Development of Schizochytrium sp. strain HS01 with high-DHA and low-saturated fatty acids production by multi-pronged adaptive evolution.

PURPOSE: Docosahexaenoic acid (DHA) is an important omega-3 unsaturated fatty acid and has been widely applied in medicine, food additives, and feed ingredients. The fermentative production of DHA using microorganisms, including Schizochytrium sp., attracted much attention due to its high production efficiency and environment friendly properties. An efficient laboratory evolution approach was used to improve the strain's performance in this study. METHODS: A multi-pronged laboratory evolution approach was applied to evolve high-yield DHA-producing Schizochytrium strain. We further employed comparative transcriptional analysis to identify transcriptional changes between the screened strain HS01 and its parent strain GS00. RESULTS: After multiple generations of ALE, a strain HS01 with higher DHA content and lower saturated fatty acids content was obtained. Low nitrogen conditions were important for enhancing DHA biosynthesis in HS01. The comparative transcriptional analysis results indicated that during the fermentation process of HS01, the expression of key enzymes in the glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle were up-regulated, while the expression of polyketide synthase genes and fatty acid synthesis genes were similar to those in GS00. CONCLUSION: The results suggest that the improved DHA production capacity of HS01 is not due to enhancement of the DHA biosynthesis pathway, but rather related to modulation of central metabolism pathways.

Comparison of Deep-Sea Picoeukaryotic Composition Estimated from the V4 and V9 Regions of 18S rRNA Gene with a Focus on the Hadal Zone of the Mariana Trench.

Diversity of microbial eukaryotes is estimated largely based on sequencing analysis of the hypervariable regions of 18S rRNA genes. But the use of different regions of 18S rRNA genes as molecular markers may generate bias in diversity estimation. Here, we compared the differences between the two most widely used markers, V4 and V9 regions of the 18S rRNA gene, in describing the diversity of epipelagic, bathypelagic, and hadal picoeukaryotes in the Challenger Deep of the Mariana Trench, which is a unique and little explored environment. Generally, the V9 region identified more OTUs in deeper waters than V4, while the V4 region provided greater Shannon diversity than V9. In the epipelagic zone, where Alveolata was the dominant group, picoeukaryotic community compositions identified by V4 and V9 markers are similar at different taxonomic levels. However, in the deep waters, the results of the two datasets show clear differences. These differences were mainly contributed by Retaria, Fungi, and Bicosoecida. The primer targeting the V9 region has an advantage in amplifying Bicosoecids in the bathypelagic and hadal zone of the Mariana Trench, and its high abundance in V9 dataset pointed out the possibility of Bicosoecids as a dominant group in this environment. Chrysophyceae, Fungi, MALV-I, and Retaria were identified as the dominant picoeukaryotes in the bathypelagic and hadal zone and potentially play important roles in deep-sea microbial food webs and biogeochemical cycling by their phagotrophic, saprotrophic, and parasitic life styles. Overall, the use of different markers of 18S rRNA gene allows a better assessment and understanding of the picoeukaryotic diversity in deep-sea environments.

Spatiotemporal Variations in Antarctic Protistan Communities Highlight Phytoplankton Diversity and Seasonal Dominance by a Novel Cryptophyte Lineage.

