Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia.

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Can Acropora tenuis larvae attract native Symbiodiniaceae cells by green fluorescence at the initial establishment of symbiosis?

Most corals acquire symbiodiniacean symbionts from the surrounding environment to initiate symbiosis. The cell densities of Symbiodiniaceae in the environment are usually low, and mechanisms may exist by which new coral generations attract suitable endosymbionts. Phototaxis of suitable symbiodiniacean cells toward green fluorescence in corals has been proposed as one such mechanism. In the present study, we observed the phototaxis action wavelength of various strains of Symbiodiniaceae and the fluorescence spectra of aposymbiotic Acropora tenuis larvae at the time of endosymbiont uptake. The phototaxis patterns varied among the Symbiodiniaceae species and "native" endosymbionts-commonly found in Acropora juveniles present in natural environments; that is, Symbiodinium microadriaticum was attracted to blue light rather than to green light. Another native endosymbiont, Durusdinium trenchii, showed no phototaxis specific to any wavelength. Although the larvae exhibited green and broad orange fluorescence under blue-violet excitation light, the maximum green fluorescence peak did not coincide with that of the phototaxis action spectrum of S. microadriaticum. Rather, around the peak wavelength of larval green fluorescence, this native endosymbiont showed slightly negative phototaxis, suggesting that the green fluorescence of A. tenuis larvae may not play a role in the initial attraction of native endosymbionts. Conversely, broad blue larval fluorescence under UV-A excitation covered the maximum phototaxis action wavelength of S. microadriaticum. We also conducted infection tests using native endosymbionts and aposymbiotic larvae under red LED light that does not excite visible larval fluorescence. Almost all larvae failed to acquire S. microadriaticum cells, whereas D. trenchii cells were acquired by larvae even under red illumination. Thus, attraction mechanisms other than visible fluorescence might exist, at least in the case of D. trenchii. Our results suggest that further investigation and discussion, not limited to green fluorescence, would be required to elucidate the initial attraction mechanisms.

Circular swimming motility and disordered hyperuniform state in an algae system.

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae ([Formula: see text]), which swim in circles at the air-liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

An Alveolata secretory machinery adapted to parasite host cell invasion.

Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium (the causative agent of malaria) and Toxoplasma (one of the most widespread zoonotic pathogens). Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion1. Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function2. The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals and plants. Here, we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata and Apicomplexa that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defence for free-living unicellular eukaryotes and host cell injection in intracellular parasites.

Environmental factors drive the release of Perkinsus marinus from infected oysters.

Since the discovery of Perkinsus marinus as the cause of dermo disease in Crassostrea virginica, salinity and temperature have been identified as the main environmental drivers of parasite prevalence. However, little is known about how these variables affect the movement of the parasite from host to water column. In order to elucidate how environmental factors can influence the abundance of this parasite in the water column, we conducted a series of experiments testing the effects of time of day, temperature and salinity on the release of P. marinus cells from infected oysters. We found that P. marinus cells were released on a diurnal cycle, with most cells released during the hottest and brightest period of the day (12:00-18:00). Temperature also had a strong and immediate effect on the number of cells released, but salinity did not, only influencing the intensity of infection over the course of several months. Taken together, our results demonstrate that (1) the number of parasites in the water column fluctuates according to a diurnal cycle, (2) temperature and salinity act on different timescales to influence parasite abundance, and (3) live infected oysters may substantially contribute to the abundance of transmissive parasites in the water column under particular environmental conditions.

Centers of endemism of freshwater protists deviate from pattern of taxon richness on a continental scale.

