Evolution of myxozoan mitochondrial genomes: insights from myxobolids.

BACKGROUND: Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms. RESULTS: Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene. CONCLUSIONS: Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

Exotic myxozoan parasites establish complex life cycles in farm pond aquaculture.

Myxozoans are obligate parasites with complex life cycles, typically infecting fish and annelids. Here, we examined annelids from fish farm pond sediments in the Beit Shean Valley, in the Syrian-African Rift Valley, Israel, for myxozoan infections. We examined 1486 oligochaetes, and found 74 (5 %) were infected with actinospore stages. We used mitochondrial 16S sequencing to infer identity of 25 infected annelids as species of Potamothrix, Psammoryctides, Tubifex and Dero. We identified 7 myxozoan types from collective groups Neoactinomyxum and Sphaeractinomyxon, and characterized them by small subunit ribosomal DNA sequencing. The Neoactinomyxum type was genetically most similar (∼93 %) to cyprinid fish-infecting Myxobolus spp. The six Sphaeractinomyxon types were genetically similar (93-100 %) to Mugilid-infecting Myxobolus spp.; with one being the previously unknown actinospore stage of a myxospore that infects mullet from aquaculture from the Israeli coast of the Mediterranean Sea. As the farm pond system is artificial and geographically isolated from the Mediterranean, the presence of at least seven myxozoans in their annelid hosts demonstrates introduction and establishment of these parasites in a novel, brackish environment.

Responses to pathogen exposure in sentinel juvenile fall-run Chinook salmon in the Sacramento River, CA.

This study investigated how the deployment of juvenile Chinook salmon in ambient river conditions and the subsequent exposure to and infection by pathogens was associated with the changes in the expression of genes involved in immune system functioning, general stress and host development. Juvenile fish were deployed in sentinel cages for 21 days in the Sacramento River, CA, USA. Gill, kidney and intestinal tissue were sampled at 0, 7, 14 and 21 days post-deployment. Pathogen detection and host response were assessed by a combination of molecular and histopathological evaluation. Our findings showed that fish became infected by the parasites Ceratonova shasta, Parvicapsula minibicornis and Ichthyophthirius multifiliis, and to a lesser extent, the bacteria Flavobacterium columnare and Rickettsia-like organisms. Co-infection was common among sentinel fish. Expression of investigated genes was altered following deployment and was often associated with pathogen abundance. This study provides a foundation for future avenues of research investigating pathogens that affect out-migrating Chinook salmon in the Sacramento River, and offers crucial knowledge related to conservation efforts.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

Ceratonova shasta: a cnidarian parasite of annelids and salmonids.

The myxozoan Ceratonova shasta was described from hatchery rainbow trout over 70 years ago. The parasite continues to cause severe disease in salmon and trout, and is recognized as a barrier to salmon recovery in some rivers. This review incorporates changes in our knowledge of the parasite's life cycle, taxonomy and biology and examines how this information has expanded our understanding of the interactions between C. shasta and its salmonid and annelid hosts, and how overarching environmental factors affect this host­parasite system. Development of molecular diagnostic techniques has allowed discrimination of differences in parasite genotypes, which have differing host affinities, and enabled the measurement of the spatio-temporal abundance of these different genotypes. Establishment of the C. shasta life cycle in the laboratory has enabled studies on host­parasite interactions and the availability of transcriptomic data has informed our understanding of parasite virulence factors and host defences. Together, these advances have informed the development of models and management actions to mitigate disease.

A myxozoan genome reveals mosaic evolution in a parasitic cnidarian.