The Andvord fjord in the West Antarctic Peninsula (WAP) is known for its productivity and abundant megafauna. Nevertheless, seasonal patterns of the molecular diversity and abundance of protistan community members underpinning WAP productivity remain poorly resolved. We performed spring and fall expeditions pursuing protistan diversity, abundance of photosynthetic taxa, and the connection to changing conditions. 18S rRNA amplicon sequence variant (ASV) profiles revealed diverse predatory protists spanning multiple eukaryotic supergroups, alongside enigmatic heterotrophs like the Picozoa. Among photosynthetic protists, cryptophyte contributions were notable. Analysis of plastid-derived 16S rRNA ASVs supported 18S ASV results, including a dichotomy between cryptophytes and diatom contributions previously reported in other Antarctic regions. We demonstrate that stramenopile and cryptophyte community structures have distinct attributes. Photosynthetic stramenopiles exhibit high diversity, with the polar diatom Fragilariopsis cylindrus, unidentified Chaetoceros species, and others being prominent. Conversely, ASV analyses followed by environmental full-length rRNA gene sequencing, electron microscopy, and flow cytometry revealed that a novel alga dominates the cryptophytes. Phylogenetic analyses established that TPG clade VII, as named here, is evolutionarily distinct from cultivated cryptophyte lineages. Additionally, cryptophyte cell abundance correlated with increased water temperature. Analyses of global data sets showed that clade VII dominates cryptophyte ASVs at Southern Ocean sites and appears to be endemic, whereas in the Arctic and elsewhere, Teleaulax amphioxeia and Plagioselmis prolonga dominate, although both were undetected in Antarctic waters. Collectively, our studies provide baseline data against which future change can be assessed, identify different diversification patterns between stramenopiles and cryptophytes, and highlight an evolutionarily distinct cryptophyte clade that thrives under conditions enhanced by warming. IMPORTANCE The climate-sensitive waters of the West Antarctic Peninsula (WAP), including its many fjords, are hot spots of productivity that support multiple marine mammal species. Here, we profiled protistan molecular diversity in a WAP fjord known for high productivity and found distinct spatiotemporal patterns across protistan groups. Alongside first insights to seasonal changes in community structure, we discovered a novel phytoplankton species with proliferation patterns linked to temperature shifts. We then examined evolutionary relationships between this novel lineage and other algae and their patterns in global ocean survey data. This established that Arctic and Antarctic cryptophyte communities have different species composition, with the newly identified lineage being endemic to Antarctic waters. Our research provides critical knowledge on how specific phytoplankton at the base of Antarctic food webs respond to warming, as well as information on overall diversity and community structure in this changing polar environment.

Mediocremonas mediterraneus, a New Member within the Developea.

The stramenopiles are a large and diverse group of eukaryotes that possess various lifestyles required to thrive in a broad array of environments. The stramenopiles branch with the alveolates, rhizarians, and telonemids, forming the supergroup TSAR. Here, we present a new genus and species of aquatic nanoflagellated stramenopile: Mediocremonas mediterraneus, a free-swimming heterotrophic predator. M. mediterraneus cell bodies measure between 2.0-4.0 µm in length and 1.2-3.7 µm in width, possessing two flagella and an oval body morphology. The growth and grazing rate of M. mediterraneus in batch cultures ranges from 0.68 to 1.83 d-1 and 1.99 to 5.38 bacteria/h, respectively. M. mediterraneus was found to be 93.9% phylogenetically similar with Developayella elegans and 94.7% with Develorapax marinus, two members within the class Developea. The phylogenetic position of the Developea and the ability of M. mediterraneus to remain in culture make it a good candidate for further genomic studies that could help us to better understand phagotrophy in marine systems as well as the transition from heterotrophy to phototrophy within the stramenopiles.

Multigene Phylogeny, Morphological Observation and Re-examination of the Literature Lead to the Description of the Phaeosacciophyceae Classis Nova and Four New Species of the Heterokontophyta SI Clade.

The relationships among the Aurearenophyceae, Phaeothamniophyceae, Phaeophyceae and Xanthophyceae lineages of the Heterokontophyta SI clade are not well known. By adding previously unexamined taxa related to these classes in a five gene phylogeny (SSU rRNA, atpB, psaA, psaB, rbcL), we recovered an assemblage of taxa previously unrecognized. We propose the class Phaeosacciophyceae class. nov., that includes Phaeosaccion collinsii, Phaeosaccion multiseriatum sp. nov., Phaeosaccion okellyi sp. nov., Antarctosaccion applanatum, Tetrasporopsis fuscescens, Tetrasporopsis moei sp. nov., and Psammochrysis cassiotisii gen. & sp. nov. We re-examine the literature for Chrysomeris, Nematochrysis, Chrysowaernella and the invalid name "Giraudyopsis" and conclude some taxa in previous studies are misidentified or misnamed, i.e. Chrysomeris and Chrysowaernella, respectively. We also show that Nematochrysis sessilis var. vectensis and Nematochrysis hieroglyphica may belong in the recently described class Chrysoparadoxophyceae. The phylogenetic relationships of Phaeobotrys solitaria and Pleurochloridella botrydiopsis are not clearly resolved, but they branch near the Xanthophyceae. Here we describe a new class Phaeosacciophyceae, a new order Phaeosacciales, a new family Tetrasporopsidaceae, a new genus Psammochrysis and four new species.

