Life-cycle-coupled evolution of mitosis in close relatives of animals.

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.

The nuclear-to-cytoplasmic ratio drives cellularization in the close animal relative Sphaeroforma arctica.

The ratio of nuclear content to cytoplasmic volume (N/C ratio) is a key regulator driving the maternal-to-zygotic transition in most animal embryos. Altering this ratio often impacts zygotic genome activation and deregulates the timing and outcome of embryogenesis.1,2,3 Despite being ubiquitous across animals, little is known about when the N/C ratio evolved to control multicellular development. Such capacity either originated with the emergence of animal multicellularity or was co-opted from the mechanisms present in unicellular organisms.4 An effective strategy to tackle this question is to investigate the close relatives of animals exhibiting life cycles with transient multicellular stages.5 Among these are ichthyosporeans, a lineage of protists undergoing coenocytic development followed by cellularization and cell release.6,7,8 During cellularization, a transient multicellular stage resembling animal epithelia is generated, offering a unique opportunity to examine whether the N/C ratio regulates multicellular development. Here, we use time-lapse microscopy to characterize how the N/C ratio affects the life cycle of the best-studied ichthyosporean model, Sphaeroforma arctica. We uncover that the last stages of cellularization coincide with a significant increase in the N/C ratio. Increasing the N/C ratio by reducing the coenocytic volume accelerates cellularization, whereas decreasing the N/C ratio by lowering the nuclear content halts it. Moreover, centrifugation and pharmacological inhibitor experiments suggest that the N/C ratio is locally sensed at the cortex and relies on phosphatase activity. Altogether, our results show that the N/C ratio drives cellularization in S. arctica, suggesting that its capacity to control multicellular development predates animal emergence.

Time-resolved small RNA transcriptomics of the ichthyosporean Sphaeroforma arctica.

Ichthyosporea, a clade of holozoans, represent a clade closely related to animals, and thus hold a key phylogenetic position for understanding the origin of animals. We have previously discovered that an ichthyosporean, Sphaeroforma arctica, contains microRNAs (miRNAs) as well as the miRNA processing machinery. This was the first discovery of miRNAs among the closest single-celled relatives of animals and raised intriguing questions about the roles of regulatory small RNAs in cell development and differentiation in unicellular eukaryotes. Like many ichthyosporeans, S. arctica also undergoes a transient multicellular developmental life cycle. As miRNAs are, among other roles, key regulators of gene expression during development in animals, we wanted to investigate the dynamics of miRNAs during the developmental cycle in S. arctica. Here we have therefore collected a comprehensive time-resolved small RNA transcriptome linked to specific life stages with a substantially higher sequencing depth than before, which can enable further discovery of functionally relevant small RNAs. The data consists of Illumina-sequenced small RNA libraries from two independent biological replicates of the entire life cycle of S. arctica with high temporal resolution. The dataset is directly linked and comes from the same samples as a previously published mRNA-seq dataset, thus enabling direct cross-functional analyses.

Infection by a eukaryotic gut parasite in wild Daphnia sp. associates with a distinct bacterial community.

Host-associated bacterial communities play an important role in host fitness and resistance to diseases. Yet, few studies have investigated tripartite interaction between a host, parasite and host-associated bacterial communities in natural settings. Here, we use 16S rRNA gene amplicon sequencing to compare gut- and body- bacterial communities of wild water fleas belonging to the Daphnia longispina complex, between uninfected hosts and those infected with the common and virulent eukaryotic gut parasite Caullerya mesnili (Family: Ichthyosporea). We report community-level changes in host-associated bacteria with the presence of the parasite infection; namely decreased alpha diversity and increased beta diversity at the site of infection, i.e. host gut (but not host body). We also report decreased abundance of bacterial taxa proposed elsewhere to be beneficial for the host, and an appearance of taxa specifically associated with infected hosts. Our study highlights the host-microbiota-infection link in a natural system and raises questions about the role of host-associated microbiota in natural disease epidemics as well as the functional roles of bacteria specifically associated with infected hosts.

A phylogeny based on cytochrome-c oxidase gene sequences identifies sympatric Ichthyophonus genotypes in the NE Pacific Ocean.

