Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

The new chytridiomycete Paradinomyces triforaminorum gen. et sp. nov. co-occurs with other parasitoids during a Kryptoperidinium foliaceum (Dinophyceae) bloom in the Baltic Sea.

A new chytrid genus and species was isolated and cultured from samples obtained in the Baltic Sea during a dinoflagellate bloom event. This species is characterized by having a spherical sporangium without papillae and zoospores of 2-3 µm in diameter that are released through 3 discharge pores. Molecular phylogeny based on ribosomal operon showed its sister position to the Dinomyces cluster in Rhizophydiales. Zoospores lack fenestrated cisternae but contain a paracrystalline inclusion, found in a Rhizophydiales representative for the first time. Additionally, the kinetid features are uncommon for Rhizophydiales and only observed in Dinomyces representatives so far. These morphological features and its phylogenetic relationships justify the description of the new genus and speciesParadinomyces triforaminorum gen. nov. sp. nov. belonging to the family Dinomycetaceae. The chytrid was detected during a high-biomass bloom of the dinoflagellate Kryptoperidinium foliaceum. Laboratory experiments suggest this species is highly specific and demonstrate the impact it can have on HAB development. The chytrid co-occurred with three other parasites belonging to Chytridiomycota (Fungi) and Perkinsea (Alveolata), highlighting that parasitic interactions are common during HABs in brackish and marine systems, and these multiple parasites compete for similar hosts.

A diversified and segregated mRNA spliced-leader system in the parasitic Perkinsozoa.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5' exon, giving different mRNAs identical 5' sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

A cell-cell atlas approach for understanding symbiotic interactions between microbes.

Natural environments are composed of a huge diversity of microorganisms interacting with each other to form complex functional networks. Our understanding of the operative nature of host-symbiont associations is limited because propagating such associations in a laboratory is challenging. The advent of single-cell technologies applied to, for example, animal cells and apicomplexan parasites has revolutionized our understanding of development and disease. Such cell atlas approaches generate maps of cell-specific processes and variations within cellular populations. These methods can now be combined with cellular-imaging so that interaction stage versus transcriptome state can be quantized for microbe-microbe interactions. We predict that the combination of these methods applied to the study of symbioses will transform our understanding of many ecological interactions, including those sampled directly from natural environments.

New Perkinsea Parasitoids of Dinoflagellates Distantly Related to Parviluciferaceae Members.

Perkinsea is a phylogenetic group of protists that includes parasites of distantly related hosts. However, its diversity is still mainly composed of environmental sequences, mostly obtained from freshwater environments. Efforts to isolate and culture parasitoids of dinoflagellates have led to the description of several phylogenetically closely related species constituting the Parviluciferaceae family. In this study, two new parasitoid species infecting dinoflagellates during recurrent coastal blooms are reported. Using the ribosomal RNA (rRNA) gene phylogenies, we show that both cluster within Perkinsea, one of them at the base of Parviluciferaceae and the other in a distinct branch unrelated to other described species. The establishment of host-parasite lab cultures of the latter allowed its morphological characterization, resulting in the formal description of Maranthos nigrum gen. nov., sp. nov. The life-cycle development of the two parasitoids is generally the same as that of other members of the Parviluciferaceae family but they differ in the features of the trophont and sporont stages, including the arrangement of zoospores during the mature sporangium stage and the lack of specialized structures that release the zoospores into the environment. Laboratory cross-infection experiments showed that the parasitoid host range is restricted to dinoflagellates, although it extends across several different genera. The maximum prevalence reached in the tested host populations was lower than in other Parviluciferaceae members. The findings from this study suggest that Perkinsea representatives infecting dinoflagellates are more widespread than previously thought.

Host preferences of coexisting Perkinsea parasitoids during coastal dinoflagellate blooms.