Comprehensive analysis of metabolic alterations in Schizochytrium sp. strains with different DHA content.

Along with the daily growth of the market requirements for docosahexaenoic acid (DHA) algae oil, a large DHA ingredients are needed to ensure worldwide supply. Undoubtedly a high-productive strain would be the prerequisite for high quality and yield. A comprehensive understanding of the processes of DHA synthesis from glycolysis to the lipid accumulation would be benefit to achieve the final optimization of DHA production. In this study, we comprehensively characterized the metabolic profiles of a Schizochytrium sp. strain, which has higher DHA content and different biomass amino acid composition compared with the wild type to explore the affected pathways and underlying mechanism. Combined with the multivariate statistical analysis, twenty-two differential metabolites were screened as relevant to the discrepancy between two strains. The results showed relatively downregulated glycolysis and saturated fatty acids (SFA) synthesis, and upregulated TCA cycle, amino acids and polyunsaturated fatty acids (PUFA) synthesis in DHA high yield strain. The current study provide a terminal picture of gene regulation from downstream metabolism and demonstrate the advantage of metabolomics in characterizing metabolic status which in turn could provide effective information for the metabolic engineering.

Shotgun metagenomics reveal a diverse assemblage of protists in a model Antarctic soil ecosystem.

The soils of the McMurdo Dry Valleys (MDV) of Antarctica are established models for understanding fundamental processes in soil ecosystem functioning (e.g. ecological tipping points, community structuring and nutrient cycling) because the extreme physical environment drastically reduces biodiversity and ecological complexity. Understanding the functioning of MDV soils requires in-depth knowledge of the diversity of MDV soil species. Protists, which contribute significantly to soil ecosystem functioning worldwide, remain poorly characterized in the MDV. To better assess the diversity of MDV protists, we performed shotgun metagenomics on 18 sites representing a variety of landscape features and edaphic variables. Our results show MDV soil protists are diverse at both the genus (155 of 281 eukaryote genera) and family (120) levels, but comprise only 6% of eukaryotic reads. Protists are structured by moisture, total N and distance from the local coast and possess limited richness in arid (< 5% moisture) and at high elevation sites, known drivers of communities in the MDV. High relative diversity and broad distribution of protists in our study promotes these organisms as key members of MDV soil microbiomes and the MDV as a useful system for understanding the contribution of soil protists to the structure of soil microbiomes.

Assessing the response of micro-eukaryotic diversity to the Great Acceleration using lake sedimentary DNA.

Long-term time series have provided evidence that anthropogenic pressures can threaten lakes. Yet it remains unclear how and the extent to which lake biodiversity has changed during the Anthropocene, in particular for microbes. Here, we used DNA preserved in sediments to compare modern micro-eukaryotic communities with those from the end of the 19th century, i.e., before acceleration of the human imprint on ecosystems. Our results obtained for 48 lakes indicate drastic changes in the composition of microbial communities, coupled with a homogenization of their diversity between lakes. Remote high elevation lakes were globally less impacted than lowland lakes affected by local human activity. All functional groups (micro-algae, parasites, saprotrophs and consumers) underwent significant changes in diversity. However, we show that the effects of anthropogenic changes have benefited in particular phototrophic and mixotrophic species, which is consistent with the hypothesis of a global increase of primary productivity in lakes.

Evolution of the genetic code in the mitochondria of Labyrinthulea (Stramenopiles).

