The Organellar Genomes of Chromera and Vitrella, the Phototrophic Relatives of Apicomplexan Parasites.

Apicomplexa are known to contain greatly reduced organellar genomes. Their mitochondrial genome carries only three protein-coding genes, and their plastid genome is reduced to a 35-kb-long circle. The discovery of coral-endosymbiotic algae Chromera velia and Vitrella brassicaformis, which share a common ancestry with Apicomplexa, provided an opportunity to study possibly ancestral forms of organellar genomes, a unique glimpse into the evolutionary history of apicomplexan parasites. The structurally similar mitochondrial genomes of Chromera and Vitrella differ in gene content, which is reflected in the composition of their respiratory chains. Thus, Chromera lacks respiratory complexes I and III, whereas Vitrella and apicomplexan parasites are missing only complex I. Plastid genomes differ substantially between these algae, particularly in structure: The Chromera plastid genome is a linear, 120-kb molecule with large and divergent genes, whereas the plastid genome of Vitrella is a highly compact circle that is only 85 kb long but nonetheless contains more genes than that of Chromera. It appears that organellar genomes have already been reduced in free-living phototrophic ancestors of apicomplexan parasites, and such reduction is not associated with parasitism.

The transcriptome of the rumen ciliate Entodinium caudatum reveals some of its metabolic features.

BACKGROUND: Rumen ciliates play important roles in rumen function by digesting and fermenting feed and shaping the rumen microbiome. However, they remain poorly understood due to the lack of definitive direct evidence without influence by prokaryotes (including symbionts) in co-cultures or the rumen. In this study, we used RNA-Seq to characterize the transcriptome of Entodinium caudatum, the most predominant and representative rumen ciliate species. RESULTS: Of a large number of transcripts, > 12,000 were annotated to the curated genes in the NR, UniProt, and GO databases. Numerous CAZymes (including lysozyme and chitinase) and peptidases were represented in the transcriptome. This study revealed the ability of E. caudatum to depolymerize starch, hemicellulose, pectin, and the polysaccharides of the bacterial and fungal cell wall, and to degrade proteins. Many signaling pathways, including the ones that have been shown to function in E. caudatum, were represented by many transcripts. The transcriptome also revealed the expression of the genes involved in symbiosis, detoxification of reactive oxygen species, and the electron-transport chain. Overall, the transcriptomic evidence is consistent with some of the previous premises about E. caudatum. However, the identification of specific genes, such as those encoding lysozyme, peptidases, and other enzymes unique to rumen ciliates might be targeted to develop specific and effective inhibitors to improve nitrogen utilization efficiency by controlling the activity and growth of rumen ciliates. The transcriptomic data will also help the assembly and annotation in future genomic sequencing of E. caudatum. CONCLUSION: As the first transcriptome of a single species of rumen ciliates ever sequenced, it provides direct evidence for the substrate spectrum, fermentation pathways, ability to respond to various biotic and abiotic stimuli, and other physiological and ecological features of E. caudatum. The presence and expression of the genes involved in the lysis and degradation of microbial cells highlight the dependence of E. caudatum on engulfment of other rumen microbes for its survival and growth. These genes may be explored in future research to develop targeted control of Entodinium species in the rumen. The transcriptome can also facilitate future genomic studies of E. caudatum and other related rumen ciliates.

High light acclimation of Chromera velia points to photoprotective NPQ.

It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761-2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.

Genetic and epigenetic mechanisms underlying cell-surface variability in protozoa and fungi.

Eukaryotic microorganisms have evolved ingenious mechanisms to generate variability at their cell surface, permitting differential adherence, rapid adaptation to changing environments, and evasion of immune surveillance. Fungi such as Saccharomyces cerevisiae and the pathogen Candida albicans carry a family of mucin and adhesin genes that allow adhesion to various surfaces and tissues. Trypanosoma cruzi, T. brucei, and Plasmodium falciparum likewise contain large arsenals of different cell surface adhesion genes. In both yeasts and protozoa, silencing and differential expression of the gene family results in surface variability. Here, we discuss unexpected similarities in the structure and genomic location of the cell surface genes, the role of repeated DNA sequences, and the genetic and epigenetic mechanisms-all of which contribute to the remarkable cell surface variability in these highly divergent microbes.

Updating Biodiversity Studies in Loricate Protists: The Case of the Tintinnids (Alveolata, Ciliophora, Spirotrichea).

Species determination is crucial in biodiversity research. In tintinnids, identification is based almost exclusively on the lorica, despite its frequent intraspecific variability and interspecific similarity. We suggest updated procedures for identification and, depending on the aim of the study, further steps to obtain morphological, molecular, and ecological data. Our goal is to help improving the collection of information (e.g. species re-/descriptions and DNA barcodes) that is essential for generating a natural tintinnid classification and a reliable reference for environmental surveys. These suggestions are broadly useful for protistologists because they exemplify data integration, quality/effort compromise, and the need for scientific collaborations.

