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.

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.

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.

Tuberlatum coatsi gen. n., sp. n. (Alveolata, Perkinsozoa), a New Parasitoid with Short Germ Tubes Infecting Marine Dinoflagellates.

Perkinsozoa is an exclusively parasitic group within the alveolates and infections have been reported from various organisms, including marine shellfish, marine dinoflagellates, freshwater cryptophytes, and tadpoles. Despite its high abundance and great genetic diversity revealed by recent environmental rDNA sequencing studies, Perkinsozoa biodiversity remains poorly understood. During the intensive samplings in Korean coastal waters during June 2017, a new parasitoid of dinoflagellates was detected and was successfully established in culture. The new parasitoid was most characterized by the presence of two to four dome-shaped, short germ tubes in the sporangium. The opened germ tubes were biconvex lens-shaped in the top view and were characterized by numerous wrinkles around their openings. Phylogenetic analyses based on the concatenated SSU and LSU rDNA sequences revealed that the new parasitoid was included in the family Parviluciferaceae, in which all members were comprised of two separate clades, one containing Parvilucifera species (P. infectans, P. corolla, and P. rostrata), and the other containing Dinovorax pyriformis, Snorkelia spp., and the new parasitoid from this study. Based on morphological, ultrastructural, and molecular data, we propose to erect a new genus and species, Tuberlatum coatsi gen. n., sp. n., from the new parasitoid found in this study. Further, we examined and discussed the validity of some diagnostic characteristics reported for parasitoids in the family Parviluciferaceae at both the genus and species levels.

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.

Violaxanthin inhibits nonphotochemical quenching in light-harvesting antenna of Chromera velia.

Non-photochemical quenching (NPQ) is a photoprotective mechanism in light-harvesting antennae. NPQ is triggered by chloroplast thylakoid lumen acidification and is accompanied by violaxanthin de-epoxidation to zeaxanthin, which further stimulates NPQ. In the present study, we show that violaxanthin can act in the opposite direction to zeaxanthin because an increase in the concentration of violaxanthin reduced NPQ in the light-harvesting antennae of Chromera velia. The correlation overlapped with a similar relationship between violaxanthin and NPQ as observed in isolated higher plant light-harvesting complex II. The data suggest that violaxanthin in C. velia can act as an inhibitor of NPQ, indicating that violaxanthin has to be removed from the vicinity of the protein to reach maximal NPQ.

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.

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.

New Insights into the Parasitoid Parvilucifera sinerae Life Cycle: The Development and Kinetics of Infection of a Bloom-forming Dinoflagellate Host.

Parvilucifera sinerae is a parasitoid of dinoflagellates, the major phytoplankton group responsible for harmful algal bloom events. Here we provide a detailed description of both the life cycle of P. sinerae, based on optical, confocal, and transmission electron microscopy observations, and its infection kinetics and dynamics. P. sinerae completes its life cycle in 3-4 days. The zoospore encounters and penetrates the host cell within 24h after its addition to the host culture. Inside the host, the parasitoid develops a trophocyte, which constitutes the longest stage of its life cycle. The trophocyte replicates and divides by schizogony to form hundreds of new zoospores contained within a sporangium. Under laboratory conditions, P. sinerae has a short generation time, a high rate of asexual reproduction, and is highly prevalent (up to 80%) in the Alexandrium minutum population. Prevalence was shown to depend on both the parasitoid inoculum size and host density, which increase the encounter probability rate. The parasitoid infection parameters described in this study are the first reported for the genus Parvilucifera. They show that P. sinerae is well-adapted to its dinoflagellate hosts and may be an important factor in the termination of A. minutum blooms in the natural environment.

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.

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.

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."

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.

Polykrikos tanit sp. nov., a new mixotrophic unarmoured pseudocolonial dinoflagellate from the NW Mediterranean Sea.

Pigmented pseudocolonies initially identified as Polykrikos hartmannii Zimmermann were detected at several locations of the Catalan coast (NW Mediterranean Sea) in April-June of 2012 and April-May of 2013. To further explore the several remarkable morphological discrepancies between these organisms and P. hartmannii, we carried out a detailed morphological study and used single-cell PCR to obtain partial LSU and SSU rDNA sequences. The resulting phylogenies showed that our isolates occupy a basal position within the Polykrikos clade, close to P. hartmannii, but do not correspond to any described polykrikoid species. P. barnegatensis Martin is controversially considered to be synonymous with P. hartmannii. The organisms studied in this work were similar to P. barnegatensis but showed significant morphological differences with its original description such as the torsion of the pseudocolony, more pronounced overhanging of the cingula, stepped fusion border of the zooids, and number and shape of nuclei. Consequently, we propose that the isolates constitute a new species, which we named Polykrikos tanit sp. nov. The observed characters, pigmented, same number of zooids and nuclei, sulci not fused, and its phylogeny suggest that the species is an early evolutionary Polykrikos species.

