogeny and declare the phylogenetic placement of the Myxozoa within the Cnidaria. Rns was found to be a new promissing phylogenetic marker in the cnidarian phylogeny which may resolve the problem of myxozoan placement

Bioinformatická a funkční charakterizace taxonomicky omezených genů

Evolutionary dynamics, generating novel phenotypic traits, embody an essential route to either lineage-specific adaptations or to the creation of endless forms in organisms. Understanding the evolution of such ultimate novelties requires the study of the origin and the role of the functional units in generating unique traits, which represent the taxon-specific genes (TRGs). Myxozoa, the endoparasitic cnidarians, have become a suitable model for the study of evolutionary innovations, with their example of single-celled novelty: polar capsules. This uniquelly evolved structure within the spore stage represents a lineage-specific analogy of cnidarian stinging cells (nematocyst). In Cnidaria, nematocysts contain several uniquely emerged genes, rendering the assembly of this complex structure. The prominent fraction represents minicollagens, extremely shortened structural peptides forming the wall and tubule during the nematocyst development. Unlike in Cnidaria, the role of TRGs such as minicollagens in myxozoan development is unknown. The thesis describes the evolution and functional characterization of minicollagens in Myxozoa. Using the bioinformatics and molecular approaches, we explored the diversity and genomic organization and comprehensively characterized minicollagen gene expressions during myxospore development and localization of minicollagens in the polar capsule

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

Abstract 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

Agrobacterium phylogeny analysis

<p>Complete dataset for phylogeny analysis of new Agrobacterium species proposed as <em>Agrobacterium bohemicum </em>sp. nov.. </p

The gel-shift assay of the <i>xdo</i> operon.

<p>The gel-shift assay of the <i>xdo</i> operon.</p