Here, we analyzed patterns of taxon richness and endemism of freshwater protists in Europe. Even though the significance of physicochemical parameters but also of geographic constraints for protist distribution is documented, it remains unclear where regional areas of high protist diversity are located and whether areas of high taxon richness harbor a high proportion of endemics. Further, patterns may be universal for protists or deviate between taxonomic groups. Based on amplicon sequencing campaigns targeting the SSU and ITS region of the rDNA we address these patterns at two different levels of phylogenetic resolution. Our analyses demonstrate that protists have restricted geographical distribution areas. For many taxonomic groups the regions of high taxon richness deviate from those having a high proportion of putative endemics. In particular, the diversity of high mountain lakes as azonal habitats deviated from surrounding lowlands, i.e. many taxa were found exclusively in high mountain lakes and several putatively endemic taxa occurred in mountain regions like the Alps, the Pyrenees or the Massif Central. Beyond that, taxonomic groups showed a pronounced accumulation of putative endemics in distinct regions, e.g. Dinophyceae along the Baltic Sea coastline, and Chrysophyceae in Scandinavia. Many other groups did not have pronounced areas of increased endemism but geographically restricted taxa were found across Europe.

Scanning electron microscopic observation of the in vitro cultured protozoan, Perkinsus olseni, isolated from the Manila clam, Ruditapes philippinarum.

BACKGROUND: Perkinsosis is a major disease affecting the commercially important marine mollusk Ruditapes philippinarum (Manila clam) in Asian waters. In this study, we investigated the morphological characteristics of Perkinsus olseni, the causative agent of perkinsosis, cultured under laboratory conditions at different stages of its life cycle using a scanning electron microscope (SEM). RESULTS: The prezoosporangia formed after induction with Ray's fluid thioglycollate medium (RFTM) developed into zoosporangia. During this process, a discharge tube formed a porous sponge-like structure that detached before the zoospores were released; thus, this organelle operated as a bung. Liberated zoospores gradually transformed into immature trophozoites, during which detachment of the anterior flagella occurred, but the loss of the posterior flagella was not clearly observed in the present study. Mature trophozoites underwent schizogony by cleaving the cell forming some merozoites in schizonts, which were released by the rupturing of the cellular membrane of the schizont within a few days. CONCLUSIONS: Our morphological and ultrastructural studies contribute new information on the life cycle and propagation of P. olseni.

Comparative transcriptomic analyses of Chromera and Symbiodiniaceae.

Reef-building corals live in a mutualistic relationship with photosynthetic algae (family Symbiodiniaceae) that usually provide most of the energy required by the coral host. This relationship is sensitive to temperature stress; as little as a 1°C increase often leads to the collapse of the association. This sensitivity has led to an interest in the potential of more stress-tolerant algae to supplement or substitute for the normal Symbiodiniaceae mutualists. In this respect, the apicomplexan-like microalga Chromera is of particular interest due to its greater temperature tolerance. We generated a de novo transcriptome for a Chromera strain isolated from a GBR coral ('GBR Chromera') and compared with those of the reference strain of Chromera ('Sydney Chromera'), and to those of Symbiodiniaceae (Fugacium kawagutii, Cladocopium goreaui and Breviolum minutum), as well as the apicomplexan parasite, Plasmodium falciparum. In contrast to the high sequence divergence amongst representatives of different genera within the family Symbiodiniaceae, the two Chromera strains featured low sequence divergence at orthologous genes, implying that they are likely to be conspecifics. Although KEGG categories provide few criteria by which true coral mutualists might be identified, they do supply a molecular rationalization that explains the ecological dominance of Cladocopium spp. amongst Indo-Pacific reef corals. The presence of HSP20 genes may contribute to the high thermal tolerance of Chromera.

Confirmation of the wide host range of Parvilucifera corolla (Alveolata, Perkinsozoa).

Marine parasites of the genus Parvilucifera have been described as endoparasitoids of dinoflagellates. Recently, the species Parvilucifera corolla was described, but its host range was not examined. Here, the host selectivity of P. corolla was screened, including 110 strains of dinoflagellates (24 genera) and other microalgal groups as potential hosts. Infections and the full life cycle of the parasitoid were observed in 73 strains (16 genera) of dinoflagellates. Parvilucifera corolla did not infect most chlorophytes, cryptophytes, chrysophytes, diatoms, haptophytes and raphidophytes but one strain of Pyramimonas (chlorophyte) was infected, although without viable sporangia. In Symbiodinium natans, a transition to the coccoid stage was induced above a certain parasite:host ratio. These results confirm P. corolla as a generalist parasitoid of dinoflagellates, with important differences in host range regarding other species of the genus.