Mitochondrial translation often exhibits departures from the standard genetic code, but the full spectrum of these changes has certainly not yet been described and the molecular mechanisms behind the changes in codon meaning are rarely studied. Here we report a detailed analysis of the mitochondrial genetic code in the stramenopile group Labyrinthulea (Labyrinthulomycetes) and their relatives. In the genus Aplanochytrium, UAG is not a termination codon but encodes tyrosine, in contrast to the unaffected meaning of the UAA codon. This change is evolutionarily independent of the reassignment of both UAG and UAA as tyrosine codons recently reported from two uncultivated labyrinthuleans (S2 and S4), which we show are not thraustochytrids as proposed before, but represent the clade LAB14 previously recognised in environmental 18S rRNA gene surveys. We provide rigorous evidence that the UUA codon in the mitochondria of all labyrinthuleans serves as a termination codon instead of encoding leucine, and propose that a sense-to-stop reassignment has also affected the AGG and AGA codons in the LAB14 clade. The distribution of the different forms of sense-to-stop and stop-to-sense reassignments correlates with specific modifications of the mitochondrial release factor mtRF2a in different subsets of labyrinthuleans, and with the unprecedented loss of mtRF1a in Aplanochytrium and perhaps also in the LAB14 clade, pointing towards a possible mechanistic basis of the code changes observed. Curiously, we show that labyrinthulean mitochondria also exhibit a sense-to-sense codon reassignment, manifested as AUA encoding methionine instead of isoleucine. Furthermore, we show that this change evolved independently in the uncultivated stramenopile lineage MAST8b, together with the reassignment of the AGR codons from arginine to serine. Altogether, our study has uncovered novel variants of the mitochondrial genetic code and previously unknown modifications of the mitochondrial translation machinery, further enriching our understanding of the rules governing the evolution of one of the central molecular process in the cell.

Unveiling protist diversity associated with the Pacific oyster Crassostrea gigas using blocking and excluding primers.

BACKGROUND: Microbiome of macroorganisms might directly or indirectly influence host development and homeostasis. Many studies focused on the diversity and distribution of prokaryotes within these assemblages, but the eukaryotic microbial compartment remains underexplored so far. RESULTS: To tackle this issue, we compared blocking and excluding primers to analyze microeukaryotic communities associated with Crassostrea gigas oysters. High-throughput sequencing of 18S rRNA genes variable loops revealed that excluding primers performed better by not amplifying oyster DNA, whereas the blocking primer did not totally prevent host contaminations. However, blocking and excluding primers showed similar pattern of alpha and beta diversities when protist communities were sequenced using metabarcoding. Alveolata, Stramenopiles and Archaeplastida were the main protist phyla associated with oysters. In particular, Codonellopsis, Cyclotella, Gymnodinium, Polarella, Trichodina, and Woloszynskia were the dominant genera. The potential pathogen Alexandrium was also found in high abundances within some samples. CONCLUSIONS: Our study revealed the main protist taxa within oysters as well as the occurrence of potential oyster pathogens. These new primer sets are promising tools to better understand oyster homeostasis and disease development, such as the Pacific Oyster Mortality Syndrome (POMS) targeting juveniles.

Growth, fatty, and amino acid profiles of the soil alga Vischeria sp. E71.10 (Eustigmatophyceae) under different cultivation conditions.

In this study, a unicellular soil alga isolated from farmland in Germany was surveyed. The investigation of the hypervariable molecular markers ITS1 rDNA and ITS2 rDNA identified strain E71.10 as conspecific with Vischeria sp. SAG 51.91 (Eustigmatophyceae). The culture was tested for biomass generation and for the yield of fatty acids and amino acids. The survey included four different culture conditions (conventional, elevated CO2, nitrogen depletion, or sodium chloride stress) at room temperature. The best yield of dry biomass was achieved applying 1% CO2, whereas nitrogen-free medium resulted into least growth. The fatty acid content peaked in nitrogen-free medium at 59% per dry mass. Eicosapentaenoic acid was the most abundant fatty acid in all treatments (except for nitrogen free), accounting for 10.44 to 16.72 g/100 g dry mass. The highest content of amino acids (20%) was achieved under conventional conditions. The results show that abiotic factors strongly influence to which extent metabolites are intracellularly stored and they confirm also for this yet undescribed strain of Vischeria that Eustigmatophyceae are promising candidates for biotechnology.

A Widely Distributed Thraustochytrid Parasite of Diatoms Isolated from the Arctic Represents a gen. and sp. nov.

A unicellular, heterotrophic, eukaryotic parasite was isolated from nearshore Arctic marine sediment in association with the diatom Pleurosigma sp. The parasite possessed ectoplasmic threads that could penetrate diatom frustules. Healthy and reproducing Pleurosigma cultures would begin to collapse within a week following the introduction of this parasite. The parasite (2-10 µm diameter) could reproduce epibiotically with biflagellate zoospores, as well as binary division inside and outside the diatom host. While the parasite grew, diatom intracellular content disappeared. Evaluation of electron micrographs from co-cultures revealed the presence of hollow tubular processes and amorphic cells that could transcend the diatom frustule, generally at the girdle band, as well as typical thraustochytrid ultrastructure, such as the presence of bothrosomes. After nucleotide extraction, amplification, and cloning, database queries of DNA revealed closest molecular affinity to environmental thraustochytrid clone sequences. Testing of phylogenetic hypotheses consistently grouped this unknown parasite within the Thraustochytriidae on a distinct branch within the environmental sequence clade Lab19. Reclassification of Arctic high-throughput sequencing data, with appended reference datasets that included this diatom parasite, indicated that the majority of thraustochytrid sequences, previously binned as unclassifiable stramenopiles, are allied to this new isolate. Based on the combined information acquired from electron microscopy, life history, and phylogenetic testing, this unknown isolate is described as a novel species and genus.