BACKGROUND: Parasite evolution has been conceptualized as a process of genetic loss and simplification. Contrary to this model, there is evidence of expansion and conservation of gene families related to essential functions of parasitism in some parasite genomes, reminiscent of widespread mosaic evolution-where subregions of a genome have different rates of evolutionary change. We found evidence of mosaic genome evolution in the cnidarian Myxobolus honghuensis, a myxozoan parasite of fish, with extremely simple morphology. RESULTS: We compared M. honghuensis with other myxozoans and free-living cnidarians, and determined that it has a relatively larger myxozoan genome (206 Mb), which is less reduced and less compact due to gene retention, large introns, transposon insertion, but not polyploidy. Relative to other metazoans, the M. honghuensis genome is depleted of neural genes and has only the simplest animal immune components. Conversely, it has relatively more genes involved in stress resistance, tissue invasion, energy metabolism, and cellular processes compared to other myxozoans and free-living cnidarians. We postulate that the expansion of these gene families is the result of evolutionary adaptations to endoparasitism. M. honghuensis retains genes found in free-living Cnidaria, including a reduced nervous system, myogenic components, ANTP class Homeobox genes, and components of the Wnt and Hedgehog pathways. CONCLUSIONS: Our analyses suggest that the M. honghuensis genome evolved as a mosaic of conservative, divergent, depleted, and enhanced genes and pathways. These findings illustrate that myxozoans are not as genetically simple as previously regarded, and the evolution of some myxozoans is driven by both genomic streamlining and expansion.

Two novel myxosporean parasite species of Ceratomyxa Thélohan, 1892 from the banded cusk-eel Raneya brasiliensis (Kaup) (Ophidiiformes: Ophidiidae) off Patagonia, Argentina.

We described two novel myxozoan parasite species Ceratomyxa argentina n. sp. and Ceratomyxa raneyae n. sp. from the gall bladder of Raneya brasiliensis (Kaup) from the Patagonian coast of Argentina. Both species can be distinguished from other ceratomyxids by myxospore and polar capsule (nematocyst) morphology and morphometry, fish host and geographic locality. Phylogenetic reconstruction using ssrDNA gene sequences showed that the two new species are placed in a long-branching ceratomyxid clade which also include Ceratomyxa appendiculata Thélohan, 1892, Ceratomyxa anko Freeman, Yokoyama and Ogawa, 2008, Ceratomyxa pantherini Gunter, Burger and Adlard, 2010 and Pseudoalataspora kovalevae Kalavati, MacKenzie, Collins, Hemmingsen and Brickle, 2013. This study documents additional biodiversity of marine myxozoans in the South Atlantic, a region still largely unexplored for this group of parasitic cnidarians.

Myxobolus cerebralis Causes Presporogonic Mortality in Juvenile Mountain Whitefish.

Recent range expansions of whirling disease impelled us to understand the impacts of its causative agent, the myxozoan parasite Myxobolus cerebralis, on lesser-studied fish hosts. Mountain Whitefish Prosopium williamsoni overlap broadly with M. cerebralis across the western United States and Canada, and populations have experienced widespread declines since the 1990s. To evaluate effects of the parasite on Mountain Whitefish, we revisit formerly unpublished work of the Colorado Division of Wildlife (now Colorado Parks and Wildlife), comparing infection in age-matched Mountain Whitefish, Rainbow Trout Oncorhynchus mykiss, and Brown Trout Salmo trutta. To complement the original report, we reanalyze mortality data and include additional SEM imagery. Infection of M. cerebralis in juvenile Mountain Whitefish was characterized by a brief but heavy period of mortality in the first 2 weeks after exposure, with limited pathology. This clinical effect is unique among the known salmonid hosts of M. cerebralis.

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.

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection.

BACKGROUND: Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known. METHODS: We searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals. RESULTS: We identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish's gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a "venom" can have and represent targets for designing therapeutics against myxozoan infections.

Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts.

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

Myxosporea (Myxozoa, Cnidaria) Lack DNA Cytosine Methylation.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

Description of myxosporeans (Cnidaria: Myxozoa) infecting the popular food fish Notopterus notopterus (Pisces: Notopteridae) in Malaysia and India.

