Increased sea ice melt as a driver of enhanced Arctic phytoplankton blooming.

Phytoplankton primary production in the Arctic Ocean has been increasing over the last two decades. In 2019, a record spring bloom occurred in Fram Strait, characterized by a peak in chlorophyll that was reached weeks earlier than in other years and was larger than any previously recorded May bloom. Here, we consider the conditions that led to this event and examine drivers of spring phytoplankton blooms in Fram Strait using in situ, remote sensing, and data assimilation methods. From samples collected during the May 2019 bloom, we observe a direct relationship between sea ice meltwater in the upper water column and chlorophyll a pigment concentrations. We place the 2019 spring dynamics in context of the past 20 years, a period marked by rapid change in climatic conditions. Our findings suggest that increased advection of sea ice into the region and warmer surface temperatures led to a rise in meltwater input and stronger near-surface stratification. Over this time period, we identify large-scale spatial correlations in Fram Strait between increased chlorophyll a concentrations and increased freshwater flux from sea ice melt.

Temperature Affects the Biological Control of Dinoflagellates by the Generalist Parasitoid Parvilucifera rostrata.

The increase in emerging harmful algal blooms in the last decades has led to an extensive concern in understanding the mechanisms behind these events. In this paper, we assessed the growth of two blooming dinoflagellates (Alexandrium minutum and Heterocapsa triquetra) and their susceptibility to infection by the generalist parasitoid Parvilucifera rostrata under a temperature gradient. The growth of the two dinoflagellates differed across a range of temperatures representative of the Penzé Estuary (13 to 22 °C) in early summer. A. minutum growth increased across this range and was the highest at 19 and 22 °C, whereas H. triquetra growth was maximal at intermediate temperatures (15-18 °C). Interestingly, the effect of temperature on the parasitoid infectivity changed depending on which host dinoflagellate was infected with the dinoflagellate responses to temperature following a positive trend in A. minutum (higher infections at 20-22 °C) and a unimodal trend in H. triquetra (higher infections at 18 °C). Low temperatures negatively affected parasitoid infections in both hosts (i.e., "thermal refuge"). These results demonstrate how temperature shifts may not only affect bloom development in microalgal species but also their control by parasitoids.

First examination of sterols in the marine dinoflagellate genus Vulcanodinium.

Vulcanodinium is an ecologically relevant dinoflagellate genus due to its production of neurotoxins known as pinnatoxins. We present here the first examination of the sterols of a Vulcanodinium rugosum isolate. Sterols are ringed lipids that assist in maintaining rigidity of cellular membranes, and the Dinophyceae are well-studied for their ability to produce a diverse array of sterols, many of which have chemotaxonomic utility. We have determined that V. rugosum produces a set of major sterols, namely cholesterol, dinosterol, 4α,24-dimethyl-5α-cholest-22E-en-3ß-ol, and 4α,24-dimethyl-5α-cholestan-3ß-ol, common to the Dinophyceae. However, this displayed marked differences from those studied members of the genera Scrippsiella and Peridinium, the closest phylogenetic relatives. Included in these differences is production by V. rugosum of a much lower percentage of dinostanol, a saturated form of dinosterol.

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp.

BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

Cryptic species in the parasitic Amoebophrya species complex revealed by a polyphasic approach.

As critical primary producers and recyclers of organic matter, the diversity of marine protists has been extensively explored by high-throughput barcode sequencing. However, classification of short metabarcoding sequences into traditional taxonomic units is not trivial, especially for lineages mainly known by their genetic fingerprints. This is the case for the widespread Amoebophrya ceratii species complex, parasites of their dinoflagellate congeners. We used genetic and phenotypic characters, applied to 119 Amoebophrya individuals sampled from the same geographic area, to construct practical guidelines for species delineation that could be applied in DNA/RNA based diversity analyses. Based on the internal transcribed spacer (ITS) regions, ITS2 compensatory base changes (CBC) and genome k-mer comparisons, we unambiguously defined eight cryptic species among closely related ribotypes that differed by less than 97% sequence identity in their SSU rDNA. We then followed the genetic signatures of these parasitic species during a three-year survey of Alexandrium minutum blooms. We showed that these cryptic Amoebophrya species co-occurred and shared the same ecological niche. We also observed a maximal ecological fitness for parasites having narrow to intermediate host ranges, reflecting a high cost for infecting a broader host range. This study suggests that a complete taxonomic revision of these parasitic dinoflagellates is long overdue to understand their diversity and ecological role in the marine plankton.

