Sublethal effects on the hard clam Mercenaria mercenaria after exposure to Aureococcus anophagefferens Chinese strain isolates.

Brown tides caused by Aureococcus anophagefferens occur frequently worldwide and have contributed to the collapse of Mercenaria mercenaria farming in the United States. This economically valuable hard clam has been used in China for more than 20 years. To date, it has remained unknown whether A. anophagefferens Chinese strain has an impact on hard clam cultivation in the coastal areas of China or other sea areas worldwide if it enters through ship ballast water and other ways. In this study, a Chinese strain of A. anophagefferens isolated from the brown tide waters of Bohai Bay, China, was selected to explore its influence on the feedback of hard clams. After being fed with A. anophagefferens, hard clams showed characteristics similar to starvation. The reduced feeding efficiency of hard clams leads to reduced energy intake. However, the immune response and oxidative stress, result in increased energy consumption. An imbalance in the energy budget may be an important reason for hard clam starvation. This study has described the response characteristics of the A. anophagefferens Chinese strain to M. mercenaria, explored the reasons for the negative impact of A. anophagefferens on hard clams, and provides ideas for reducing shellfish aquaculture caused by brown tides.

Bioactives Overproduction through Operational Strategies in the Ichthyotoxic Microalga Heterosigma akashiwo Culture.

The red tide-forming microalga Heterosigma akashiwo has been associated with massive events of fish deaths, both wild and cultured. Culture conditions are responsible for the synthesis or accumulation of some metabolites with different interesting bioactivities. H. akashiwo LC269919 strain was grown in a 10 L bubble column photobioreactor artificially illuminated with multi-coloured LED lights. Growth and production of exopolysaccharides, polyunsaturated fatty acids (PUFAs), and carotenoids were evaluated under different culture modes (batch, fed-batch, semicontinuous, and continuous) at two irradiance levels (300 and 700 µE·s-1·m-2). Continuous mode at the dilution rate of 0.2·day-1 and 700 µE·s-1·m-2 provided the highest production of biomass, PUFAs (132.6 and 2.3 mg·L-1·day-1), and maximum fucoxanthin productivity (0.16 mg·L-1·day-1). The fed-batch mode accumulated exopolysaccharides in a concentration (1.02 g·L-1) 10-fold over the batch mode. An extraction process based on a sequential gradient partition with water and four water-immiscible organic solvents allowed the isolation of bioactive fucoxanthin from methanolic extracts of H. akashiwo. Metabolites present in H. akashiwo, fucoxanthin and polar lipids (i.e., eicosapentaenoic acid (EPA)), or probably such as phytosterol (ß-Sitosterol) from other microalgae, were responsible for the antitumor activity obtained.

Metabolic analysis of Schizochytrium sp. mutants with high EPA content achieved with ARTP mutagenesis screening.

Eicosapentaenoic acid (EPA) belonged to the ω-3 series of polyunsaturated fatty acids and had physiological functions lipid as regulating blood lipid and preventing cardiovascular diseases. Schizochytrium sp. was considered to be a potential industrial fermentation strain of EPA because of its fast growth, high oil content, and simple fatty acid composition. However, Schizochytrium sp. produced EPA with low production efficiency and a long synthesis path. This research aims to improve the yield of EPA in Schizochytrium sp. by ARTP mutagenesis and to reveal the mechanism of high-yield EPA through transcriptome analysis. ARTP mutagenesis screening yielded the mutant M12 that whereas the productivity of EPA increased 108% reaching 0.48 g/L, the total fatty acid concentration was 13.82 g/L with an increase of 13.7%. The transcriptomics revealed 2995 differentially expressed genes were identified between M12 and the wild-type strain and transcripts involved in carbohydrate, amino acid, energy, and lipid metabolism were up-regulated. Among them, the hexokinase (HK) and the phosphofructokinase genes (PFK), which can catalyze pyruvate to acetyl-CoA, were increased 2.23-fold and 1.78-fold. Glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GLDH), which can both generate NADPH, were increased by 1.67-fold and 3.11-fold. Furthermore, in the EPA synthesis module, the expression of 3-oxoacyl-[acyl-carrier protein] reductase(fabG) and carbonyl reductase 4 / 3-oxoacyl-[acyl-carrier protein] reductase beta subunit(CBR4), also up-regulated 1.11-fold and 2.67-fold. These may lead to increases in cell growth. The results provide an important reference for further research on promoting fatty acid and EPA accumulation in Schizochytrium sp.