This study was a co-operative investigation of myxosporean infections of Notopterus notopterus, the bronze featherback, which is a popular food fish in the South Asian region. We examined fish from Lake Kenyir, Malaysia and the River Ganga, Hastinapur, Uttar Pradesh, India, and observed infections with two myxosporeans: Myxidium cf. notopterum (Myxidiidae) and Henneguya ganapatiae (Myxobolidae), respectively. These species were identified by myxospore morphology, morphometry and host tissue affinity, and the original descriptions supplemented with small-subunit ribosomal DNA sequences and phylogenetic analysis. Free myxospores of M. cf. notopterum were found in the gallbladder, and measured 14.7 ±â€¯0.6 µm long and 6.3 ±â€¯0.6 µm wide; host, tissue and myxospore dimensions overlapped with the type, but differed in morphological details (spore shape, valve cell ridges) and locality (Malaysia versus India). Plasmodia and spores of H. ganapatiae were observed in gills, and myxospores had a spore body 9.7 ±â€¯0.4 µm long, 4.5 ±â€¯0.5 µm wide; sample locality, host, tissue, spore morphology and morphometry matched the original description. Small-subunit ribosomal DNA sequences were deposited in GenBank (M. cf. notopterum MT365527, H. ganapatiae MT365528) and both differed by >7% from congeneric species. Although the pathogenicity and clinical manifestation of myxozoan in humans are poorly understood, consumption of raw fish meat with myxozoan infection was reported to be associated with diarrhea. Identification of current parasite fauna from N. notopterus is an essential first step in assessing pathogen risks to stocks of this important food fish.

A comparison of the structure and function of nematocysts in free-living and parasitic cnidarians (Myxozoa).

Myxozoans are obligate parasites that have complex life cycles requiring alternate vertebrate and invertebrate hosts, with transmission via microscopic waterborne spores. Unusually for parasites, they belong to the phylum Cnidaria, alongside thousands of free-living corals, sea anemones, jellyfish and hydrozoans. Their cnidarian affinity is affirmed by genetic relatedness and the presence of nematocysts, historically called "polar capsules" in myxozoan research. Free-living cnidarians utilise this cellular weaponry for defence, predation and adhesion, whereas myxozoans use it to anchor to their hosts as the first step in infection. Despite the ~650 million years of divergence between free-living cnidarians and myxozoans, their nematocysts retain many shared morphological and molecular characters. Both are intra-cellular capsules with a single opening, and contain a coiled, evertable tubule. They are composed of unique nematocyst proteins, nematogalectin and minicollagen, and both likely contain an internal matrix of metal cations covalently bound to the anionic polymer poly-gamma glutamate. The rapid dissociation of this matrix and the resulting increase in internal osmotic potential is the driving force behind tubule elongation during discharge. In this review, we compare the structure and function of nematocysts in Myxozoa and free-living Cnidaria, incorporating recent molecular characterizations. We propose that terminology for homologous myxozoan structures be synonymized with those from other Cnidaria, hence, "polar capsule" as a taxon-specific nematocyst morphotype and "polar filament" as "tubule." Despite taxonomic divergence, genome reduction and an evolution to parasitism, myxozoans maintain nematocysts that are structurally and functionally homologous to those of their free-living cnidarian relatives.

In vitro and in vivo assays reveal that cations affect nematocyst discharge in Myxobolus cerebralis (Cnidaria: Myxozoa).

Myxozoans are parasitic, microscopic cnidarians that have retained the phylum-characteristic stinging capsules called nematocysts. Free-living cnidarians, like jellyfish and corals, utilize nematocysts for feeding and defence, with discharge powered by osmotic energy. Myxozoans use nematocysts to anchor to their fish hosts in the first step of infection, however, the discharge mechanism is poorly understood. We used Myxobolus cerebralis, a pathogenic myxozoan parasite of salmonid fishes, and developed two assays to explore the nature of its nematocyst discharge. Using parasite actinospores, the infectious stage to fish, we stimulated discharge of the nematocysts with rainbow trout mucus in vitro, in solutions enriched with chloride salts of Na+, K+, Ca2+ and Gd3+, and quantified discharge using microscopy. We then used quantitative polymerase chain reaction to evaluate the in vivo effects of these treatments, plus Mg2+ and the common aquaculture disinfectant KMnO4, on the ability of M. cerebralis actinospores to infect fish. We found that Mg2+ and Gd3+ reduced infection in vivo, whereas Na+ and K+ over-stimulated nematocyst discharge in vitro and reduced infection in vivo. These findings align with nematocyst discharge behaviour in free-living Cnidaria, and suggest phylum-wide commonalties, which could be exploited to develop novel approaches for controlling myxozoan diseases in aquaculture.