Parasites in aquatic systems are highly diverse and ubiquitous. In marine environments, parasite-host interactions contribute substantially to shaping microbial communities, but their nature and complexity remain poorly understood. In this study, we examined the relationship between Perkinsea parasitoids and bloom-forming dinoflagellate species. Our aim was to determine whether parasite-host species interactions are specific and whether the diversity and distribution of parasitoids are shaped by their dinoflagellate hosts. Several locations along the Catalan coast (NW Mediterranean Sea) were sampled during the blooms of five dinoflagellate species and the diversity of Perkinsea was determined by combining cultivation-based methods with metabarcoding of the V4 region of 18S rDNA. Most known species of Parviluciferaceae, and others not yet described, were detected, some of them coexisting in the same coastal location, and with a wide distribution. The specific parasite-host interactions determined for each of the studied blooms demonstrated the host preferences exhibited by parasitoids in nature. The dominance of a species within the parasitoid community is driven by the presence and abundances of its preferred host(s). The absence of parasitoid species, often associated with a low abundance of their preferred hosts, suggested that high infection rates are reached only under conditions that favour parasitoid propagation, especially dinoflagellate blooms.

Description of two new coexisting parasitoids of blooming dinoflagellates in the Baltic sea: Parvilucifera catillosa sp. nov. and Parvilucifera sp. (Perkinsea, Alveolata).

Perkinsea are a group of intracellular protist parasites that inhabit all types of aquatic environments and cause significant population declines of a wide variety of hosts. However, the diversity of this lineage is mostly represented by environmental rDNA sequences. Complete descriptions of Perkinsea that infect marine dinoflagellates have increased in recent literature due to the identification, isolation and culturing of representatives during bloom events, contributing to expand the knowledge on the diversity and ecology of the group. Shallow coastal areas in the Baltic Sea suffer seasonal dinoflagellate blooms. In summer 2016, two parasitoids were isolated during a Kryptoperidinium foliaceum bloom in the Baltic Sea. Morphological features and sequences of the small and large subunit of the ribosomal DNA gene revealed these two parasitoids were new species that belong to the genus Parvilucifera. This is the first time that Parvilucifera infections are reported in the Inner Baltic Sea. The first species, Parvilucifera sp. has some morphological and phylogenetic features in common with P. sinerae and P. corolla, although its ultrastructure could not be studied and the formal description could not be done. The second new species, named Parvilucifera catillosa, has several distinct morphological features in its zoospores (e.g. the presence of a rostrum), and in the shape and size of the apertures in the sporangium stage, which are larger and more protuberant than in the other species of the genus. Infections observed in the field and cross-infection experiments determined that the host range of both Parvilucifera species was restricted to dinoflagellates, each one showing a different host preference. The coexistence in the same environment by the two closely related parasitoids with very similar life cycles suggests that their niche separation is the preferred host.

Evolutionary Trends of Perkinsozoa (Alveolata) Characters Based on Observations of Two New Genera of Parasitoids of dinoflagellates, Dinovorax gen. nov. and Snorkelia gen. nov.

Parasites are one of the ecologically most relevant groups of marine food webs, but their taxonomic and biological complexity hampers the assessment of their diversity and evolutionary trends. Moreover, the within-host processes that govern parasitoid infection, development and reproduction are often unknown. In this study, we describe a new species of a perkinsozoan endoparasitoid that infects the toxic dinoflagellate Dinophysis sacculus, by including observations of its morphology, ultrastructure, life-cycle development and phylogeny. The SSU rDNA sequence and main morphological features were also obtained for a second parasitoid species infecting the bloom-forming dinoflagellate Levanderina fissa. Phylogenetic analyses including the sequences obtained show that all known Perkinsozoa species infecting dinoflagellates cluster together. However, sequences of Parvilucifera prorocentri and those obtained in this study cluster at the base of the clade, while the rest of Parvilucifera representatives form a separated highly-supported cluster. These results, together with differing morphological characters like the formation of a germ-tube, the presence of trichocysts, or the heterochromatin presence in zoospores nucleus justify the erection of Dinovorax pyriformis gen. nov. et sp. nov., and Snorkelia prorocentri gen. nov. et comb. nov. (=Parvilucifera prorocentri). The morphological features and phylogenetic position of these parasitoids represent ancestral characters for the Perkinsozoa phylum, and also for Dinozoa clade, allowing the inference of the evolutionary framework of these Alveolata members.