Parvilucifera rostrata sp. nov. (Perkinsozoa), a novel parasitoid that infects planktonic dinoflagellates.

The diversity and ecological roles of protists in marine plankton are still poorly known. In 2011, we made a substantial effort to isolate parasites into cultures during the course of blooms of the toxic microalga Alexandrium minutum (Dinophyceae) in two estuaries (the Penzé and the Rance, Brittany coast, north-west of France). In total, 99 parasitic strains were obtained. Screening of ribosomal internal transcribed spacer regions (including ITS1, 5.8S and ITS2) revealed the existence of two ribotypes. Small subunit and partial large subunit rRNA genes revealed that these two ribotypes belong to different species of the genus Parvilucifera. The first ribotype was tentatively affiliated to the species Parvilucifera infectans, whilst the second represents a new species, Parvilucifera rostrata sp. nov. The new species has several distinct morphological features in the general organization of its zoospore and in the shape and size of processes covering the sporangium. Both Parvilucifera species are generalist parasitoids with similar generation times, and this study thus raises the question of how two parasitoids exploiting similar ecological resources and infection strategies can coexist in the same ecosystem. Taxonomic relationships between Parvilucifera spp. and other closely related marine parasitoids, such as syndinians, are discussed.

Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalves.

Perkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation at micromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanism of bioactivation in P. olseni sharing similarities with the one proposed in P. falciparum parasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuole while the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus and may also be important in the development of new strategies for Perkinsosis prevention and control.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.

Parvilucifera sinerae (Alveolata, Myzozoa) is a generalist parasitoid of dinoflagellates.

This study begins with a description of the infective process in the dinoflagellate type host Alexandrium minutum by a strain of the parasitoid, Parvilucifera sinerae, including the morphologies of the various dinoflagellate and parasitoid stages during the infection. Then, the susceptibility of 433 microalgal strains to P. sinerae infection was studied. The parasitoid was found to be capable of infecting several dinoflagellate species of the genera Alexandrium, Coolia, Dinophysis, Fragilidium, Gambierdiscus, Gymnodinium, Gyrodinium, Heterocapsa, Kryptoperidinium, Lepidodinium, Ostreopsis, Pentapharsodinium, Protoceratium, Scrippsiella, and Woloszynskia. Intra-strain variability was observed as well, such that within the same dinoflagellate species some strains were infected whereas others were not. Likewise, species of other dinoflagellate genera were not infected, such as Akashiwo, Amphidinium, Barrufeta, Bysmatrum, Karenia, Karlodinium, Prorocentrum, and Takayama. Moreover, P. sinerae was not able to infect any of the tested haptophyte, diatom, and chlorophyte species. In natural samples screened for P. sinerae infectivity, several dinoflagellate species of the genera Alexandrium, Coolia, Gonyaulax, Gymnodinium, Phalacroma, Protoperidinium, and Scrippsiella were identified as susceptible. Sporangia size was found to be proportional to the size of the host, and variations in the sporangia size were observed to influence their maturation time.

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.

Photosynthesis in Chromera velia represents a simple system with high efficiency.

Chromera velia (Alveolata) is a close relative to apicomplexan parasites with a functional photosynthetic plastid. Even though C. velia has a primitive complement of pigments (lacks chlorophyll c) and uses an ancient type II form of RuBISCO, we found that its photosynthesis is very efficient with the ability to acclimate to a wide range of irradiances. C. velia maintain similar maximal photosynthetic rates when grown under continual light-limited (low light) or light-saturated (high light) conditions. This flexible acclimation to continuous light is provided by an increase of the chlorophyll content and photosystem II connectivity under light limited conditions and by an increase in the content of protective carotenoids together with stimulation of effective non-photochemical quenching under high light. C. velia is able to significantly increase photosynthetic rates when grown under a light-dark cycle with sinusoidal changes in light intensity. Photosynthetic activities were nonlinearly related to light intensity, with maximum performance measured at mid-morning. C. velia efficiently acclimates to changing irradiance by stimulation of photorespiration and non-photochemical quenching, thus avoiding any measurable photoinhibition. We suggest that the very high CO(2) assimilation rates under sinusoidal light regime are allowed by activation of the oxygen consuming process (possibly chlororespiration) that maintains high efficiency of RuBISCO (type II). Despite the overall simplicity of the C. velia photosynthetic system, it operates with great efficiency.