Effects of salinity and temperature on in vitro cell cycle and proliferation of Perkinsus marinus from Brazil.

Field and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

RNA-Mediated Epigenetic Programming of Genome Rearrangements.

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.

Cryptic sex in Symbiodinium (Alveolata, Dinoflagellata) is supported by an inventory of meiotic genes.

Symbiodinium encompasses a diverse clade of dinoflagellates that are ecologically important as symbionts of corals and other marine organisms. Despite decades of study, cytological evidence of sex (karyogamy and meiosis) has not been demonstrated in Symbiodinium, although molecular population genetic patterns support the occurrence of sexual recombination. Here, we provide additional support for sex in Symbiodinium by uncovering six meiosis-specific and 25 meiosis-related genes in three published genomes. Cryptic sex may be occurring in Symbiodinium's seldom-seen free-living state while being inactive in the symbiotic state.

Genetics and morphology characterize the dinoflagellate Symbiodinium voratum, n. sp., (Dinophyceae) as the sole representative of Symbiodinium Clade E.

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free-living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well-separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) µm. Unique among species in the genus, a high proportion (approximately 10-20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.

Amoebophrya spp. from the bloom-forming dinoflagellate Cochlodinium polykrikoides: parasites not nested in the "Amoebophrya ceratii complex".

Members of Amoebophrya ceratii complex are known to infect a number of free-living dinoflagellates including harmful algal bloom species. In August and October 2012, Amoebophrya infections during two bloom events of the dinoflagellate Cochlodinium polykrikoides were observed along southern coastal waters of Korea. Microscopic observations and molecular data revealed that two different Amoebophrya parasites infected the same host species. In addition, while one developed in the host's nucleus, the other in the host's cytoplasm. Phylogenetic analyses showed that the two parasites were not nested in the previously recognized "Amoebophrya ceratii complex clade", which contained sequences of parasites infecting numerous dinoflagellate species. Instead, they branched as sister taxa to the isolate (possibly Amoebophrya) from radiolarians Hexacontium gigantheum. Our result indicates that the two Amoebophrya parasites infecting C. polykrikoides may be different species from those inside the "complex."

Communicative functions of GPI-anchored surface proteins in unicellular eukaryotes.

Research on several unicellular eukaryotes has identified communicative surface proteins, which are anchored to the outer membrane by glycosylphosphatidylinositol (GPI). Surprisingly, these surface proteins are also released into the environment, raising questions regarding the underlying adaptive advantages and the physical mechanisms that allow for this shedding. This article reviews the current knowledge on several GPI-proteins of different protist species, assembles the puzzling data on the different functions of surface bound and released forms of these proteins, and summarizes their contribution to intra- and interspecific signaling. Recent advances in biochemistry and glycobiology indicate that the GPI-anchor is one of the prerequisites of protein function of membrane bound as well as of released proteins, and hence is a crucial invention for microbial molecular communication. The sensitivity of GPI-anchors (e.g. to phospholipase C) requires consideration of environmental lipase activity of different sources in microbial communities, as these may represent exogenous factors involved in surface protein release. We hypothesize a complex surface protein based communication network and discuss the known facts on protist GPIs in an evolutionary context.

Morphological and molecular characterization of Hematodinium perezi (Dinophyceae: Syndiniales), a dinoflagellate parasite of the harbour crab, Liocarcinus depurator.

Hematodinium perezi Chatton and Poisson 1931 (Dinophyceae: Syndiniales) is reported from one of its type hosts, Liocarcinus depurator, from Rye Bay in the English Channel, a site in a similar geographical location to that of the type description. The histology and ultrastructure of vegetative trophont stages, and rDNA sequences of the parasite infecting this host are reported for the first time. Ultrastructurally, H. perezi was confirmed by the presence of condensed chromatin profiles, trichocysts, an alveolar membrane, and micropores. The pathology of H. perezi was similar to other Hematodinium descriptions with large numbers of parasites present within the haemolymph and host tissues. No host responses against the parasite were observed. Molecular analysis of the ITS rRNA regions from H. perezi infecting L. depurator suggests that Callinectes sapidus from the United States, and Portunus trituberculatus and Scylla serrata from China are infected with different genotypes of H. perezi. The morphological and molecular characterization of H. perezi in one of the type hosts from Europe will allow for a better understanding of the phylogeny of these pathogens of commercially important Crustacea.

Revision of the family Duboscquellidae with description of Euduboscquella crenulata n. gen., n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Favella panamensis Kofoid & Campbell.

Recent recognition that tintinnids are infected by dinophycean as well as syndinean parasites prompts taxonomic revision of dinoflagellate species that parasitize these ciliates. Long overlooked features of the type species Duboscquella tintinnicola are used to emend the genus and family Duboscquellidae, resulting in both taxa being moved from the Syndinea to the Dinophyceae. Syndinean species previously classified as Duboscquella are relocated to Euduboscquella n. gen., with Euduboscquella crenulata n. sp. as the type. As an endoparasitic species, E. crenulata shares with its congeners processes associated with intracellular development and sporogenesis, but differs from closely related species in nuclear and cortical morphology of the trophont, including a distinctively grooved shield (= episome) that imparts a crenulated appearance in optical section. In addition, E. crenulata produces three morphologically distinct spore types, two of which undergo syngamy to form a uninucleate zygote. The zygote undergoes successive division to produce four daughter cells of unequal size, but that resemble the nonmating spore type.