In situ Occurrence, Prevalence and Dynamics of Parvilucifera Parasitoids during Recurrent Blooms of the Toxic Dinoflagellate Alexandrium minutum.

Dinoflagellate blooms are natural phenomena that often occur in coastal areas, which in addition to their large number of nutrient-rich sites are characterized by highly restricted hydrodynamics within bays, marinas, enclosed beaches, and harbors. In these areas, massive proliferations of dinoflagellates have harmful effects on humans and the ecosystem. However, the high cell density reached during blooms make them vulnerable to parasitic infections. Under laboratory conditions parasitoids are able to exterminate an entire host population. In nature, Parvilucifera parasitoids infect the toxic dinoflagellate Alexandrium minutum during bloom conditions but their prevalence and impact remain unexplored. In this study, we evaluated the in situ occurrence, prevalence, and dynamics of Parvilucifera parasitoids during recurrent blooms of A. minutum in a confined site in the NW Mediterranean Sea as well as the contribution of parasitism to bloom termination. Parvilucifera parasitoids were recurrently detected from 2009 to 2013, during seasonal outbreaks of A. minutum. Parasitic infections in surface waters occurred after the abundance of A. minutum reached 104-105 cells L-1, suggesting a density threshold beyond which Parvilucifera transmission is enhanced and the number of infected cells increases. Moreover, host and parasitoid abundances were not in phase. Instead, there was a lag between maximum A. minutum and Parvilucifera densities, indicative of a delayed density-dependent response of the parasitoid to host abundances, similar to the temporal dynamics of predator-prey interactions. The highest parasitoid prevalence was reached after a peak in host abundance and coincided with the decay phase of the bloom, when a maximum of 38% of the A. minutum population was infected. According to our estimates, Parvilucifera infections accounted for 5-18% of the total observed A. minutum mortality, which suggested that the contribution of parasitism to bloom termination is similar to that of other biological factors, such as encystment and grazing.

Integrating chytrid fungal parasites into plankton ecology: research gaps and needs.

Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.

Life-cycle, ultrastructure, and phylogeny of Parvilucifera corolla sp. nov. (Alveolata, Perkinsozoa), a parasitoid of dinoflagellates.

Recent studies of marine protists have revealed parasites to be key components of marine communities. Here we describe a new species of the parasitoid genus Parvilucifera that was observed infecting the dinoflagellate Durinskia baltica in salt marshes of the Catalan coast (NW Mediterranean). In parallel, the same species was detected after the incubation of seawater from the Canary Islands (Lanzarote, NE Atlantic). The successful isolation of strains from both localities allowed description of the life cycle, ultrastructure, and phylogeny of the species. Its infection mechanism consists of a free-living zoospore that penetrates a dinoflagellate cell. The resulting trophont gradually degrades the dinoflagellate cytoplasm while growing in size. Once the host is consumed, schizogony of the parasitoid yields a sporocyte. After cytokinesis is complete, the newly formed zoospores are released into the environment and are ready to infect new host cells. A distinguishing feature of the species is the radial arrangement of its zoospores around the central area of the sporocyte during their formation. The species shows a close morphological similarity with other species of the genus, including P. infectans, P. sinerae, and P. rostrata.

A Game of Russian Roulette for a Generalist Dinoflagellate Parasitoid: Host Susceptibility Is the Key to Success.

