Ivan Fiala

Avian schistosomes (AS) are trematodes of significant medical importance as the causative agents of cercarial dermatitis, a re-emerging waterborne human disease. Their monitoring has traditionally relied on examination of snail hosts; however, this approach is time-consuming, requires taxonomic expertise and often underestimates their diversity due to low prevalence of infection. Although environmental DNA (eDNA)-based methods have shown promise in detecting AS, most studies have employed species-specific assays that restrict the discovery of novel genetic lineages. This study integrated three complementary methodologies to comprehensively assess AS diversity in Central Europe: (i) metabarcoding of environmental water samples from various localities; (ii) metabarcoding of naturally infected snail hosts to detect pre-patent stages and (iii) standard parasitological examination and collection of cercariae released from snail hosts. In total, 34 species/genetic lineages were detected at 19 localities. Of these, three species were identified by all three techniques, two lineages by both water sample and snail tissue metabarcoding; 17 exclusively through water sample metabarcoding, 10 only in snail tissues and two solely by the standard method. Notably, metabarcoding of environmental water samples was the most powerful tool for capturing most of the AS diversity, while snail tissue metabarcoding uncovered early pre-patent infections and additional unique lineages not detected in water. These findings underscore the critical value of a multi-method approach to minimise detection bias associated with any single technique.

Microsporidians are highly specialised intracellular parasites adapted to an obligate parasitic lifestyle. Although more than 1,500 species have been described to date, their true diversity is likely much higher, particularly in poorly-studied aquatic environments. Here, we applied an optimised tissue-based metabarcoding approach to investigate the diversity and distribution of microsporidians in aquatic invertebrates from three nearby fish ponds in South Bohemia in the Czech Republic. We analysed more than two thousand individuals of chironomids and oligochaetes, which represent important aquatic invertebrate hosts of microsporidians, and compared microsporidian communities across ponds and host morphogroups. In total, 66 microsporidian OTUs were detected across both host groups. Metabarcoding and phylogenetic analyses revealed broad sequence diversity within the studied localities, with parasite sequences clustering into four main clades. The high proportion of OTUs without close matches to described taxa (more than 95%) highlights substantial undocumented diversity of microsporidians in freshwater ecosystems. The occurrence of shared OTUs amongst different invertebrate taxa is consistent with multi-host strategies and/or complex life cycles. Overall, our findings emphasise the ecological complexity of microsporidians in aquatic habitats. Further experimental work, including parasite localisation and transmission studies, is needed to determine whether the observed host associations represent active infections or incidental detection.

Common carp (Cyprinus carpio) aquaculture is a traditional, socioeconomically vital farming system in Central and Eastern Europe. Lacking intensive controls, it remains highly vulnerable to environmental fluctuations, making the understanding of microbial impacts on fish health a major challenge. In this preliminary study, we designed a high-resolution framework using full-length 16S rRNA Nanopore sequencing to track microbial flow across four key compartments during a single production cycle: chicken manure fertilizer, sediment, water, and fish tissues (gut and gills). Results demonstrate that chicken manure serves as a critical "founding" microbial vector, introducing allochthonous genera like Acinetobacter and Geofilum into the aquatic system. While the water column acted as a stable microbial buffer dominated by pelagic specialists (Limnohabitans), the sediment and fish tissues were highly sensitive to environmental stress. During an acute summer heatwave (30 °C), sediment microbial richness collapsed by 53%, and fish mucosal diversity reached its lowest levels, accompanied by an enrichment of opportunistic Pantoea. However, the system exhibited remarkable resilience; by drawdown, diversity recovered, and communities re-stabilized with environment-derived taxa, including Polynucleobacter and Lysinibacillus. This pilot monitoring underscores the direct impact of organic management practices and highlights the inherent capacity of the pond holobiome to regain stability following acute thermal disruption. Overall, these preliminary findings establish a crucial baseline supporting sustainable aquaculture strategies amid increasing climate instability.

Background: Myxozoans are ancient cnidarian parasites with highly derived genomes characterized by an extremely accelerated rate of nucleotide substitution, abundant orphan and lineage-specific genes without further characterization. Genome data are limited to two out of the four main evolutionary lineages, assemblies are highly fragmented and often show significant levels of host contamination. Results: We present a near chromosome-scale myxozoan genome, based on Oxford Nanopore-reads of Sphaerospora molnari, a member of the previously uncharacterized group of blood-feeding myxozoans, thereby addressing a key gap in the subphylum’s phylogeny. The haploid genome assembly spans 40.17 Mb in 40 contigs, harbors 14,957 genes and shows the smallest mitogenome of myxozoans (14,015 bp). Gene gain/gene loss analyses showed that myxozoans have small ancestral gene repertoires and show highly lineage-specific genome compositions. Using comparative analyses focusing on identifying unique but diversified gene compartments in S. molnari, we discovered taxonomically restricted protist genes related to red blood cell attachment ( Plasmodium ETRAMPs) and surface protein variation ( Plasmodium variant surface antigen families RIFIN and STEVOR, as well as Metamonada variant-specific surface proteins, VSPs), raising questions about their origins and evolution. A genomic trait shared between several myxozoans is the significant expansion of DNA transposable elements belonging to the mutator-like elements (MULEs), and while the simple copy-paste mechanism of these transposases may suggest frequent uncontrolled mutation, we demonstrate domestication of MULEs into transcription factors. Analyses of the gene fragments of chimeric MULEs (pack-MULES) of S. molnari show that these coincide with highly diversified gene groups in this lineage, including alien genes, suggesting MULEs as a driving force for gene evolution in myxozoans. Conclusions: Parasitic lifestyle shifts drive exceptionally rapid genome evolution in myxozoans, primarily through nonadaptive mutation and gene transfer via MULEs, with adaptive refinement through MULE domestication and selection. In S. molnari , these processes underpin unique erythrocyte exploitation and immune evasion strategies essential for survival in the hosts’ bloodstream.