One Perkinsus species may hide another: characterization of Perkinsus species present in clam production areas of France.

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

Development and application of a novel histological multichrome technique for clam histopathology.

A novel and expedient histological tetrachrome technique was developed and applied to whole-body sections of the clam Ruditapes decussatus (L. 1758). The technique involves fixation in Carnoy's fluid followed by immediate embedding in paraffin with staining with a combination of Alcian Blue, Periodic Acid-Schiff's, Haematoxylin and Picric Acid. Fixation and staining was perfect for all tissues and resolved good identification of Perkinsus sp. infection and high structural detail. Among the surveyed fixatives, Bouin-Hollande's fluid also provided good results, however, fixation is potentially longer, polysaccharide staining was less intense and fibres appeared to be better preserved by Carnoy's.

Comparative study on the toxic effects of red tide flagellates Heterocapsa circularisquama and Chattonella marina on the short-necked clam (Ruditapes philippinarum).

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition.

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell-cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.

Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata.

BACKGROUND: Dinophysis is exceptional among dinoflagellates, possessing plastids derived from cryptophyte algae. Although Dinophysis can be maintained in pure culture for several months, the genus is mixotrophic and needs to feed either to acquire plastids (a process known as kleptoplastidy) or obtain growth factors necessary for plastid maintenance. Dinophysis does not feed directly on cryptophyte algae, but rather on a ciliate (Myrionecta rubra) that has consumed the cryptophytes and retained their plastids. Despite the apparent absence of cryptophyte nuclear genes required for plastid function, Dinophysis can retain cryptophyte plastids for months without feeding. RESULTS: To determine if this dinoflagellate has nuclear-encoded genes for plastid function, we sequenced cDNA from Dinophysis acuminata, its ciliate prey M. rubra, and the cryptophyte source of the plastid Geminigera cryophila. We identified five proteins complete with plastid-targeting peptides encoded in the nuclear genome of D. acuminata that function in photosystem stabilization and metabolite transport. Phylogenetic analyses show that the genes are derived from multiple algal sources indicating some were acquired through horizontal gene transfer. CONCLUSIONS: These findings suggest that D. acuminata has some functional control of its plastid, and may be able to extend the useful life of the plastid by replacing damaged transporters and protecting components of the photosystem from stress. However, the dearth of plastid-related genes compared to other fully phototrophic algae suggests that D. acuminata does not have the nuclear repertoire necessary to maintain the plastid permanently.

Effect of nutrient concentration and salinity on immotile-motile transformation of Chromera velia.

Chromera velia (Chromerida: Alveolata) is a photosynthetic, unicellular organism closely related to parasitic apicomplexa. Diurnal rhythmicity of an immotile-motile transformation has been observed but its role in the life cycle remains largely unknown. Using a multiwell system, we show that salinity and f-medium concentration significantly affect the percentage of motile C. velia cells. An inverse relationship between salinity and motility in C. velia occurred, and flagellation was also suppressed at high nutrient levels. These results suggest a low salinity environment with relatively low nutrient levels enables flagellate transformation during the diurnal cycle of C. velia.

Discovery of a New Clade Nested Within the Genus Alexandrium (Dinophyceae): Morpho-molecular Characterization of Centrodinium punctatum (Cleve) F.J.R. Taylor.

Investigation of phytoplankton from East China Sea of the Pacific Ocean, offshore Réunion Island of the Indian Ocean, and the French Atlantic coast revealed a species of poorly known armored fusiform dinoflagellate. To clarify this species, morphology and phylogeny based on mitochondrial and nuclear protein gene sequence (Cox1, Cob and Hsp90) concatenated with the SSU, ITS region and LSU rDNA sequences were analysed. Epifluorescence and scanning electron microscopy observations revealed that the nucleus of the specimen was elongated, sausage-shaped and located equatorially on the left lateral side of the cell, and that the plate formula is Po, 3', 1a, 6″, 6C, 8S, 5‴, 1p, 2'‴. These morphological features indicate that the species can be assigned to Centrodinium punctatum. Interestingly, the phylogenetic analyses placed this species within the Alexandrium clade, with Alexandrium affine being its closest relative. This indicates that genus Alexandrium is not monophyletic. The most similar morphological traits between C. punctatum and Alexandrium species were the shape of apical pore plate and the arrangement of the sulcal plates. However, since there are significant morphological differences between C. punctatum and Alexandrium species, further studies are needed to clarify the relation between the morphology and molecular phylogeny of other Centrodinium-related fusiform species.