Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria.

BACKGROUND: Ascetosporea (Endomyxa, Rhizaria) is a group of unicellular parasites infecting aquatic invertebrates. They are increasingly being recognized as widespread and important in marine environments, causing large annual losses in invertebrate aquaculture. Despite their importance, little molecular data of Ascetosporea exist, with only two genome assemblies published to date. Accordingly, the evolutionary origin of these parasites is unclear, including their phylogenetic position and the genomic adaptations that accompanied the transition from a free-living lifestyle to parasitism. Here, we sequenced and assembled three new ascetosporean genomes, as well as the genome of a closely related amphizoic species, to investigate the phylogeny, origin, and genomic adaptations to parasitism in Ascetosporea. RESULTS: Using a phylogenomic approach, we confirm the monophyly of Ascetosporea and show that Paramyxida group with Mikrocytida, with Haplosporida being sister to both groups. We report that the genomes of these parasites are relatively small (12-36 Mb) and gene-sparse (~ 2300-5200 genes), while containing surprisingly high amounts of non-coding sequence (~ 70-90% of the genomes). Performing gene-tree aware ancestral reconstruction of gene families, we demonstrate extensive gene losses at the origin of parasitism in Ascetosporea, primarily of metabolic functions, and little gene gain except on terminal branches. Finally, we highlight some functional gene classes that have undergone expansions during evolution of the group. CONCLUSIONS: We present important new genomic information from a lineage of enigmatic but important parasites of invertebrates and illuminate some of the genomic innovations accompanying the evolutionary transition to parasitism in this lineage. Our results and data provide a genetic basis for the development of control measures against these parasites.

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Diversity of myxozoans (Cnidaria) infecting Neotropical fishes in southern Mexico.

Myxozoans are a unique group of microscopic parasites that infect mainly fishes. These extremely reduced cnidarians are highly diverse and globally distributed in freshwater and marine habitats. Myxozoan diversity dimension is unknown in Mexico, a territory of an extraordinary biological diversity. This study aimed to explore, for the first time, myxozoan parasite diversity from fishes of the Neotropical region of Mexico. We performed a large morphological and molecular screening using host tissues of 22 ornamental and food fish species captured from different localities of Veracruz, Oaxaca and Chiapas. Myxozoan infections were detected in 90% of the fish species, 65% of them had 1 or 2 and 35% had 3 and up to 8 myxozoan species. Forty-one putative new species were identified using SSU rDNA phylogenetic analyses, belonging to two main lineages: polychaete-infecting (5 species) and oligochaete-infecting (36 species) myxozoans; from those we describe 4 new species: Myxidium zapotecus sp. n., Zschokkella guelaguetza sp. n., Ellipsomyxa papantla sp. n. and Myxobolus zoqueus sp. n. Myxozoan detection increased up to 6 × using molecular screening, which represents 3.7 × more species detected than by microscopy. This study demonstrated that Neotropical fishes from Mexico are hosts of a multitude of myxozoans, representing a source of emerging diseases with large implications for economic and conservation reasons.

Correlated evolution of fish host length and parasite spore size: a tale from myxosporeans inhabiting elasmobranchs.

Myxozoa represent a diverse group of microscopic cnidarian endoparasites alternating between invertebrate and vertebrate hosts. Of the approximately 2,600 species described predominantly from teleost fish, only 1.8% have been reported from cartilaginous fishes (Elasmobranchii). As ancestral vertebrate hosts of myxozoans, elasmobranchs may have played an important role in myxozoan evolution, however, they are also some of the largest vertebrate hosts known for this group of parasites. We screened 50 elasmobranchs belonging to nine species and seven families, from various geographical areas, for myxozoan infection. We found a 22% overall prevalence of myxozoans in elasmobranchs and describe five species new to science. We investigated, for the first known time, the evolution of spore size within three phylogenetic clades, Ceratomyxa, Sphaerospora sensu stricto and Parvicapsula. We found that spores from elasmobranch-infecting myxozoans were on average 4.8× (Ceratomyxa), 2.2× (Parvicapsula clade) and 1.8× (Sphaerospora sensu stricto except polysporoplasmic Sphaerospora spp.) larger than those from teleosts. In all analysed clades, spore size was correlated with phylogenetic position. In ceratomyxids, it was further strongly positively correlated with fish body size and habitat depth, independent of cellular composition of the spores and phylogenetic position in the tree. While in macroparasites a host size-correlated increase in parasite size occurs on a large scale and is often related to improved exploitation of host resources, in microscopic parasites size ranges vary at the scale of a few micrometres, disproportionate to the available additional space in a large host. We discuss the ecological role of these changes with regard to transmission under high pressure and an invertebrate fauna that is adapted to deeper marine habitats.