Myxozoans are microscopical parasites widely distributed in fish, with over 2,600 described species, but their actual diversity is still underestimated. Among salmonids, more than 70 myxozoan species have been identified. This study focuses on species of Chloromyxum Mingazzini, 1890 that infect salmonid kidneys, particularly C. majori Yasutake et Wood, 1957 and C. schurovi Shulman et Ieshko, 2003. Despite their similar spore morphology, they exhibit distinct host preferences, tissue affinities and geographical distributions. Chloromyxum schurovi predominantly infects the renal tubules of Salmo salar Linnaues and S. trutta Linnaeus in Europe, while C. majori targets the glomeruli of Oncorhynchus mykiss (Walbaum) and O. tshawytscha (Walbaum) in North America. The sequence data for C. majori and C. schurovi have been either missing or questionable. In our study, we examined the kidneys of two salmonid species for chloromyxid infections, using both morphological and molecular data to characterise Chloromyxum species in salmonids. The sequence of C. schurovi obtained in our study did not match the previously published parasite data. Instead, it clustered as an independent lineage sister to the Paramyxidium Freeman et Kristmundsson, 2018 clade gathering the species from various fish organs, including the urinary tract. Our findings clarified the taxonomic origin of the previous C. schurovi sequence as Myxidium giardi C?pède, 1906, highlighting the risks associated with the presence of myxozoan blood stages in the bloodstream of their fish host and the challenges of non-specific PCR amplification. We redescribe C. schurovi, thus contributing to a better understanding of the diversity and phylogeny of kidney-infecting species of Chloromyxum.

European eel, Anguilla anguilla (Linnaeus) (Elopomorpha: Anguilliformes), is a critically endangered fish of ecological and economic importance, hosting numerous parasites, including myxozoans (Cnidaria). Since its initial discovery in the kidney of European eel, Myxidium giardi C?pède, 1906 has been reported with numerous spore sizes and shapes from various tissues of multiple anguillid species. Morphological variability, wide host and tissue spectrum, and lack of sequence data raised doubts about the conspecificity of reported isolates. Subsequent studies provided 18S rDNA sequences of several isolates from anguillids and other elopiform fish, and demonstrated a split of parasite data into two distinct phylogenetic lineages, one comprising the M. giardi sequence, and the other all species infecting elopiform fishes classified under the recently established genus Paramyxidium Freeman et Kristmundsson, 2018. Myxidium giardi was, however, transferred to this genus as Paramyxidium giardi n. comb. and designated as the type species of the genus. In line with this change, the sequence originally identified as M. giardi was considered to have been incorrectly associated with this species. To shed light on the status of M. giardi originally described by C?pède (1906), we conducted microscopic and molecular examinations of various organs of 24 individuals of European eel, originating from diverse Czech habitats. Through morphometric and molecular analyses, we demonstrated that spore and polar capsule morphology, morphometry and tissue tropism of our European eel kidney parasite isolates matched the features of the original M. giardi description. Our isolates clustered in the lineage encompassing the first published M. giardi sequence. Thus, the originally described M. giardi indeed represents an existing species within the genus Myxidium B?tschli, 1882, which we formally resurrect and redescribe. Due to the morphological and molecular differences between M. giardi and P. giardi of Freeman et Kristmundsson (2018), we additionally rename the latter species as Paramyxidium freemani nom. nov.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.

The environmental DNA (eDNA) metabarcoding approach has become a useful tool for detecting the species diversity of different animal groups, including parasites. Myxozoa (Malacosporea and Myxosporea) represent a unique group of morphologically simplified endoparasites that mainly infest fish and whose diversity remains largely unexplored. Metabarcoding of DNA from the aquatic environment is a promising non‐invasive method that allows us to assess myxozoan biodiversity at a given site. This is essential not only for describing myxozoan communities, but also for the development of disease control methods. Using an alignment of 330 sequences, we employed in silico PCR to score primer pairs, designed to amplify the V4 region of the SSU rDNA of different myxosporean sublineages comprising the entire diversity of oligochaete‐infecting (freshwater) myxosporeans. We selected eight clade‐specific primer sets for metabarcoding, avoiding amplification of DNA from other organisms present in eutrophic freshwaters. The metabarcoding approach used in the analysis of eDNA sediment samples detected a high myxosporean diversity even in small sample volumes (in total 44 OTUs). Furthermore, metabarcoding analysis of myxosporeans in fish tissue samples selected for primer testing revealed 91 different myxosporean OTUs, more than double the number obtained by classical PCR screening and Sanger sequencing with general myxozoan primers and almost seven times higher detection than by microscopic examination. Our results further suggest quantitative sampling requirements for realistic future diversity estimates by comparing OTUs from fish tissue metabarcoding and eDNA samples. The use of specific primer sets enabled the detection of a high proportion of myxosporean reads (63–100%) in all datasets, even in highly eutrophic habitats. This shows our metabarcoding approach as an excellent tool for non‐invasive and sensitive detection of myxosporean biodiversity in aquatic sediments, potentially useful for monitoring myxozoan disease agents that threaten economically important fish in aquaculture.