Emerging Parasitic Protists: The Case of Perkinsea

The last century has witnessed an increasing rate of new disease emergence across the world leading to permanent loss of biodiversity. Perkinsea is a microeukaryotic parasitic phylum composed of four main lineages of parasitic protists with broad host ranges. Some of them represent major ecological and economical threats because of their geographically invasive ability and pathogenicity (leading to mortality events). In marine environments, three lineages are currently described, the Parviluciferaceae, the Perkinsidae, and the Xcellidae, infecting, respectively, dinoflagellates, mollusks, and fish. In contrast, only one lineage is officially described in freshwater environments: the severe Perkinsea infectious agent infecting frog tadpoles. The advent of high-throughput sequencing methods, mainly based on 18S rRNA assays, showed that Perkinsea is far more diverse than the previously four described lineages especially in freshwater environments. Indeed, some lineages could be parasites of green microalgae, but a formal nature of the interaction needs to be explored. Hence, to date, most of the newly described aquatic clusters are only defined by their environmental sequences and are still not (yet) associated with any host. The unveiling of this microbial black box presents a multitude of research challenges to understand their ecological roles and ultimately to prevent their most negative impacts. This review summarizes the biological and ecological traits of Perkinsea—their diversity, life cycle, host preferences, pathogenicity, and highlights their diversity and ubiquity in association with a wide range of hosts.

Triangulopteris lacunata gen. et sp. nov. (Centroplasthelida), a New Centrohelid Heliozoan from Soil †

A new genus and species of centrohelid heliozoans, Triangulopteris lacunata gen. et sp. nov. (Pterocystidae Cavalier-Smith and Heyden, 2007), from four geographically remote locations (the Crimean Peninsula, the Dnieper Lowland (the East European Plain), Franz Josef Land, and the Kolyma Lowland (North–Eastern Siberia) was examined using light and electron microscopy. The novel centrohelid is characterized by round shape, 4.3–16.3 μm in diameter, covered with two types of scales: 1.06–4.54 μm long triangular spine scales and 1.22–2.05 μm oval plate scales. Studied centrohelid heliozoan possesses a unique spine scale morphology. The base of scales is represented by a horse hoof-shaped basal plate. The inner surface and lateral wings of spine scales have numerous radial ribs with two ‘pockets’ that are located on both sides of the spine shaft. These pockets are formed by the lateral wings and ends of the basal plate. The cyst formation and transition to a spicules-bearing stage were noted. Additionally, phylogenetic tree was constructed based on SSU rRNA sequences including the strain HF-25 from the permafrost of Kolyma Lowland. The resulting phylogeny recovered it within the clade Pterista, while forming a separate sister lineage to H2 clade, which only had included freshwater environmental sequences.

Diversity Patterns of Protists are Highly Affected by Methods Disentangling Inter-specific Variants: A Case Study in Oligotrich (s.l.) Ciliates

An enormous amount of environmental sequences produced by high-throughput sequencing (HTS) is popular for inferring diversity and distribution patterns of protists, which are widely distributed and playing important roles in energy flux and nutrient cycling. However, study testing whether methods disentangling inter-specific variants affect the diversity and distribution patterns of protists using field samples is insufficient. Using oligotrich (s.l.) ciliates, one group of abundant and dominate planktonic protists, in field samples as an example, the present study indicates that DADA2 performs better than UCLUST and UPARSE for inferring diversity patterns of oligotrich (s.l.) ciliates in Pearl River Estuary and surrounding regions. UPARSE, as an OTU-construction method might underestimate species richness and produce less reliable beta diversity pattern than DADA2. UCLUST with 97% and 99% clustering thresholds overestimate species richness, and the beta diversity pattern inferred by the former one is unreasonable. Salinity is shown to be one of the key factors responsible for variations in community distribution of ciliates, but infrequent marine-freshwater transitions occurred during evolutionary terms of this group.

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.

The long-time orphan protist Meringosphaera mediterranea Lohmann, 1902 [1903] is a centrohelid heliozoan

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophytes-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exists so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the West Coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 μm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S-V9 metabarcoding dataset which revealed four OTUs with 95.5-98.5% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily.