Natural Feed Additives Modulate Immunity and Mitigate Infection with Sphaerospora molnari (Myxozoa:Cnidaria) in Common Carp: A Pilot Study.

Myxozoans are a diverse group of cnidarian parasites, including important pathogens in different aquaculture species, without effective legalized treatments for fish destined for human consumption. We tested the effect of natural feed additives on immune parameters of common carp and in the course of a controlled laboratory infection with the myxozoan Sphaerospora molnari. Carp were fed a base diet enriched with 0.5% curcumin or 0.12% of a multi-strain yeast fraction, before intraperitoneal injection with blood stages of S. molnari. We demonstrate the impact of these treatments on respiratory burst, phagocytosis, nitric oxide production, adaptive IgM+ B cell responses, S. molnari-specific antibody titers, and on parasite numbers. Both experimental diets enriched B cell populations prior to infection and postponed initial parasite proliferation in the blood. Curcumin-fed fish showed a decrease in reactive oxygen species, nitric oxide production and B cell density at late-stage infection, likely due to its anti-inflammatory properties, favoring parasite propagation. In contrast, multi-strain yeast fraction (MsYF)-fed fish harbored the highest S. molnari-specific antibody titer, in combination with the overall lowest parasite numbers. The results demonstrate that yeast products can be highly beneficial for the outcome of myxozoan infections and could be used as effective feed additives in aquaculture.

Transcriptome of Sphaerospora molnari (Cnidaria, Myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp.

BACKGROUND: Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such as Sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of a S. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify S. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. RESULTS: An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. CONCLUSIONS: In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study of S. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

Mechanisms and Drivers for the Establishment of Life Cycle Complexity in Myxozoan Parasites.

It is assumed that complex life cycles in cnidarian parasites belonging to the Myxozoa result from incorporation of vertebrates into simple life cycles exploiting aquatic invertebrates. However, nothing is known about the driving forces and implementation of this event, though it fostered massive diversification. We performed a comprehensive search for myxozoans in evolutionary ancient fishes (Chondrichthyes), and more than doubled existing 18S rDNA sequence data, discovering seven independent phylogenetic lineages. We performed cophylogenetic and character mapping methods in the largest monophyletic dataset and demonstrate that host and parasite phylogenies are strongly correlated, and that tectonic changes may explain phylogeographic clustering in recent skates and softnose skates, in the Atlantic. The most basal lineages of myxozoans inhabit the bile of chondrichthyans, an immunologically privileged site and protective niche, easily accessible from the gut via the bile duct. We hypothesize that feed-integration is a likely mechanism of host acquisition, an idea supported by feeding habits of chimaeras and ancient sharks and by multiple entries of different parasite lineages from invertebrates into the new host group. We provide exciting first insights into the early evolutionary history of ancient metazoan parasites in a host group that embodies more evolutionary distinctiveness than most other vertebrates.

The description of two new species of Chloromyxum from skates in the Argentine Sea reveals that a limited geographic host distribution causes phylogenetic lineage separation of myxozoans in Chondrichthyes.

During a survey on the myxosporean fauna of Rajiformes from the Atlantic coast of Argentina, in waters off Buenos Aires Province (34°-42°S; 53°-62°W), the gall bladders of 217 specimens belonging to seven species of skates, representatives of two families, were examined. As a result, three species of Chloromyxum Mingazzini, 1890, namely C. atlantoraji n. sp., C. zearaji n. sp. and C. riorajum Azevedo, Casal, Garcia, Matos, Teles-Grilo and Matos, 2009 were found infecting three endemic host species, the spotback skate Atlantoraja castelnaui (Arhynchobatidae), the yellownose skate Zearaja chilensis (Rajidae) and the Rio skate Rioraja agassizii (Arhynchobatidae), respectively. These species were described based on myxospore morphology and morphometry characterization, as well as by providing their small subunit ribosomal DNA (SSU rDNA) sequences. The SSU rDNA-based phylogenetic analyses showed that these three species constituted a well-established monophyletic subclade within the marine Chloromyxum clade, while branches subtending the other Chloromyxum species were poorly resolved or unresolved, independently of the host taxonomic identities (Carchariniformes, Myliobatiformes, Orectolobiformes, Pristiophoriformes, Rajiformes, Squaliformes and Torpediniformes) and/or host geographic distribution (Atlantic coast of Portugal, Atlantic coast of the USA, Australian waters or Mediterranean Sea). The possible causes of these discrepancies are discussed, providing new insights into the phylogeny of the marine Chloromyxum clade.

Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa).

BACKGROUND: Myxozoa are extremely diverse microscopic parasites belonging to the Cnidaria. Their life-cycles alternate between vertebrate and invertebrate hosts, predominantly in aquatic habitats. Members of the phylogenetically well-defined Sphaerospora (sensu stricto) clade predominantly infect the urinary system of marine and freshwater fishes and amphibians. Sphaerosporids are extraordinary due to their extremely long and unique insertions in the variable regions of their 18S and 28S rDNA genes and due to the formation of motile proliferative stages in the hosts' blood. To date, DNA sequences of only 19 species have been obtained and information on the patterns responsible for their phylogenetic clustering is limited. METHODS: We screened 549 fish kidney samples from fish of various geographical locations, mainly in central Europe, to investigate sphaerosporid biodiversity microscopically and by 18S rDNA sequences. We performed multiple phylogenetic analyses to explore phylogenetic relationships and evolutionary trends within the Sphaerospora (s.s.) clade, by matching host and habitat features to the resultant 18S rDNA trees. The apparent co-clustering of species from related fish hosts inspired us to further investigate host-parasite co-diversification, using tree-based (CoRE-PA) and distance-based (ParaFit) methods. RESULTS: Our study considerably increased the number of 18S rDNA sequence data for Sphaerospora (s.s.) by sequencing 17 new taxa. Eight new species are described and one species (Sphaerospora diminuta Li & Desser, 1985) is redescribed, accompanied by sufficient morphological data. Phylogenetic analyses showed that sphaerosporids cluster according to their vertebrate host order and habitat, but not according to geography. Cophylogenetic analyses revealed a significant congruence between the phylogenetic trees of sphaerosporids and of their vertebrate hosts and identified Cypriniformes as a host group of multiple parasite lineages and with high parasite diversity. CONCLUSIONS: This study significantly contributed to our knowledge of the biodiversity and evolutionary history of the members of the Sphaerospora (s.s.) clade. The presence of two separate phylogenetic lineages likely indicates independent historical host entries, and the remarkable overlap of the larger clade with vertebrate phylogeny suggests important coevolutionary adaptations. Hyperdiversification of sphaerosporids in cypriniform hosts, which have undergone considerable radiations themselves, points to host-driven diversification.

Repatriation of an old fish host as an opportunity for myxozoan parasite diversity: The example of the allis shad, Alosa alosa (Clupeidae), in the Rhine.

BACKGROUND: Wildlife repatriation represents an opportunity for parasites. Reintroduced hosts are expected to accumulate generalist parasites via spillover from reservoir hosts, whereas colonization with specialist parasites is unlikely. We address the question of how myxozoan parasites, which are characterized by a complex life-cycle alternating between annelids and fish, can invade a reintroduced fish species and determine the impact of a de novo invasion on parasite diversity. We investigated the case of the anadromous allis shad, Alosa alosa (L.), which was reintroduced into the Rhine approximately 70 years after its extinction in this river system. METHODS: We studied parasites belonging to the Myxozoa (Cnidaria) in 196 allis shad from (i) established populations in the French rivers Garonne and Dordogne and (ii) repatriated populations in the Rhine, by screening the first adults returning to spawn in 2014. Following microscopical detection of myxozoan infections general myxozoan primers were used for SSU rDNA amplification and sequencing. Phylogenetic analyses were performed and cloned sequences were analyzed from individuals of different water sources to better understand the diversity and population structure of myxozoan isolates in long-term coexisting vs recently established host-parasite systems. RESULTS: We describe Hoferellus alosae n. sp. from the renal tubules of allis shad by use of morphological and molecular methods. A species-specific PCR assay determined that the prevalence of H. alosae n. sp. is 100 % in sexually mature fish in the Garonne/Dordogne river systems and 22 % in the first mature shad returning to spawn in the Rhine. The diversity of SSU rDNA clones of the parasite was up to four times higher in the Rhine and lacked a site-specific signature of SNPs such as in the French rivers. A second myxozoan, Ortholinea sp., was detected exclusively in allis shad from the Rhine. CONCLUSIONS: Our data demonstrate that the de novo establishment of myxozoan infections in rivers is slow but of great genetic diversity, which can only be explained by the introduction of spores from genetically diverse sources, predominantly via straying fish or by migratory piscivorous birds. Long-term studies will show if and how the high diversity of a de novo introduction of host-specific myxozoans succeeds into the establishment of a local successful strain in vertebrate and invertebrate hosts.

