cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA

The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.

Linking extreme seasonality and gene expression in arctic marine protists

At high latitudes, strong seasonal differences in light availability affect marine organisms and restrict the timing of ecosystem processes. Marine protists are key players in Arctic aquatic ecosystems, yet little is known about their ecological roles over yearly cycles. This is especially true for the dark polar night period, which up until recently was assumed to be devoid of biological activity. A 12 million transcripts catalogue was built from 0.45-10 μm protist assemblages sampled over 13 months in a time series station in an arctic fjord in Svalbard. Community gene expression was correlated with seasonality, with light as the main driving factor. Transcript diversity and evenness were higher during polar night compared to polar day. Light-dependent functions had higher relative expression during polar day, except phototransduction. 64% of the most expressed genes could not be functionally annotated, yet up to 78% were identified in arctic samples from Tara Oceans, suggesting that arctic marine assemblages are distinct from those from other oceans. Our study increases understanding of the links between extreme seasonality and biological processes in pico- and nanoplanktonic protists. Our results set the ground for future monitoring studies investigating the seasonal impact of climate change on the communities of microbial eukaryotes in the High Arctic.

An Oil Hyper-Accumulator Mutant Highlights Peroxisomal ATP Import as a Regulatory Step for Fatty Acid Metabolism in Aurantiochytrium limacinum

Thraustochytrids are marine protists that naturally accumulate triacylglycerol with long chains of polyunsaturated fatty acids, such as ω3-docosahexaenoic acid (DHA). They represent a sustainable response to the increasing demand for these “essential” fatty acids (FAs). Following an attempt to transform a strain of Aurantiochytrium limacinum, we serendipitously isolated a clone that did not incorporate any recombinant DNA but contained two to three times more DHA than the original strain. Metabolic analyses indicated a deficit in FA catabolism. However, whole transcriptome analysis did not show down-regulation of genes involved in FA catabolism. Genome sequencing revealed extensive DNA deletion in one allele encoding a putative peroxisomal adenylate transporter. Phylogenetic analyses and yeast complementation experiments confirmed the gene as a peroxisomal adenylate nucleotide transporter (AlANT1), homologous to yeast ScANT1 and plant peroxisomal adenylate nucleotide carrier AtPNC genes. In yeast and plants, a deletion of the peroxisomal adenylate transporter inhibits FA breakdown and induces FA accumulation, a phenotype similar to that described here. In response to this metabolic event, several compensatory mechanisms were observed. In particular, genes involved in FA biosynthesis were upregulated, also contributing to the high FA accumulation. These results support AlANT1 as a promising target for enhancing DHA production in Thraustochytrids.

C4-monomethylsterol β-glucoside and its synthase in Aurantiochytrium limacinum mh0186

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and two-dimensional nuclear magnetic resonance to be 3-O-β-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-β-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol β-glucosides (β-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these β-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol β-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from UDP-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in total SG and almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of β-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

Eco-Evolutionary Perspectives on Mixoplankton

Mixotrophy, i.e., the capability of both phototrophy and phagotrophy within a single organism, is a prominent trophic mode in aquatic ecosystems. Mixotrophic strategies can be highly advantageous when feeding or photosynthesis alone does not sustain metabolic needs. In the current review, we discuss the functional types of mixotrophic marine protists (herein mixoplankton) within the context of evolution. Permanent plastids have been established in large due to gene transfer from prey and/or endosymbionts to the host cell. In some kleptoplastidic mixoplankton, prior gene transfers and active transcription of plastid related genes in the host can help maintain and extend retention of the current kleptoplast. In addition to kleptoplasts, the prey nucleus is also sometimes retained and actively transcribed to help maintain and even replicate the kleptoplasts. Endosymbiotic relations vary considerably in the extent to which hosts affect symbionts. For example, some endosymbionts are heavily modified to increase photosynthetic efficiency, or are controlled in their cell division. It can be proposed that many kleptoplasts and endosymbionts are in fact en route to becoming permanent plastids. Conditions such as increased temperature and limiting nutrients seem to favor phagotrophy in mixoplankton. However, responses of mixoplankton to changing environmental conditions like light irradiance, temperature, nutrient, and prey availability are variable and species-specific. Studying mixotrophs with temporary plastids could elucidate past and future evolutionary mechanisms and dynamics of processes such as phagotrophy and the establishment of (secondary) permanent plastids.

Coiling directions in the planktonic foraminifer Pulleniatina: A complex eco-evolutionary dynamic spanning millions of years

Planktonic foraminifera are heterotrophic sexually reproducing marine protists with an exceptionally complete fossil record that provides unique insights into long-term patterns and processes of evolution. Populations often exhibit strong biases towards either right (dextral) or left (sinistral) shells. Deep-sea sediment cores spanning millions of years reveal that some species show large and often rapid fluctuations in their dominant coiling direction through time. This is useful for biostratigraphic correlation but further work is required to understand the population dynamical processes that drive these fluctuations. Here we address the case of coiling fluctuations in the planktonic foraminifer genus Pulleniatina based on new high-resolution counts from two recently recovered sediment cores from either side of the Indonesian through-flow in the tropical west Pacific and Indian Oceans (International Ocean Discovery Program Sites U1486 and U1483). We use single-specimen stable isotope analyses to show that dextral and sinistral shells from the same sediment samples can show significant differences in both carbon and oxygen isotopes, implying a degree of ecological separation between populations. In one case we detect a significant difference in size between dextral and sinistral specimens. We suggest that major fluctuations in coiling ratio are caused by cryptic populations replacing one another in competitive sweeps, a mode of evolution that is more often associated with asexual organisms than with the classical ‘biological species concept’.

Resolving cryptic species complexes in marine protists: phylogenetic haplotype networks meet global DNA metabarcoding datasets

Marine protists have traditionally been assumed to be lowly diverse and cosmopolitan. Yet, several recent studies have shown that many protist species actually consist of cryptic complexes of species whose members are often restricted to particular biogeographic regions. Nonetheless, detection of cryptic species is usually hampered by sampling coverage and application of methods (e.g. phylogenetic trees) that are not well suited to identify relatively recent divergence and ongoing gene flow. In this paper, we show how these issues can be overcome by inferring phylogenetic haplotype networks from global metabarcoding datasets. We use the Chaetoceros curvisetus (Bacillariophyta) species complex as study case. Using two complementary metabarcoding datasets (Ocean Sampling Day and Tara Oceans), we equally resolve the cryptic complex in terms of number of inferred species. We detect new hypothetical species in both datasets. Gene flow between most of species is absent, but no barcoding gap exists. Some species have restricted distribution patterns whereas others are widely distributed. Closely related taxa occupy contrasting biogeographic regions, suggesting that geographic and ecological differentiation drive speciation. In conclusion, we show the potential of the analysis of metabarcoding data with evolutionary approaches for systematic and phylogeographic studies of marine protists.