Anti-Steatotic Effects of Chlorella vulgaris, Nannochloropsis gaditana and Gracilaria vermiculophylla Algae Extracts in AML-12 Hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common chronic liver alteration whose prevalence is increasing in Western countries. Microalgae and macroalgae have attracted great interest due to the high content in bioactive compounds with beneficial effects on health. The aim of the present study is to assess the potential interest of extracts rich in proteins obtained from the microalgae Chlorella vulgaris and Nannochloropsis gaditana and the macroalga Gracilaria vermiculophylla in the prevention of lipid accumulation in AML-12 hepatocytes. Toxicity was not observed at any of the tested doses. Both microalgae and the macroalga were effective in preventing triglyceride accumulation, with Nannochloropsis gaditana being the most effective one. Although the three algae extracts were able to increase different catabolic pathways involved in triglyceride metabolism, the mechanisms underlying the anti-steatotic effect were different in each algae extract. In conclusion, the present study demonstrates that Chlorella vulgaris, Nannochloropsis gaditana and Gracilaria vermiculophylla extracts are able to partially prevent the accumulation of triglycerides induced by palmitic acid in cultured hepatocytes, a model used to mimic the steatosis induced in liver by dietary patterns rich in saturated fat.

Saturated and Polyunsaturated Fatty Acids Production by Aurantiochytrium limacinum PKU#Mn4 on Enteromorpha Hydrolysate.

Thraustochytrids are unicellular marine heterotrophic protists, which have recently shown a promising ability to produce omega-3 fatty acids from lignocellulosic hydrolysates and wastewaters. Here we studied the biorefinery potential of the dilute acid-pretreated marine macroalgae (Enteromorpha) in comparison with glucose via fermentation using a previously isolated thraustochytrid strain (Aurantiochytrium limacinum PKU#Mn4). The total reducing sugars in the Enteromorpha hydrolysate accounted for 43.93% of the dry cell weight (DCW). The strain was capable of producing the highest DCW (4.32 ± 0.09 g/L) and total fatty acids (TFA) content (0.65 ± 0.03 g/L) in the medium containing 100 g/L of hydrolysate. The maximum TFA yields of 0.164 ± 0.160 g/g DCW and 0.196 ± 0.010 g/g DCW were achieved at 80 g/L of hydrolysate and 40 g/L of glucose in the fermentation medium, respectively. Compositional analysis of TFA revealed the production of equivalent fractions (% TFA) of saturated and polyunsaturated fatty acids in hydrolysate or glucose medium. Furthermore, the strain yielded a much higher fraction (2.61-3.22%) of eicosapentaenoic acid (C20:5n-3) in the hydrolysate medium than that (0.25-0.49%) in the glucose medium. Overall, our findings suggest that Enteromorpha hydrolysate can be a potential natural substrate in the fermentative production of high-value fatty acids by thraustochytrids.

Screening of a Thraustochytrid Strain Collection for Carotenoid and Squalene Production Characterized by Cluster Analysis, Comparison of 18S rRNA Gene Sequences, Growth Behavior, and Morphology.

