Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia, Heterolobosea.

The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Chlamydial seasonal dynamics and isolation of 'Candidatus†Neptunochlamydia vexilliferae' from a Tyrrhenian coastal lake.

The Chlamydiae are a phylum of obligate intracellular bacteria comprising important human and animal pathogens, yet their occurrence in the environment, their phylogenetic diversity and their host range has been largely underestimated. We investigated the seasonality of environmental chlamydiae in a Tyrrhenian coastal lake. By catalysed reporter deposition fluorescence in situ hybridization, we quantified the small planktonic cells and detected a peak in the abundance of environmental chlamydiae in early autumn with up to 5.9 × 10(4) cells ml(-1) . Super-resolution microscopy improved the visualization and quantification of these bacteria and enabled the detection of pleomorphic chlamydial cells in their protist host directly in an environmental sample. To isolate environmental chlamydiae together with their host, we applied a high-throughput limited dilution approach and successfully recovered a Vexillifera sp., strain harbouring chlamydiae (93% 16S rRNA sequence identity to Simkania negevensis), tentatively named 'Candidatus Neptunochlamydia vexilliferae'. Transmission electron microscopy in combination with fluorescence in situ hybridization was used to prove the intracellular location of these bacteria representing the first strain of marine chlamydiae stably maintained alongside with their host in a laboratory culture. Taken together, this study contributes to a better understanding of the distribution and diversity of environmental chlamydiae in previously neglected marine environments.

Vermistella arctica n. sp. Nominates the Genus Vermistella as a Candidate for Taxon with Bipolar Distribution.

A new amoebozoan species, Vermistella arctica n. sp., is described from marine habitats in the central part of Svalbard archipelago. This is the first report on Arctic amoebae belonging to the genus Vermistella Moran and Anderson, 2007, the type species of which was described from the opposite pole of the planet. Psychrophily proved in the new strains qualifies the genus Vermistella as a bipolar taxon. Molecular phylogenetic analyses based on 18S rDNA and actin sequences did not show any affinity of the genus Vermistella to Stygamoeba regulata ATCC(®) 50892(™) strain. A close phylogenetic relationship was found between Vermistella spp. and a sequence originating from an environmental sample from Cariaco basin, the largest marine permanently anoxic system in the world. Possible mechanisms of bipolar distribution are discussed.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium.

BACKGROUND: Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. RESULTS: We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. CONCLUSIONS: Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract.

Examining the Relationship Between the Testate Amoeba Hyalosphenia papilio (Arcellinida, Amoebozoa) and its Associated Intracellular Microalgae Using Molecular and Microscopic Methods.

Symbiotic relationships between heterotrophic and phototrophic partners are common in microbial eukaryotes. Among Arcellinida (Amoebozoa) several species are associated with microalgae of the genus Chlorella (Archaeplastida). So far, these symbioses were assumed to be stable and mutualistic, yet details of the interactions are limited. Here, we analyzed 22 single-cell transcriptomes and 36 partially-sequenced genomes of the Arcellinida morphospecies Hyalosphenia papilio, which contains Chlorella algae, to shed light on the amoeba-algae association. By characterizing the genetic diversity of associated Chlorella, we detected two distinct clades that can be linked to host genetic diversity, yet at the same time show a biogeographic signal across sampling sites. Fluorescence and transmission electron microscopy showed the presence of intact algae cells within the amoeba cell. Yet analysis of transcriptome data suggested that the algal nuclei are inactive, implying that instead of a stable, mutualistic relationship, the algae may be temporarily exploited for photosynthetic activity before being digested. Differences in gene expression of H. papilio and Hyalosphenia elegans demonstrated increased expression of genes related to oxidative stress. Together, our analyses increase knowledge of this host-symbiont association and reveal 1) higher diversity of associated algae than previously characterized, 2) a transient association between H. papilio and Chlorella with unclear benefits for the algae, 3) algal-induced gene expression changes in the host.

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.

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus (Ca.) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Xenoma-like formations induced by Soricimyxum fegati (Myxosporea) in three species of shrews (Soricomorpha: Soricidae), including records of new hosts.

In South Bohemia, Czech Republic, 178 shrews, including 98 common shrews, Sorex araneus L., 70 pygmy shrews, Sorex minutus L., and 10 lesser white-toothed shrews, Crocidura suaveolens (Pallas), were examined for Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 infections, using squash preparations of unfixed tissues, histological sections and molecular methods. The infection was found in 51 (52%) S. araneus, 14 (20%) S. minutus and 1 (10%) C. suaveolens. The records of the latter two species extend host range of S. fegati. Lesions associated with S. fegati infections in the liver, the organ of specific localisation of the parasite, were found to be induced by proliferative stages migrating toward lumina of bile ducts. In other organs of these three host species, xenoma-like formations (XLFs) were found that severely injured blood vessels. XLFs contained presporogonic stages of S. fegati, whose species identity was evidenced using molecular methods.