Variation in global transcriptomic response to Perkinsus marinus infection among eastern oyster families highlights potential mechanisms of disease resistance.

Dermo disease, caused by the protozoan parasite Perkinsus marinus, negatively impacts wild and cultured Eastern oyster populations, yet our knowledge of the mechanistic bases for parasite pathogenicity and the Eastern oyster's response to it is limited. To better understand host responses to the parasite and identify molecular mechanisms underlying disease-resistance phenotypes, we experimentally challenged two families exhibiting divergent Dermo-resistance phenotypes with the parasite, generated global expression profiles using RNAseq and identified differentially expressed transcripts between control and challenged oysters from each family at multiple time points post-parasite injection. The susceptible and resistant families exhibited strikingly different transcriptomic responses to the parasite over a 28-day time period. The resistant family exhibited a strong, focused, early response to P. marinus infection, where many significantly upregulated transcripts were associated with the biological processes "regulation of proteolysis" and "oxidation-reduction process." P. marinus virulence factors are mainly comprised of proteases that facilitate parasite invasion and weaken host humoral defenses, thus host upregulation of transcripts associated with negative regulation of proteolysis is consistent with a Dermo-resistant phenotype. In contrast, the susceptible family mounted a very weak, disorganized, initial response to the parasite. Few transcripts were differentially expressed between control and injected oysters, and no functional enrichment was detected among them. At the final 28 d time point 2450 differentially expressed transcripts were identified and were associated with either "G-protein coupled receptor activity" (upregulated) or "microtubule-based process" (downregulated). A handful of protease inhibitors were differentially expressed between control and injected susceptible oysters, but this function was not enriched in the susceptible data set. The differential expression patterns observed in this study provide valuable insight into the functional basis of Dermo resistance and suggest that the timing of expression is just as important as the transcripts being expressed.

Perkinsus mediterraneus infection induces oxidative stress in the mollusc Mimachlamys varia.

Perkinsus mediterraneus is a protozoan parasite that can cause marine mollusc diseases known as perkinsosis being a serious threat for clam cultures worldwide. The aim of the present study was first to determine the Perkinsus species infecting the variegated scallop Mimachlamys varia and then to evaluate the existence of oxidative stress in gills of M. varia according to different degrees of infection. DNA sequencing confirmed that P. mediterraneus was the species infecting M. varia. ROS production was progressively increasing with the degree of infection although the differences were only significant in the high-infected group. Low degree of infection significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and nitrite levels with respect to the control group. In the high-infected group, a significant increase was evidenced in all analysed enzymes, catalase, SOD, glutathione reductase and GST. Non-significant differences in MDA levels were observed between the control and low-infected groups; however, a significant increase in MDA levels was observed in the high-infected group. In conclusion, the infection by Perkinsus mediterraneus in M. varia induces oxidative stress and an antioxidant response directly related to the infection degree that can contribute to the pathogenicity of the infection.

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.

Spatial and temporal disease dynamics of the parasite Hematodinium sp. in shore crabs, Carcinus maenas.

BACKGROUND: The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. METHODS: Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. RESULTS: Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P < 0.002) and sex (P < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs (Cancer pagurus) and langoustines (Nephrops norvegicus). CONCLUSIONS: Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

Differential bleaching and recovery pattern of southeast Indian coral reef to 2016 global mass bleaching event: Occurrence of stress-tolerant symbiont Durusdinium (Clade D) in corals of Palk Bay.

Information about coral community response to bleaching on Indian reefs is much more limited compared with Indo-Pacific reefs, with no understanding of algal symbionts. We investigated a reef in Palk Bay to understand the coral community response to 2016 bleaching event and to reveal dominant symbiont type association in four common coral genera. Out of 508 colonies surveyed, we found 20.9% (106) mortality in 53.8% (n = 290) of bleached corals. We found differential bleaching and recovery pattern among coral genera. Bleaching was most prevalent in Acropora (86.36%), followed by Porites (65.45%), while moderate to no bleaching was recorded in Favites 5.88%, Symphyllia 51.11% and Favia 55.77%, Platygyra 41.67%, Goniastrea 41.83%. Pre-bleaching and post bleaching samplings revealed changes in dominant symbiont type following bleaching only in Acropora (Cladocopium, Clade C to Durusdinium Clade D) while no such changes were found in other coral genera hosted Clade D. This is the first observation of coral symbiont diversity in the Indian reef.