In recent decades, evidence has accumulated to suggest that the widespread and highly variable parasite Ichthyophonus hoferi is actually a species complex. Highly plastic morphology and a general lack of defining structures has contributed to the likely underestimate of biodiversity within this group. Molecular methods are a logical next step in the description of these parasites, but markers used to date have been too conserved to resolve species boundaries. Here we use mitochondrial encoded cytochrome-c oxidase (MTCO1) gene sequences and phylogenic analysis to compare Ichthyophonus spp. isolates from several marine and anadromous fish hosts. The resulting phylogeny displays lineage separation among isolates and possible host/niche segregation not previously described. The parasite type that infects Pacific herring Clupea pallasii, Atlantic herring C. harengus, Atlantic salmon Salmo salar, and Pacific staghorn sculpin Oligocottus maculosus (Clade A) is different from that which infects Chinook salmon Oncorhynchus tshawytscha, walleye pollock Gadus chalcogrammus, Greenland halibut Reinhardtius hippoglossoides, and Pacific halibut Hippoglossus stenolepsis (Clade B). MTCO1 sequences confirmed the presence of a more divergent Ichthyophonus sp. isolated from American shad Alosa sapidissima in rivers of eastern North America (Clade C), while American shad introduced to the Pacific Ocean are infected with the same parasite that infects Pacific herring (Clade A). Currently there are no consensus criteria for delimiting species within Ichthyophonidae, but MTCO1 sequences hold promise as a potential species identifying marker and useful epizootiological tool.

Surveillance of densoviruses and mesomycetozoans inhabiting grossly normal tissues of three Aotearoa New Zealand asteroid species.

Asteroid wasting events and mass mortality have occurred for over a century. We currently lack a fundamental understanding of the microbial ecology of asteroid disease, with disease investigations hindered by sparse information about the microorganisms associated with grossly normal specimens. We surveilled viruses and protists associated with grossly normal specimens of three asteroid species (Patiriella regularis, Stichaster australis, Coscinasterias muricata) on the North Island / Te Ika-a-Maui, Aotearoa New Zealand, using metagenomes prepared from virus and ribosome-sized material. We discovered several densovirus-like genome fragments in our RNA and DNA metagenomic libraries. Subsequent survey of their prevalence within populations by quantitative PCR (qPCR) demonstrated their occurrence in only a few (13%) specimens (n = 36). Survey of large and small subunit rRNAs in metagenomes revealed the presence of a mesomycete (most closely matching Ichthyosporea sp.). Survey of large subunit prevalence and load by qPCR revealed that it is widely detectable (80%) and present predominately in body wall tissues across all 3 species of asteroid. Our results raise interesting questions about the roles of these microbiome constituents in host ecology and pathogenesis under changing ocean conditions.

Ichthyophonosis in Peruvian rainbow trout Oncorhynchus mykiss: identification of endemic areas using molecular and histopathological tools.

Ichthyophonus infection was first detected in Peruvian Oncorhynchus mykiss in 1986, but the occurrence of ichthyophonosis disease in the region is unknown. This study investigated the presence and distribution of Ichthyophonus sp. in Peruvian rainbow trout using traditional and DNA sequencing tools. Between 2007 and 2008, 205 rainbow trout from 13 hatcheries in the Mantaro river basin were examined for the presence of Ichthyophonus, and at that time only 3 farms were positive. This early study confirmed the presence of Ichthyophonus sp. in the Tranca Grande lagoon for the first time, at a prevalence of 50%. In 2012, examination of 240 trout from 24 fish farms in 2 Peruvian Departments found 9 infected farms. More recently, in 2018, Ichthyophonus sp. was found in Lake Titicaca, infecting a trout in the Ichu area (in the Department of Puno). Our molecular analysis of the infected trout showed that ichthyophonosis disease in the Peruvian trout was caused by Ichthyophonus sp. Clade C. The finding of this pathogen in Lake Titicaca should be an alert for nearby farms and entities dealing with fish of economic importance in the rivers of Peru.

Infection of the Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) with a dermocystid parasite in the Tisza River Basin, Hungary.

The Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) is an endemic protected species of Cephalaspidomorphi in the Carpathian Basin. No parasites have become known from these jawless vertebrates to date. Here, the authors describe an infection from a single specimen manifesting in protuberant skin cysts 7-10 mm in diameter, scattered on the body surface. Similar dermal infection was observed in 25 of the 274 lampreys recorded in the population survey. Skin cysts filled with round spore-like structures of a dermocystid parasite were found. These particles measured 8-14 µm in diameter and had an about 0.5 µm thick wall, and containing mainly a granular mass and a relatively scarce plasma. No hyphae were recorded. Despite conspicuous morphological changes in the skin, no inflammatory reactions were found. The molecular analysis of 18S rDNA showed similarity to dermocystid species of several fish species but differed from them approximately by 2%. This is the first record of a dermocystid parasite infecting a jawless vertebrate.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

The complete mitochondrial genome of a parasite at the animal-fungal boundary.

BACKGROUND: Sphaerothecum destruens is an obligate intracellular fish parasite which has been identified as a serious threat to freshwater fishes. Taxonomically, S. destruens belongs to the order Dermocystida within the class Ichthyosporea (formerly referred to as Mesomycetozoea), which sits at the animal-fungal boundary. Mitochondrial DNA (mtDNA) sequences can be valuable genetic markers for species detection and are increasingly used in environmental DNA (eDNA) based species detection. Furthermore, mtDNA sequences can be used in epidemiological studies by informing detection, strain identification and geographical spread. METHODS: We amplified the entire mitochondrial (mt) genome of S. destruens in two overlapping long fragments using primers designed based on the cox1, cob and nad5 partial sequences. The mt-genome architecture of S. destruens was then compared to close relatives to gain insights into its evolution. RESULTS: The complete mt-genome of Sphaerothecum destruens is 23,939 bp in length and consists of 47 genes including 21 protein-coding genes, 2 rRNA, 22 tRNA and two unidentified open reading frames. The mitochondrial genome of S. destruens is intronless and compact with a few intergenic regions and includes genes that are often missing from animal and fungal mt-genomes, such as, the four ribosomal proteins (small subunit rps13 and 14; large subunit rpl2 and 16), tatC (twin-arginine translocase component C), and ccmC and ccmF (cytochrome c maturation protein ccmC and heme lyase). CONCLUSIONS: We present the first mt-genome of S. destruens which also represents the first mt-genome for the order Dermocystida. The availability of the mt-genome can assist the detection of S. destruens and closely related parasites in eukaryotic diversity surveys using eDNA and assist epidemiological studies by improving molecular detection and tracking the parasite's spread. Furthermore, as the only representative of the order Dermocystida, its mt-genome can be used in the study of mitochondrial evolution of the unicellular relatives of animals.

Ichthyophonus sp. Infection in Opaleye (Girella nigricans).

Over a 3-year-period, 17 wild-caught opaleye (Girella nigricans) housed in a public display aquarium were found dead without premonitory signs. Grossly, 4 animals had pinpoint brown or black foci on coelomic adipose tissue. Histologically, liver, spleen, heart, and posterior kidney had mesomycetozoan granulomas in all cases; other organs were less commonly infected. Four opaleye had goiter; additional substantial lesions were not identified. Granulomas surrounded melanized debris, leukocytes, and mesomycetozoa represented by folded membranes (collapsed schizont walls), intact schizonts (50- to >200 µm in diameter with a multilaminate membrane), plasmodia (budding from schizonts or free in tissue), or rarely germinal tubes (budding from schizonts). Ichthyophonus was grown from fresh tissues in tissue explant broth cultures of the heart, liver, and/or spleen. Polymerase chain reaction using 18S ribosomal DNA primers amplified a 1730-bp region, and the DNA sequence was most similar to Ichthyophonus hoferi, which is often associated with freshwater aquaculture fish.

Characterization of Dermotheca sp. Infection in a midwestern state-endangered salamander (Ambystoma platineum) and a co-occurring common species (Ambystoma texanum).