Morphological description of Opalina obtrigonoidea Metcalf, 1923 (Heterokonta, Opalinea) from Duttaphrynus melanostictus and evaluation of the ITS region as a suitable genetic marker for inter-species identification in Opalina.

The redescription of Opalina obtrigonoidea Metcalf, 1923, collected from the rectum of the toads Duttaphrynus melanostictus, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that O. obtrigonoidea varies greatly in body dimensions. Its morphological characteristics allow its differentiation from Opalina undulata. Surprisingly, we sequenced its SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found the SSU rDNA of O. obtrigonoidea is nearly identical to that of O. undulata. However, there are differences in both the ITS1 and ITS2 regions that allow their distinction and confirm the morphological differences. Our results indicate that O. obtrigonoidea and O. undulata are closely related species in which morphological and genetic markers have evolved at different speeds. Due to this, the SSU rDNA gene may not be a valid marker for inter-species identification in Opalina, but the ITS is a valid marker for differentiating species in this genus.

Global comparison of bicosoecid Cafeteria-like flagellates from the deep ocean and surface waters, with reorganization of the family Cafeteriaceae.

Cafeteria is one of the most common and ecologically significant genera of heterotrophic nanoflagellates in marine plankton. We could isolate and cultivate 29 strains morphologically similar to Cafeteria obtained from surface waters and the deep sea all over the world's ocean. Morphological characterization obtained by high resolution microscopy revealed only small differences between the strains. Sequencing the type material of the type species C. roenbergensis (CCAP 1900/1) and molecular analyses (18S rDNA, 28S rDNA) of newly isolated strains resulted in a revision and separation of the Cafeteriaceae into two known species (C. roenbergensis, C. mylnikovii) and six new species (C. maldiviensis, C. biegae, C. loberiensis, C. chilensis, C. graefeae, C. burkhardae). Many deposited Cafeteria sequences at GenBank and most of our own sequences clustered within one clade (C. burkhardae) with a p-distance of 5% to strain CCAP 1900/1. Only C. maldiviensis clustered together with the type species C. roenbergensis. While C. burkhardae seems to have a cosmopolitan distribution, the distribution of the other species seems to be more restricted. A strain from the Angola Basin had a p-distance of 10% to Cafeteria species and clustered separately within the Anoecales requiring the erection of a new genus, Bilabrum gen. nov., with B. latius sp. nov. as type species.

Ancient Adaptive Lateral Gene Transfers in the Symbiotic Opalina-Blastocystis Stramenopile Lineage.

Lateral gene transfer is a very common process in bacterial and archaeal evolution, playing an important role in the adaptation to new environments. In eukaryotes, its role and frequency remain highly debated, although recent research supports that gene transfer from bacteria to diverse eukaryotes may be much more common than previously appreciated. However, most of this research focused on animals and the true phylogenetic and functional impact of bacterial genes in less-studied microbial eukaryotic groups remains largely unknown. Here, we have analyzed transcriptome data from the deep-branching stramenopile Opalinidae, common members of frog gut microbiomes, and distantly related to the well-known genus Blastocystis. Phylogenetic analyses suggest the early acquisition of several bacterial genes in a common ancestor of both lineages. Those lateral gene transfers most likely facilitated the adaptation of the free-living ancestor of the Opalinidae-Blastocystis symbiotic group to new niches in the oxygen-depleted animal gut environment.

Antarctic thraustochytrids: Producers of long-chain omega-3 polyunsaturated fatty acids.

Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses.

Unexpected mitochondrial genome diversity revealed by targeted single-cell genomics of heterotrophic flagellated protists.