Dinoflagellate Host Chloroplasts and Mitochondria Remain Functional During Amoebophrya Infection.

Dinoflagellates are major components of phytoplankton that play critical roles in many microbial food webs, many of them being hosts of countless intracellular parasites. The phototrophic dinoflagellate Scrippsiella acuminata (Dinophyceae) can be infected by the microeukaryotic parasitoids Amoebophrya spp. (Syndiniales), some of which primarily target and digest the host nucleus. Early digestion of the nucleus at the beginning of the infection is expected to greatly impact the host metabolism, inducing the knockout of the organellar machineries that highly depend upon nuclear gene expression, such as the mitochondrial OXPHOS pathway and the plastid photosynthetic carbon fixation. However, previous studies have reported that chloroplasts remain functional in swimming host cells infected by Amoebophrya. We report here a multi-approach monitoring study of S. acuminata organelles over a complete infection cycle by nucleus-targeting Amoebophrya sp. strain A120. Our results show sustained and efficient photosystem II activity as a hallmark of functional chloroplast throughout the infection period despite the complete digestion of the host nucleus. We also report the importance played by light on parasite production, i.e., the amount of host biomass converted to parasite infective propagules. Using a differential gene expression analysis, we observed an apparent increase of all 3 mitochondrial and 9 out of the 11 plastidial genes involved in the electron transport chains (ETC) of the respiration pathways during the first stages of the infection. The longer resilience of organellar genes compared to those encoded by the nucleus suggests that both mitochondria and chloroplasts remain functional throughout most of the infection. This extended organelle functionality, along with higher parasite production under light conditions, suggests that host bioenergetic organelles likely benefit the parasite Amoebophrya sp. A120 and improve its fitness during the intracellular infective stage.

Identification of Portimine B, a New Cell Permeable Spiroimine That Induces Apoptosis in Oral Squamous Cell Carcinoma.

Spiroimines are a class of compounds produced by marine dinoflagellates with a wide range of toxicity and therapeutic potential. The smallest of the cyclic imines, portimine, is far less toxic than other known members in several animal models. Portimine has also been shown to induce apoptosis and reduce the growth of a variety of cancer cell lines at low nanomolar concentrations. In an effort to discover new spiroimines, the current study undertook a metabolomic analysis of cultures of cyclic imine-producing dinoflagellates, and a new analog of portimine was discovered in which the five-membered cyclic ether is open. Further scrutiny with human oral cavity squamous cell carcinoma (OCSCC) cell lines revealed that the open ring congener was less potent than portimine A but could still lead to the accumulation of apoptotic gene transcripts, fragment genomic DNA, and reduce cancer cell proliferation in the range of 100-200 nM.

A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum.

Harmful blooms formed by planktonic microalgae (HABs) in both freshwater and coastal waters regularly lead to severe mortalities of fish and invertebrates causing substantial economic losses of marine products worldwide. The mixotrophic haptophyte Prymnesium parvum is one of the most important microalgae associated with fish kills. Here 26 strains of P. parvum with a wide geographical distribution were screened for the production of prymnesins, the suspected causative allelochemical toxins. All investigated strains produced prymnesins, indicating that the toxins play an important role for the organism. The prymnesins can be classified into three types based on the length of the carbon backbone of the compound and each algal strain produced only one of these types. Biogeographical mapping of the prymnesin distribution indicated a global distribution of each type. In addition, phylogenetic analyses based on internal transcribed spacer (ITS) sequences revealed monophyletic origin of all prymnesin types and clades could therefore be defined based on the toxic compound. It might be that evolution of new species within the P. parvum species complex is driven by changes in toxin type or that they are a result of it. Such a correlation between chemotype and phylotype has never been documented before for a harmful microalga. Chemotaxonomy and ITS-type classification may thus be used to further delimit the P. parvum species complex.