Transcriptome-Wide Comparisons and Virulence Gene Polymorphisms of Host-Associated Genotypes of the Cnidarian Parasite Ceratonova shasta in Salmonids.

Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. Ceratonova shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the data sets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed single-nucleotide polymorphism-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.

The invertebrate host of salmonid fish parasites Ceratonova shasta and Parvicapsula minibicornis (Cnidaria: Myxozoa), is a novel fabriciid annelid, Manayunkia occidentalis sp. nov. (Sabellida: Fabriciidae).

Myxosporea (Cnidaria: Myxozoa) are common fish parasites with complex life cycles that involve annelid hosts. Two economically important salmonid-infecting myxosporeans from rivers of the northwestern United States, Ceratonova shasta (Noble, 1950) and Parvicapsula minibicornis Kent et al., 1997, have life cycles that require a freshwater annelid host, identified previously as Manayunkia speciosa Leidy, 1859. This species was described originally from Pennsylvania, with subsequent records from New Jersey, the Great Lakes and west coast river basins. Despite apparent widespread distributions of both suitable fish hosts and the nominal annelid host, both parasites are restricted to river basins in the northwestern US and have never been recorded from the Great Lakes or the eastern US. In this study, we sampled 94 infected and uninfected annelids from two northwestern US rivers to confirm the identity of the host. We found these new specimens had mitochondrial COI sequences with no more than 4.5% distance from each other, but with at least 11% divergence from M. speciosa sampled from near the type locality (New Jersey) and Lake Superior. We did not recover any M. speciosa from either west coast river. The annelid from the Klamath and Willamette rivers showed marked sexual dimorphism that has not been reported in any Manayunkia described to date, though it is apparent that this had been missed in M. speciosa. Accordingly, we describe a new taxon, Manayunkia occidentalis sp. nov., and show that it can host both C. shasta and P. minibicornis. We suspect that previous records of Manayunkia from Pacific Northwest watersheds are likely to be M. occidentalis sp. nov. and not M. speciosa. Sampling of Manayunkia from additional localities is underway to test if the novel Manayunkia species is the only freshwater fabriciid annelid present across the Pacific Northwest.

A cnidarian parasite of salmon (Myxozoa: Henneguya) lacks a mitochondrial genome.

Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis, has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.

Validation of environmental DNA sampling for determination of Ceratonova shasta (Cnidaria: Myxozoa) distribution in Plumas National Forest, CA.

Ceratonova shasta is the etiological agent of myxozoan-associated enteronecrosis in North American salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and a freshwater fabriciid annelid. The success and survival of annelids can be enhanced by flow moderation by dams, and through the erosion of fine sediments into stream channels following wildfires. In this study, the presence of C. shasta environmental/ex-host DNA (eDNA) in river water and substrate samples collected from areas affected by recent fire activity in California, USA, was investigated. Additionally, DNA loads in the environment were compared to C. shasta infection in sentinel-exposed rainbow trout (Oncorhynchus mykiss). Significant associations between C. shasta detection in environmental samples and location within a wildfire perimeter (p = 0.002), between C. shasta detection in sentinel fish and exposure location within a wildfire perimeter (p = 0.015), and between C. shasta detection in fish and locations where water temperature was above the median (p < 0.001) were observed. Additionally, a higher prevalence of C. shasta infection in fish was detected where C. shasta was also detected in environmental samples (p < 0.001). Results suggest that pathogen eDNA sampling can be used as a non-invasive, rapid, specific, and sensitive method for establishing risk of C. shasta infection in wild populations. Knowledge of the complete life cycle of the target parasite, including ecology of each host, can inform the choice of eDNA sampling strategy. Environmental DNA sampling also revealed a novel species of Ceratonova, not yet observed in a host.

Mitochondrial genome of the freshwater annelid Manayunkia occidentalis (Sabellida: Fabriciidae).

Here, we report the 15,103 bp mitochondrial genome of the freshwater fabriciid tubeworm Manayunkia occidentalis. We recovered 13 protein-coding genes, 2 rRNA, and 22 tRNA. The gene order is consistent with the conserved pattern observed in most annelids.