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.

Role of host genetics and heat-tolerant algal symbionts in sustaining populations of the endangered coral Orbicella faveolata in the Florida Keys with ocean warming.

Identifying which factors lead to coral bleaching resistance is a priority given the global decline of coral reefs with ocean warming. During the second year of back-to-back bleaching events in the Florida Keys in 2014 and 2015, we characterized key environmental and biological factors associated with bleaching resilience in the threatened reef-building coral Orbicella faveolata. Ten reefs (five inshore, five offshore, 179 corals total) were sampled during bleaching (September 2015) and recovery (May 2016). Corals were genotyped with 2bRAD and profiled for algal symbiont abundance and type. O. faveolata at the inshore sites, despite higher temperatures, demonstrated significantly higher bleaching resistance and better recovery compared to offshore. The thermotolerant Durusdinium trenchii (formerly Symbiondinium trenchii) was the dominant endosymbiont type region-wide during initial (78.0% of corals sampled) and final (77.2%) sampling; >90% of the nonbleached corals were dominated by D. trenchii. 2bRAD host genotyping found no genetic structure among reefs, but inshore sites showed a high level of clonality. While none of the measured environmental parameters were correlated with bleaching, 71% of variation in bleaching resistance and 73% of variation in the proportion of D. trenchii was attributable to differences between genets, highlighting the leading role of genetics in shaping natural bleaching patterns. Notably, D. trenchii was rarely dominant in O. faveolata from the Florida Keys in previous studies, even during bleaching. The region-wide high abundance of D. trenchii was likely driven by repeated bleaching associated with the two warmest years on record for the Florida Keys (2014 and 2015). On inshore reefs in the Upper Florida Keys, O. faveolata was most abundant, had the highest bleaching resistance, and contained the most corals dominated by D. trenchii, illustrating a causal link between heat tolerance and ecosystem resilience with global change.

Accumulation of Dinophysis Toxins in Bivalve Molluscs.

Several species of the dinoflagellate genus Dinophysis produce toxins that accumulate in bivalves when they feed on populations of these organisms. The accumulated toxins can lead to intoxication in consumers of the affected bivalves. The risk of intoxication depends on the amount and toxic power of accumulated toxins. In this review, current knowledge on the main processes involved in toxin accumulation were compiled, including the mechanisms and regulation of toxin acquisition, digestion, biotransformation, compartmentalization, and toxin depuration. Finally, accumulation kinetics, some models to describe it, and some implications were also considered.

Regulation of apoptosis-related genes during interactions between oyster hemocytes and the alveolate parasite Perkinsus marinus.

The alveolate Perkinsus marinus is the most devastating parasite of the eastern oyster Crassostrea virginica. The parasite is readily phagocytosed by oyster hemocytes, but instead of intracellular killing and digestion, P. marinus can survive phagocytosis and divide in host cells. This intracellular parasitism is accompanied by a regulation of host cell apoptosis. This study was designed to gain a better understanding of the molecular mechanisms of apoptosis regulation in oyster hemocytes following exposure to P. marinus. Regulation of apoptosis-related genes in C. virginica, and apoptosis-regulatory genes in P. marinus, were investigated via qPCR to assess the possible pathways involved during these interactions. In vitro experiments were also carried out to evaluate the effect of chemical inhibitors of P. marinus antioxidant processes on hemocyte apoptosis. Results indicate the involvement of the mitochondrial pathway (Bcl-2, anamorsin) of apoptosis in C. virginica exposed to P. marinus. In parallel, the antioxidants peroxiredoxin and superoxide dismutase were regulated in P. marinus exposed to C. virginica hemocytes suggesting that apoptosis regulation in infected oysters may be mediated by anti-oxidative processes. Chemical inhibition of P. marinus superoxide dismutase resulted in a marked increase of reactive oxygen species production and apoptosis in infected hemocytes. The implication of oxygen-dependent apoptosis during P. marinus infection and disease development in C. virginica is discussed.