Diat.barcode: a DNA tool to decipher diatom communities for the evaluation environmental pressures

Diatoms (Bacillariophyta) are ubiquitous microalgae, which present a huge taxonomic diversity, changing in correlation with differing environmental conditions. This makes them excellent ecological indicators for various ecosystems and ecological problematics (ecotoxicology, biomonitoring, paleo-environmental reconstruction …). Current standardized methodologies for diatoms are based on microscopic determinations, which is time consuming and prone to identification uncertainties. DNA metabarcoding has been proposed as a way to avoid these flaws, enabling the sequencing of a large quantity of barcodes from natural samples. A taxonomic identity is given to these barcodes by comparing their sequences to a barcoding reference library. However, to identify environmental sequences correctly, the reference database should contain a representative number of reference sequences to ensure a good coverage of diatom diversity. Moreover, the reference database needs to be carefully taxonomically curated by experts, as its content has an obvious impact on species detection. Diat.barcode is an open-access library for diatoms linking diatom taxonomic identities to rbcL barcode sequences (a chloroplast marker suitable for species-level identification of diatoms), which has been maintained since 2012. Data are accumulated from three sources: (1) the NCBI nucleotide database, (2) unpublished sequencing data of culture collections and more recently (3) environmental sequences. Since 2017, an international network of experts in diatom taxonomy curate this library. The last version of the database (version 9.2), includes 8066 entries that correspond to more than 280 different genera and 1490 different species. In addition to the taxonomic information, morphological features (e.g. biovolumes, chloroplasts, etc.), life-forms (mobility, colony-type) and ecological features (taxa preferences to pollution) are given. The database can be downloaded from the website (www6.inrae.fr/carrtel-collection/Barcoding-database/) or directly through the R package diatbarcode. Ready-to-use files for commonly used metabarcoding pipelines (Mothur and DADA2) are also available.

First finding of free-living representatives of Prokinetoplastina and their nuclear and mitochondrial genomes

Kinetoplastids are heterotrophic flagellated protists, including important parasites of humans and animals (trypanosomatids), and ecologically important free-living bacterial consumers (bodonids). Phylogenies have shown that the earliest-branching kinetoplastids are all parasites or obligate endosymbionts, whose highly-derived state makes reconstructing the ancestral state of the group challenging. We have isolated new strains of unusual free-living flagellates that molecular phylogeny shows to be most closely related to endosymbiotic and parasitic Perkinsela and Ichthyobodo species that, together with unidentified environmental sequences, form the clade at the base of kinetoplastids. These strains are therefore the first described free-living prokinetoplastids, and potentially very informative in understanding the evolution and ancestral states of morphological and molecular characteristics described in other kinetoplastids. Overall, we find that these organisms morphologically and ultrastructurally resemble some free-living bodonids and diplonemids, and possess nuclear genomes with few introns, polycistronic mRNA expression, high coding density, and derived traits shared with other kinetoplastids. Their genetic repertoires are more diverse than the best-studied free-living kinetoplastids, which is likely a reflection of their higher metabolic potential. Mitochondrial RNAs of these new species undergo the most extensive U insertion/deletion editing reported so far, and limited deaminative C-to-U and A-to-I editing, but we find no evidence for mitochondrial trans-splicing.

Txikispora philomaios n. sp., n. g., a Micro-Eukaryotic Pathogen of Amphipods, Reveals Parasitism and Hidden Diversity in Class Filasterea

ABSTRACTThis study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly haemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host haemocytes, resulting in haemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Comparative genomics reveals new functional insights in uncultured MAST species

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.

Fig. S1 & Tables S1-S4

Figure S1. Overview map of the biogeographical regions used for Table 1. Biogeographic regions are based on biogeographic realms (https://ecoregions2017.appspot.com/), with three major differences: Western Palearctic (Western part of the Palearctic realm), Asia (Eastern part of the Palearctic realm combined with the Indo-Malay realm), and Australasia (Australasian realm combined with the Oceanian realm). The Palearctic realm was spilt into Western Palearctic and Eastern Palearctic, Eastern Palearctic and the Indo-Malay realm form together the Asia region, and the Australasian realm is combined with the Oceania realm to form the Australasian region.Table S1. List of described Lactifluus species, together with the year of description, taxonomical classification (subgenus, section), the indication of how this taxonomical position was defined, the source(s) of this classification, and notes.Table S2. Extra information on the preliminary study of metabarcoding data of the genus Lactifluus, retrieved from the GlobalFungi website.Table S3. Overview of the results of the preliminary study of metabarcoding data of the genus Lactifluus, retrieved from the GlobalFungi website. Due to the generally shorter length and lower quality of environmental sequence data, the numbers in the table are to be considered an estimate.Table S4. List of the putative new species found in the environmental sequences. References of studies cited are given in S3.