Species complexes and phylogenetic lineages of Hoferellus (Myxozoa, Cnidaria) including revision of the genus: A problematic case for taxonomy.

BACKGROUND: Myxozoans are metazoan parasites whose traditional spore morphology-based taxonomy conflicts DNA based phylogenies. Freshwater species of the genus Hoferellus are parasites of the excretory system, with several members infecting food and ornamental fish species, as well as amphibians. This study aims to increase our understanding of their molecular diversity and development, aspects about which little is known, and to generate a molecular diagnostic tool to discriminate between different pathogenic and non-pathogenic Hoferellus spp. METHODS: SSU and ITS rDNA phylogeny, along with morphological descriptions using light and electron microscopy were used to identify and characterize Hoferellus species collected from the urinary system of fishes and frogs. A PCR-based diagnostic assay was designed to differentiate between cryptic Hoferellus spp in cyprinid fishes commonly cultured in Central Europe. RESULTS: Our phylogenetic results separate the species of Hoferellus into two phylogenetic sublineages which are indistinguishable on the basis of generic morphological traits: 1) The Hoferellus sensu stricto sublineage, which is composed of the type species Hoferellus cyprini, Hoferellus carassii and a cryptic species, Hoferellus sp. detected only molecularly in common carp. 2) The Hoferellus sensu lato sublineage into which the new species we described in this study, Hoferellus gnathonemi sp. n. from the kidney of the elephantnose fish and Hoferellus anurae from reed frogs, are placed together with Hoferellus gilsoni previously sequenced from European eel. Apart from phylogenetic analyses, we also provide novel ultrastructural data on the phagocytotic nature of some Hoferellus plasmodia and on the elusive intracellular stages ascribed to the presporogonic development of this genus. CONCLUSIONS: We provide molecular evidence of the polyphyly of the genus Hoferellus and provide novel morphological details of its members. Based on the presented data, we revise and propose emendation of the genus Hoferellus.

Morphology and phylogeny of two new species of Sphaeromyxa Thélohan, 1892 (Cnidaria: Myxozoa) from marine fish (Clinidae and Trachichthyidae).

Our survey of marine fish from South Africa and Indonesia revealed the presence of two new myxosporean species of the genus Sphaeromyxa for which we provide morphological and sequence data. Sphaeromyxa clini n. sp. detected in three Clinus spp. and Muraenoclinus dorsalis from South Africa is morphologically similar to Sphaeromyxa noblei previously described from Heteroclinus whiteleggii from Australia and to several other sphaeromyxids with arcuate spores and rounded ends. This similarity is reflected by phylogenetic positioning of S. clini n. sp. which clusters within the 'incurvata' group of the Sphaeromyxa clade. It differs from morphologically similar species by spore and polar capsule dimensions, host specificity and geographic distribution. Sphaeromyxa limocapitis n. sp., described from Gephyroberyx darwinii from Java, is morphologically similar to sphaeromyxids with straight spores and to marine Myxidium species with spindle-shaped spores but differs from them by spore and polar capsule dimensions, host specificity and geographic distribution. S. limocapitis n. sp. represents a separate lineage of the Sphaeromyxa clade and appears to be a missing link in the evolution of sphaeromyxids.

Molecular fingerprinting of the myxozoan community in common carp suffering swim bladder inflammation (SBI) identifies multiple etiological agents.

BACKGROUND: Swim bladder inflammation (SBI) is an important disease of common carp fingerlings in Central Europe. In the 1980s, its etiology was ascribed to multicellular proliferative stages of the myxozoan parasite Sphaerospora dykovae (formerly S. renicola). S. dykovae was reported to proliferate in the blood and in the swim bladder prior to the invasion of the kidney, where sporogony takes place. Due to the presence of emerging numbers of proliferative myxozoan blood stages at different carp culture sites in recent years we analysed cases of SBI, for the first time, using molecular diagnostics, to identify the myxozoan parasites present in diseased swim bladders. METHODS: We amplified myxozoan SSU rDNA in a non-specific approach and compared the species composition in swim bladders at culture sites where carp demonstrated 1. No signs of SBI, 2. Minor pathological changes, and 3. Heavy SBI. Based on DNA sequences, we determined the localisation and distribution of the most frequent species by in situ hybridisation, thereby determining which myxozoans are involved in SBI. RESULTS: Large multicellular myxozoan swim bladder stages characterised heavy SBI cases and were identified as S. dykovae, however, blood stages were predominantly represented by Sphaerospora molnari, whose numbers were greatly increased in carp with mild and heavy SBI, compared with SBI-free fish. S. molnari was found to invade different organs and cause inflammatory changes also in the absence of S. dykovae. One site with mild SBI cases was characterised by Buddenbrockia sp. infection in different organs and a general granulomatous response. CONCLUSIONS: We provide evidence that the etiology of SBI can vary in relation to culture site and disease severity and that emerging numbers of S. molnari in the blood represent an important co-factor or precondition for SBI.