Parasitic flatworms infecting thorny skate, Amblyraja radiata: Infection by the monogeneans Acanthocotyle verrilli and Rajonchocotyle emarginata in Svalbard.

Parasite diversity above the Arctic circle remains understudied even for commercially valuable host taxa. Thorny skate, Amblyraja radiata, is a common bycatch species with a growing commercial value. Its natural range covers both sides of the North Atlantic including the Arctic zone. Svalbard is a Norwegian archipelago located on the northwest corner of the Barents Shelf which sustains a spectacular species diversity. So far, several monogenean species have been reported infecting thorny skate across the Atlantic Ocean. In the present study, we intend to fill in the knowledge gap on monogenean parasites infecting thorny skate in the northern part of its range and thus indirectly assess the connectivity between the thorny skate populations off the Svalbard coast and from previously studied locations. 46 monogenean individuals were recovered from 11 specimens of thorny skate. Following morphological and molecular assessment, two species of monogeneans, Acanthocotyle verrilli and Rajonchocotyle emarginata, were identified. The results serve as the northernmost record for both parasite genera and the first record of monogenean species off Svalbard. Detailed morphometric evaluation revealed a relatively high level of morphological variation in A. verrilli compared to its congeners. Phylogenetic reconstruction placed A. verrilli in a well-supported clade with A. imo. Our study also suggests high diagnostic significance of sclerotised structures in the identification of Rajonchocotyle. Even though the occurrence of two directly transmitted parasite species supports the previously suggested long-distance migration of A. radiata, future studies employing highly variable genetic markers are needed to assess the ongoing and historical migration patterns.

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.

A single-cell genome perspective on studying intracellular associations in unicellular eukaryotes.

Single-cell genomics (SCG) methods provide a unique opportunity to analyse whole genome information at the resolution of an individual cell. While SCG has been extensively used to investigate bacterial and archaeal genomes, the technique has been rarely used to access the genetic makeup of uncultivated microbial eukaryotes. In this regard, the use of SCG can provide a wealth of information; not only do the methods allow exploration of the genome, they can also help elucidate the relationship between the cell and intracellular entities extant in nearly all eukaryotes. SCG enables the study of total eukaryotic cellular DNA, which in turn allows us to better understand the evolutionary history and diversity of life, and the physiological interactions that define complex organisms. This article is part of a discussion meeting issue 'Single cell ecology'.

Population co-divergence in common cuttlefish (Sepia officinalis) and its dicyemid parasite in the Mediterranean Sea.

Population structure and biogeography of marine organisms are formed by different drivers than in terrestrial organisms. Yet, very little information is available even for common marine organisms and even less for their associated parasites. Here we report the first analysis of population structure of both a cephalopod host (Sepia officinalis) and its dicyemid parasite, based on a homologous molecular marker (cytochrome oxidase I). We show that the population of common cuttlefish in the Mediterranean area is fragmented into subpopulations, with some areas featuring restricted level of gene flow. Amongst the studied areas, Sardinia was genetically the most diverse and Cyprus the most isolated. At a larger scale, across the Mediterranean, the population structure of the parasite shows co-diversification pattern with its host, but a slower rate of diversification. Differences between the two counterparts are more obvious at a finer scale, where parasite populations show increased level of fragmentation and lower local diversities. This discrepancy can be caused by local extinctions and replacements taking place more frequently in the dicyemid populations, due to their parasitic lifestyle.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

A Perspective Around Cephalopods and Their Parasites, and Suggestions on How to Increase Knowledge in the Field.

Although interest in several areas of cephalopod research has emerged over the last decades (e.g., neurobiology, aquaculture, genetics, and welfare), especially following their 2010 inclusion in the EU Directive on the use of animals for experimental purposes, knowledge regarding the parasites of cephalopods is lacking. Cephalopods can be intermediate, paratenic, or definitive hosts to a range of parasites with a wide variety of life cycle strategies. Here, we briefly review the current knowledge in cephalopod parasitological research, summarizing the main parasite groups that affect these animals. We also emphasize some topics that, in our view, should be addressed in future research, including: (i) better understanding of life cycles and transmission pathways of common cephalopod parasites; (ii) improve knowledge of all phases of the life cycle (i.e., paralarvae, juveniles, adults and senescent animals) and on species from polar deep sea regions; (iii) exploration of the potential of using cephalopod-parasite specificity to assess population boundaries of both, hosts and parasites; (iv) risk evaluation of the potential of standard aquacultural practices to result in parasite outbreaks; (v) evaluation and description of the physiological and behavioral effects of parasites on their cephalopod hosts; (vi) standardization of the methods for accurate parasite sampling and identification; (vii) implementation of the latest molecular methods to facilitate and enable research in above mentioned areas; (viii) sharing of information and samples among researchers and aquaculturists. In our view, addressing these topics would allow us to better understand complex host-parasite interactions, yield insights into cephalopod life history, and help improve the rearing and welfare of these animals in captivity.