Marine microbial interactions involving eukaryotes and their parasites play an important role in shaping the structure of phytoplankton communities. These interactions may alter population densities of the main host, which in turn may have consequences for the other concurrent species. The effect generalist parasitoids exert on a community is strongly dependent on the degree of host specificity. Parvilucifera sinerae is a generalist parasitoid able to infect a wide range of dinoflagellates, including toxic-bloom-forming species. A density-dependent chemical cue has been identified as the trigger for the activation of the infective stage. Together these traits make Parvilucifera-dinoflagellate hosts a good model to investigate the degree of specificity of a generalist parasitoid, and the potential effects that it could have at the community level. Here, we present for the first time, the strategy by which a generalist dinoflagellate parasitoid seeks out its host and determine whether it exhibits host preferences, highlighting key factors in determining infection. Our results demonstrate that in its infective stage, P. sinerae is able to sense potential hosts, but does not actively select among them. Instead, the parasitoids contact the host at random, governed by the encounter probability rate and once encountered, the chance to penetrate inside the host cell and develop the infection strongly depends on the degree of host susceptibility. As such, their strategy for persistence is more of a game of Russian roulette, where the chance of survival is dependent on the susceptibility of the host. Our study identifies P. sinerae as a potential key player in community ecology, where in mixed dinoflagellate communities consisting of hosts that are highly susceptible to infection, parasitoid preferences may mediate coexistence between host species, reducing the dominance of the superior competitor. Alternatively, it may increase competition, leading to species exclusion. If, however, highly susceptible hosts are absent from the community, the parasitoid population could suffer a dilution effect maintaining a lower parasitoid density. Therefore, both host community structure and host susceptibility will determine infectivity in the field.

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.

Dinomyces arenysensis gen. et sp. nov. (Rhizophydiales, Dinomycetaceae fam. nov.), a chytrid infecting marine dinoflagellates.

Environmental 18S rRNA gene surveys of microbial eukaryotes have recently revealed the diversity of major parasitic agents in pelagic freshwater systems, consisting primarily of chytrid fungi. To date, only a few studies have reported the presence of chydrids in the marine environment and a limited number of marine chytrids have been properly identified and characterized. Here, we report the isolation and cultivation of a marine chytrid from samples taken during a bloom of the toxic dinoflagellate Alexandrium minutum in the Arenys de Mar harbour (Mediterranean Sea, Spain). Cross-infections using cultures and natural phytoplankton communities revealed that this chytrid is only able to infect certain species of dinoflagellates, with a rather wide host range but with a relative preference for Alexandrium species. Phylogenetic analyses showed that it belongs to the order Rhizophydiales, but cannot be included in any of the existing families within this order. Several ultrastructural characters confirmed the placement of this taxon within the Rhizophydiales as well its novelty notably in terms of zoospore structure. This marine chytridial parasitoid is described as a new genus and species, Dinomyces arenysensis, within the Dinomycetaceae fam. nov.

The potential for arms race and Red Queen coevolution in a protist host-parasite system.

The dynamics and consequences of host-parasite coevolution depend on the nature of host genotype-by-parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies ("Red Queen" dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps ("arms race" dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host-parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host-parasite system.

Host-released dimethylsulphide activates the dinoflagellate parasitoid Parvilucifera sinerae.

Parasitoids are a major top-down cause of mortality of coastal harmful algae, but the mechanisms and strategies they have evolved to efficiently infect ephemeral blooms are largely unknown. Here, we show that the generalist dinoflagellate parasitoid Parvilucifera sinerae (Perkinsozoa, Alveolata) is activated from dormancy, not only by Alexandrium minutum cells but also by culture filtrates. We unequivocally identified the algal metabolite dimethylsulphide (DMS) as the density-dependent cue of the presence of potential host. This allows the parasitoid to alternate between a sporangium-hosted dormant stage and a chemically-activated, free-living virulent stage. DMS-rich exudates of resistant dinoflagellates also induced parasitoid activation, which we interpret as an example of coevolutionary arms race between parasitoid and host. These results further expand the involvement of dimethylated sulphur compounds in marine chemical ecology, where they have been described as foraging cues and chemoattractants for mammals, turtles, birds, fish, invertebrates and plankton microbes.

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.