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics.

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.

Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain.

A total of 109 sea urchins from 3 species collected in 2 localities off the coast of Tenerife Island, Spain, were examined for the presence of free-living amoebae in their coelomic fluid. Amoeba trophozoites were isolated exclusively from moribund individuals of long-spined sea urchins Diadema aff. antillarum (Philippi) (Echinoidea, Echinodermata) that manifested lesions related to sea urchin bald disease on their tests (16 out of 56 examined). No amoebae were detected in Arbacia lixula (L.) and Paracentrotus lividus (Lamarck). From the former sea urchin species, 8 strains, established from 10 primary isolates, were identified as Neoparamoeba branchiphila Dyková et al., 2005 using morphological and molecular methods. Results of this study (limited to the screening for free-living amoebae) together with data on agents of sea urchin mortalities reported to date justify the hypothesis that free-living amoebae play an opportunistic role in D. aff. antillarum mortality. The enlargement of the dataset of SSU rDNA sequences brought new insight into the phylogeny of Neoparamoeba species.

New data on aetiology of nodular gill disease in rainbow trout, Oncorhynchus mykiss.

We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.

Strains of Uronema marinum (Scuticociliatia) co-isolated with amoebae of the genus Neoparamoeba.

Mixed infections of histophagous ciliates and Neoparamoeba spp. Page, 1987 were diagnosed in gill tissue of farmed turbot Psetta maxima (synonym: Scophthalmus maximus) and Atlantic salmon Salmo salar during a study of amoebic gill disease. Ciliates co-isolated from lesions grossly visible on gills of both fish hosts and from 2 species of red algae Lithophyllum racemus and Palmaria palmata were characterized morphologically and by using molecular markers. Sequences of small subunit (SSU) rDNA were determined for 6 strains of ciliates isolated from hosts collected in geographically distant localities. Of these, sequences of 5 strains revealed a surprisingly high level of similarity and identified the corresponding strains with Uronema marinum Lynn et Small, 1997. Thus, the set of environmental sequences of U. marinum available in the GenBank database to date was supplemented with the first sequences of potentially histophagous strains. On the basis of SSU rDNA, the 6th strain, also isolated from affected fish gills, was identified as Aristerostoma sp.

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov, Nassonova et Cavalier-Smith, 2008.

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.

Living together: the marine amoeba Thecamoeba hilla Schaeffer, 1926 and its endosymbiont Labyrinthula sp.

Two protists isolated simultaneously from the same sample of gill tissue of Psetta maxima (L.) were identified as Thecamoeba hilla Schaeffer, 1926 and Labyrinthula sp. A Labyrinthula strain (LTH) derived from a mixed culture of both organisms was well established in a short time, while subcultures of T. hilla continued to be associated with Labyrinthula cells despite all efforts to eliminate them. Ultrastructural examination, repeated several times in the course of long-lasting subculturing of amoebae, revealed that trophozoites of T. hilla host in their cytoplasm multiplying labyrinthulid cells. Comparison of SSU rDNA sequences of the Labyrinthula strain LTH and those from labyrinthulid endosymbionts from T. hilla verified the assumption that the extra- and intra-cellularly multiplying Labyrinthula cells are identical organisms. The association of the marine amoeba T. hilla and Labyrinthula sp. displayed signs of mutualistic symbiosis.

Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): coevolution demonstrated by SSU rRNA gene phylogenies.

The molecular phylogeny of Neoparamoeba spp. based on SSU rDNA was updated by including new sequences of strains isolated from an invertebrate and an alga. In total, 59 sequences of strains representating N. pemaquidensis, N. branchiphila, N. aestuarina and N. perurans were analysed. Sequences of SSU rDNA of eukaryotic endosymbionts (Perkinsela amoebae-like organisms) were prepared from 34 samples of genomic DNA of strain-representatives of N. pemaquidensis, N. branchiphila and N. aestuarina. Comparison of phylograms reconstructed from corresponding SSU rDNA sequences of host amoebae and their symbionts revealed a high level of congruence, which argues very strongly for coevolution of these two eukaryotic organisms.