Interannual Variability of Dinophysis acuminata and Protoceratium reticulatum in a Chilean Fjord: Insights from the Realized Niche Analysis.

Here, we present the interannual distribution of Dinophysis acuminata and Protoceratium reticulatum over a 10-year period in the Reloncaví Fjord, a highly stratified fjord in southern Chile. A realized subniche approach based on the Within Outlying Mean Index (WitOMI) was used to decompose the species' realized niche into realized subniches (found within subsets of environmental conditions). The interannual distribution of both D. acuminata and P. reticulatum summer blooms was strongly influenced by climatological regional events, i.e., El Niño Southern Oscillation (ENSO) and the Southern Annual Mode (SAM). The two species showed distinct niche preferences, with blooms of D. acuminata occurring under La Niña conditions (cold years) and low river streamflow whereas P. reticulatum blooms were observed in years of El Niño conditions and positive SAM phase. The biological constraint exerted on the species was further estimated based on the difference between the existing fundamental subniche and the realized subniche. The observed patterns suggested that D. acuminata was subject to strong biological constraint during the studied period, probably as a result of low cell densities of its putative prey (the mixotrophic ciliate Mesodinium cf. rubrum) usually observed in the studied area.

First report of vampyrellid predator-prey dynamics in a marine system.

We report for the first time the in situ dynamics of a vampyrellid in a marine system. A high sampling frequency (twice-weekly) was applied in a tropical eutrophic lagoon (Rio de Janeiro, Brazil) for 5 years (2012-2016). The vampyrellid Hyalodiscus sp. specifically fed on the diatom Chaetoceros minimus during a short time window (~3 months), although the prey was intermittently detected as the dominant phytoplanktonic species over a longer period (~1 year). A classic Lotka-Volterra predator-prey dynamic was observed between the two partners, with a significant modification of the short-term oscillations of the prey. Specific abiotic preferences (i.e., relatively low temperature, intermediate salinity, and stratified conditions) associated with prey availability seemed to define this narrow temporal window of occurrence. Our results suggest that vampyrellids can be ecologically relevant in marine pelagic systems, with their impact on planktonic dynamics strongly depending on complex interactions between both biotic and abiotic factors.

Production of extracellular superoxide and hydrogen peroxide by five marine species of harmful bloom-forming algae.

Harmful bloom-forming algae include some of the most prolific microbial producers of extracellular reactive oxygen species (ROS). However, the taxonomic diversity of ROS production, the underlying physiological mechanisms and ecophysiological roles of ROS cycling are not completely characterized among phytoplankton taxa that form harmful algal blooms (HABs). This study examines the extracellular production of the ROS superoxide and hydrogen peroxide by five marine HAB species: Chattonella marina, Heterosigma akashiwo, Karenia brevis, Pseudo-nitzschia sp. and Aureococcus anophagefferens. All species produced extracellular superoxide and hydrogen peroxide. Rates of ROS production per cell spanned several orders of magnitude and varied inversely with cell density, suggesting a potential signaling role for extracellular ROS. ROS production was also detected in the spent media of all cultures except K. brevis, indicating the presence of cell-free ROS-generating constituents, such as enzymes or metabolites, which could be further investigated as molecular targets for tracking ROS production in laboratory and field settings. Finally, ratios of superoxide to hydrogen peroxide production could not be accounted for by superoxide dismutation alone, except in the case of K. brevis, indicating a diversity of ROS production and degradation pathways that may ultimately help illuminate the functions of HAB-derived ROS.

Morphology and phylogeny of Prorocentrum caipirignum sp. nov. (Dinophyceae), a new tropical toxic benthic dinoflagellate.

A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37-44µm long and 29-36µm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.

An evaluation of the genus Amphidinium (Dinophyceae) combining evidence from morphology, phylogenetics, and toxin production, with the introduction of six novel species.