Most eukaryotic microbial diversity is uncultivated, under-studied and lacks nuclear genome data. Mitochondrial genome sampling is more comprehensive, but many phylogenetically important groups remain unsampled. Here, using a single-cell sorting approach combining tubulin-specific labelling with photopigment exclusion, we sorted flagellated heterotrophic unicellular eukaryotes from Pacific Ocean samples. We recovered 206 single amplified genomes, predominantly from underrepresented branches on the tree of life. Seventy single amplified genomes contained unique mitochondrial contigs, including 21 complete or near-complete mitochondrial genomes from formerly under-sampled phylogenetic branches, including telonemids, katablepharids, cercozoans and marine stramenopiles, effectively doubling the number of available samples of heterotrophic flagellate mitochondrial genomes. Collectively, these data identify a dynamic history of mitochondrial genome evolution including intron gain and loss, extensive patterns of genetic code variation and complex patterns of gene loss. Surprisingly, we found that stramenopile mitochondrial content is highly plastic, resembling patterns of variation previously observed only in plants.

Taxon-rich Multigene Phylogenetic Analyses Resolve the Phylogenetic Relationship Among Deep-branching Stramenopiles.

Stramenopiles are one of the major eukaryotic assemblages. This group comprises a wide range of species including photosynthetic unicellular and multicellular algae, fungus-like osmotrophic organisms and many free-living phagotrophic flagellates. However, the phylogeny of the Stramenopiles, especially relationships among deep-branching heterotrophs, has not yet been resolved because of a lack of adequate transcriptomic data for representative lineages. In this study, we performed multigene phylogenetic analyses of deep-branching Stramenopiles with improved taxon sampling. We sequenced transcriptomes of three deep-branching Stramenopiles: Incisomonas marina, Pseudophyllomitus vesiculosus and Platysulcus tardus. Phylogenetic analyses using 120 protein-coding genes and 56 taxa indicated that Pl. tardus is sister to all other Stramenopiles while Ps. vesiculosus is sister to MAST-4 and form a robust clade with the Labyrinthulea. The resolved phylogenetic relationships of deep-branching Stramenopiles provide insights into the ancestral traits of the Stramenopiles.

Redescription of Opalina triangulata (Heterokonta, Opalinea) from Fejervarya limnocharis based on morphological and molecular data.

Opalinids are a large group of anaerobic protists, mainly inhabiting the cloacae of amphibians (frogs and toads). The classification of this group has not been fully resolved, because of a lack of molecular information. Here, we give a redescription of Opalina triangulata Metcalf, 1923, collected from the rectum of the frog Fejervarya limnocharis, based on detailed morphological and molecular data. Our phylogenetic analyses confirmed the monophyly of Opalinata. Within it, Opalinea were monophyletic with O. triangulata and O. undulata as well as two Protoopalina species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea. Meanwhile, the ITS2 secondary structural similarities as well as G-C content revealed greater similitudes between Opalina species and P. lacertae than with Blastocystis hominis, which is in accordance with their position as sister clades in the SSU rDNA-based phylogenies.

Genomics, Biology and Phylogeny Aurantiochytrium acetophilum sp. nov. (Thraustrochytriaceae), Including First Evidence of Sexual Reproduction.

Strain HS-399 was isolated from a mangrove swamp in Biscayne Bay (Florida, USA) and selected for its capacity to accumulate lipids (84.0±1.0% DW), particularly docosahexaenoic acid (DHA; 22:6 n-3) (28.3±0.1% DW). Molecular phylogenetic analysis demonstrated that the new organism belonged to the genus Aurantiochytrium, and when the whole nuclear genome was blasted against the type species (and only described species), A. limacinum SR21, there was a 5.38% difference at the protein level. We described our new organism as Aurantiochytrium acetophilum sp. nov. (Thraustochytriaceae, Thraustochytriales) using light microscopy, electron microscopy, substrate assimilation, biochemical composition and nuclear genomic data. We found some characteristics of biotechnological relevance that were not previously described in this family. First, strain HS-399 of A. acetophilum was extremely tolerant to acetate toxicity, and it used this substrate as a sole carbon source. Second, we observed putative gametes that fused together to form a zygote. Zygote fate and the life stage with meiosis were not determined; however, we found several meiosis genes in the genome, further supporting the possibility of breeding for these industrially relevant organisms.