Current view on phylogeny within the genus Flabellula Schaeffer, 1926 (Amoebozoa: Leptomyxida).

The molecular phylogeny of Flabellula Schaeffer, 1926 has been updated by analysing 18S rRNA and actin gene sequences of 19 new strains collected and characterised by the authors over the past ten years. The genus Flabellula Schaeffer, 1926 (Amoebozoa: Leptomyxida) is a taxon in which species delineation based on morphological data by themselves is insufficient or even misleading. The description of two novel species, F. schaefferi n. sp. and F. sawyeri n. sp., is justified by the congruence of morphological data with 18S rRNA and actin gene sequence phylogenies, in-silico secondary structure prediction of the V2 region in the 18S rRNA, and by recognition of species-specific sequential motifs within this region.

New data on Soricimyxum fegati (Myxozoa) including analysis of its phylogenetic position inferred from the SSU rRNA gene sequence.

A myxosporean species found to develop in the liver of 10 out of 24 common shrews, Sorex araneus L., caught in South Bohemia, Czech Republic, was identified as Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007, the unique representative of the genus and the first myxosporean species known to develop from plasmodia to spores in a terrestrial mammal. The original description of this species, based on fixed material, is supplemented with new data based on fresh material and with partial sequence of SSU rDNA (GenBank Acc. No. EU232760). Phylogenetic analysis of SSU rDNA revealed that S. fegati is closely related to myxosporeans infecting gall bladders of freshwater fish.

Phylogeny and taxonomy of new and re-examined strains of Tubulinea (Amoebozoa).

Morphological and molecular characterizations of three newly isolated tubulinean strains and re-examination of five strains formerly considered representatives of Saccamoeba and one strain formerly considered as Trichamoeba resulted in (a) the determination of strain BA02, isolated from a dripping rock ledge in Skansbukta (Billefjorden, Svalbard), as a new representative of Ptolemeba bulliensis Brown et al., 2014; (b) identification of strain ATCC® 50249™, deposited in the American Type Culture Collection as Trichamoeba, as the same species (P. bulliensis); (c) characterization of the new strain POHL into the Saccamoeba clade as a member closely related to S. lacustris; and (d) changing the generic residence of three strains formerly considered as representatives of Saccamoeba (strain PV67 to the P. bulliensis clade, and W187G and DP7 into the sister group of Ptolemeba noxubium Brown et al., 2014) whereas two other strains (MSED6, NTSHR) retain their original Saccamoeba clade position. Within the individual clades, the ultrastructure (especially the inner architecture of mitochondria) is congruent and thus of superior taxonomic value to that of light microscopic (morphometric) features.

Heterolobosean amoebae from Arctic and Antarctic extremes: 18 novel strains of Allovahlkampfia, Vahlkampfia and Naegleria.

The diversity of heterolobosean amoebae, important members of soil, marine and freshwater microeukaryote communities in the temperate zones, is greatly under-explored in high latitudes. To address this imbalance, we studied the diversity of this group of free-living amoebae in the Arctic and the Antarctic using culture dependent methods. Eighteen strain representatives of three heterolobosean genera, Allovahlkampfia Walochnik et Mulec, 2009 (1 strain), Vahlkampfia Chatton et Lalung-Bonnaier, 1912 (2) and Naegleria Alexeieff, 1912 (15) were isolated from 179 samples of wet soil and fresh water with sediments collected in 6 localities. The Allovahkampfia strain is the first representative of the genus from the Antarctic; 14 strains (7 from the Arctic, 7 from the Antarctic) of the highly represented genus Naegleria complete the 'polar' cluster of five Naegleria species previously known from the Arctic and Sub-Antarctic regions, whereas one strain enriches the 'dobsoni' cluster of Naegleria strains of diverse origin. Present isolations of Naegleria polarisDe Jonckheere, 2006 from Svalbard, in the Arctic and Vega Island, in the Antarctic and N. neopolarisDe Jonckheere, 2006 from Svalbard and Greenland in the Arctic, and James Ross Island, the Antarctic demonstrate their bipolar distribution, which in free-living amoebae has so far only been known for Vermistella Morand et Anderson, 2007.