The genus Amphidinium is an important group of athecated dinoflagellates because of its high abundance in marine habitats, its member's ability to live in a variety of environmental conditions and ability to produce toxins. Furthermore, the genus is of particular interest in the biotechnology field for its potential in the pharmaceutical arena. Taxonomically the there is a history of complication and confusion over the proper identities and placements of Amphidinium species due to high genetic variability coupled with high morphological conservation. Thirteen years has passed since the most recent review of the group, and while many issues were resolved, some remain. The present study used microscopy, phylogenetics of the 28S region of rDNA, secondary structure of the ITS2 region of rDNA, compensatory base change data, and cytotoxicity data from Amphidinium strains collected world-wide to elucidate remaining confusion. This holistic approach using multiple lines of evidence resulted in a more comprehensive understanding of the morphological, ecological, and genetic characteristics that are attributed to organisms belonging to Amphidinium, including six novel species: A. fijiensis, A. magnum, A. paucianulatum, A. pseudomassartii, A. theodori, and A. tomasii.

Evidence for parasite-mediated selection during short-lasting toxic algal blooms.

Parasites play a role in the control of transient algal blooms, but it is not known whether parasite-mediated selection results in coevolution of the host and the parasites over this short time span. We investigated the presence of coevolution between the toxic dinoflagellate Alexandrium minutum and two naturally occurring endoparasites during blooms lasting a month in two river estuaries, using cross-inoculation experiments across time and space. Higher parasite abundance was associated with a large daily reduction in relative A. minutum abundances, demonstrating strong parasite-mediated selection. There was genetic variability in infectivity in both parasite species, and in resistance in the host. We found no evidence for coevolution in one estuary; however, in the other estuary, we found high genetic diversity in the two parasite species, fluctuations in infectivity and suggestion that the two parasites are well adapted to their host, as in 'Red Queen' dynamics. Thus, coevolution is possible over the short time span of a bloom, but geographically variable, and may feedback on community dynamics.

Significance of plankton community structure and nutrient availability for the control of dinoflagellate blooms by parasites: a modeling approach.

Dinoflagellate blooms are frequently observed under temporary eutrophication of coastal waters after heavy rains. Growth of these opportunistic microalgae is believed to be promoted by sudden input of nutrients and the absence or inefficiency of their natural enemies, such as grazers and parasites. Here, numerical simulations indicate that increasing nutrient availability not only promotes the formation of dinoflagellate blooms but can also stimulate their control by protozoan parasites. Moreover, high abundance of phytoplankton other than dinoflagellate hosts might have a significant dilution effect on the control of dinoflagellate blooms by parasites, either by resource competition with dinoflagellates (thus limiting the number of hosts available for infection) or by affecting numerical-functional responses of grazers that consume free-living parasite stages. These outcomes indicate that although both dinoflagellates and their protozoan parasites are directly affected by nutrient availability, the efficacy of the parasitic control of dinoflagellate blooms under temporary eutrophication depends strongly on the structure of the plankton community as a whole.

Interplay between the parasite Amoebophrya sp. (Alveolata) and the cyst formation of the red tide dinoflagellate Scrippsiella trochoidea.

Syndiniales (Alveolata) are marine parasites of a wide range of hosts, from unicellular organisms to Metazoa. Many Syndiniales obligatorily kill their hosts to accomplish their life cycle. This is the case for Amoebophrya spp. infecting dinoflagellates. However, several dinoflagellate species known to be infected by these parasites produce diploid resting cysts as part of their life history. These resting cysts may survive several seasons in the sediment before germinating. How these parasites survive during the dormancy of their host remained an open question. We successfully established infections by Amoebophrya sp. in the red tide dinoflagellate Scrippsiella trochoidea. This host strain was homothallic and able to continuously produce typical calcified cysts covered by calcareous spines. Presence of the parasite significantly speeded up the host cyst production, and cysts produced were the only cells to resist infections. However, some of them were clearly infected, probably earlier in their formation. After 10 months, cysts produced in presence of the parasite were able to germinate and new infective cycles of the parasite were rapidly observed. Thus, a very novel relationship for protists is demonstrated, one in which parasite and host simultaneously enter dormancy, emerging months later to propagate both species.