Ichthyosporean parasites (order Dermocystida) can cause morbidity and mortality in amphibians, but their ecology and epidemiology remain understudied. We investigated the prevalence, gross and histologic appearance, and molecular phylogeny of a novel dermocystid in the state-endangered silvery salamander (Ambystoma platineum) and the co-occurring, non-threatened small-mouthed salamander (Ambystoma texanum) from Illinois. Silvery salamanders (N = 610) were sampled at six ephemeral wetlands from 2016 to 2018. Beginning in 2017, 1-3 mm raised, white skin nodules were identified in 24 silvery salamanders and two small-mouthed salamanders from five wetlands (prevalence = 0-11.1%). Skin biopsy histology (N = 4) was consistent with dermocystid sporangia, and necropsies (N = 3) identified infrequent hepatic sporangia. Parasitic 18S rRNA sequences (N = 5) from both salamander species were identical, and phylogenetic analysis revealed a close relationship to Dermotheca viridescens. Dermocystids were not identified in museum specimens from the same wetlands (N = 125) dating back to 1973. This is the first report of Dermotheca sp. affecting caudates in the Midwestern United States. Future research is needed to determine the effects of this pathogen on individual and population health, and to assess whether this organism poses a threat to the conservation of ambystomatid salamanders.

Longitudinal study of Amphibiocystidium sp. infection in a natural population of the Italian stream frog (Rana italica).

Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.

Unicellular Origin of the Animal MicroRNA Machinery.

The emergence of multicellular animals was associated with an increase in phenotypic complexity and with the acquisition of spatial cell differentiation and embryonic development. Paradoxically, this phenotypic transition was not paralleled by major changes in the underlying developmental toolkit and regulatory networks. In fact, most of these systems are ancient, established already in the unicellular ancestors of animals [1-5]. In contrast, the Microprocessor protein machinery, which is essential for microRNA (miRNA) biogenesis in animals, as well as the miRNA genes themselves produced by this Microprocessor, have not been identified outside of the animal kingdom [6]. Hence, the Microprocessor, with the key proteins Pasha and Drosha, is regarded as an animal innovation [7-9]. Here, we challenge this evolutionary scenario by investigating unicellular sister lineages of animals through genomic and transcriptomic analyses. We identify in Ichthyosporea both Drosha and Pasha (DGCR8 in vertebrates), indicating that the Microprocessor complex evolved long before the last common ancestor of animals, consistent with a pre-metazoan origin of most of the animal developmental gene elements. Through small RNA sequencing, we also discovered expressed bona fide miRNA genes in several species of the ichthyosporeans harboring the Microprocessor. A deep, pre-metazoan origin of the Microprocessor and miRNAs comply with a view that the origin of multicellular animals was not directly linked to the innovation of these key regulatory components.

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

Transfection of Capsaspora owczarzaki, a close unicellular relative of animals.

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.

Phylogenetic and environmental DNA insights into emerging aquatic parasites: implications for risk management.

Species translocation leads to disease emergence in native species of considerable economic importance. Generalist parasites are more likely to be transported, become established and infect new hosts, thus their risk needs to be evaluated. Freshwater systems are particularly at risk from parasite introductions due to the frequency of fish movements, lack of international legislative controls for non-listed pathogens and inherent difficulties with monitoring disease introductions in wild fish populations. Here we used one of the world's most invasive freshwater fish, the topmouth gudgeon, Pseudorasbora parva, to demonstrate the risk posed by an emergent generalist parasite, Sphaerothecum destruens. Pseudorasbora parva has spread to 32 countries from its native range in China through the aquaculture trade and has introduced S. destruens to at least five of these. We systematically investigated the spread of S. destruens through Great Britain and its establishment in native fish communities through a combination of phylogenetic studies of the host and parasite and a novel environmental DNA detection assay. Molecular approaches confirmed that S. destruens is present in 50% of the P. parva communities tested and was also detected in resident native fish communities but in the absence of notable histopathological changes. We identified specific P. parva haplotypes associated with S. destruens and evaluated the risk of disease emergence from this cryptic fish parasite. We provide a framework that can be applied to any aquatic pathogen to enhance detection and help mitigate future disease risks in wild fish populations.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum (Scotland).

Outbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infected L. helveticus in France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction from Amphibiocystidium spp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that the L. helveticus-infecting pathogen represents a single, novel species; Amphibiothecum meredithae.