Carotenoids and squalene are important terpenes that are applied in a wide range of products in foods and cosmetics. Thraustochytrids might be used as alternative production organisms to improve production processes, but the taxon is rarely studied. A screening of 62 strains of thraustochytrids sensu lato for their potential to produce carotenoids and squalene was performed. A phylogenetic tree was built based on 18S rRNA gene sequences for taxonomic classification, revealing eight different clades of thraustochytrids. Design of experiments (DoE) and growth models identified high amounts of glucose (up to 60 g/L) and yeast extract (up to 15 g/L) as important factors for most of the strains. Squalene and carotenoid production was studied by UHPLC-PDA-MS measurements. Cluster analysis of the carotenoid composition partially mirrored the phylogenetic results, indicating a possible use for chemotaxonomy. Strains in five clades produced carotenoids. Squalene was found in all analyzed strains. Carotenoid and squalene synthesis was dependent on the strain, medium composition and solidity. Strains related to Thraustochytrium aureum and Thraustochytriidae sp. are promising candidates for carotenoid synthesis. Strains closely related to Schizochytrium aggregatum might be suitable for squalene production. Thraustochytrium striatum might be a good compromise for the production of both molecule groups.

Removal of fenamiphos, imidacloprid, and oxamyl pesticides from water by microalgal Nannochloropsis oculata biomass and their determination by validated HPLC method.

The present study assessed the removal of fenamiphos, imidacloprid, and oxamyl pesticides from water using algal Nannochloropsis oculata biomass. Several factors, such as algal biomass concentration, incubation time, and pesticide concentration, were studied for their impact on pesticide removal. Analysis and quantification of pesticides by rapid HPLC have been developed and validated. The optimum conditions were obtained at 15 min, 50 mg/L of pesticide concentration, and 4,500 mg/L of the algal biomass with 92.24% and 90.43% removal for fenamiphos and imidacloprid, respectively. While optimum parameters of 10 min incubation, 250 mg/L of pesticide concentration, and 2,750 mg/L of the algal biomass exhibited 67.34% removal for oxamyl. N. oculata, marine microalgae, successively removed different concentrations of the tested pesticides from water, and the algal biomass showed a potential reduction of pesticides in polluted water samples.

Ethanol Enhances Astaxanthin Production by Aurantiochytrium sp. O5-1-1.

Two-percent ethanol increased the astaxanthin productivity of heterotrophic microalgae Aurantiochytrium sp. O5-1-1 to 2.231 mg/L, 45-fold higher than under ethanol-free condition. Ethanol in the medium decreased at the same rate as spontaneous volatilization, suggesting that it was not a transient signaling factor but a continuous stress on the cells. The triply mutated strain OM3-3 produced 5.075 mg/L astaxanthin under 2% ethanol conditions. Furthermore, the astaxanthin accumulation of the mutant OM3-9 was 0.895 mg/g, which was 150-fold higher than that of strain O5-1-1 in ethanol-free condition. These results are beneficial for the commercial exploitation of carotenoids producing Aurantiochytrium spp.

Fermentative production of astaxanthin from sorghum distillery residue by an indigenous Aurantiochytrium sp. CJ6 strain using a continuous-feeding fed-batch process.

In this study, Aurantiochytrium sp. CJ6 was cultivated heterotrophically on a waste resource, sorghum distillery residue (SDR) hydrolysate without adding any nitrogen sources. Mild sulfuric acid treatment released sugars that supported the growth of CJ6. Optimal operating parameters (salinity, 2.5%; pH, 7.5; with light exposure) determined using batch cultivation attained biomass concentration and astaxanthin content of 3.72 g/L and 69.32 µg/g dry cell weight (DCW), respectively. Using continuous-feeding fed-batch (CF-FB) fermentation, the biomass concentration of CJ6 increased to 6.3 g/L with biomass productivity and sugar utilization rate of 0.286 mg/L/d and 1.26 g/L/d, respectively. Meanwhile, CJ6 obtained maximum astaxanthin content (93.9 µg/g DCW) and astaxanthin concentration (0.565 mg/L) after 20-day cultivation. Thus, the CF-FB fermentation strategy seems to have a high potential for the cultivation of thraustochytrids to produce the high-value product (astaxanthin) using SDR as the feedstock to achieve circular economy.

Production of docosahexaenoic acid from spruce sugars using Aurantiochytrium limacinum.

In this study lignocellulosic sugars from Norway spruce were used for production of docosahexaenoic acid (DHA) by the marine thraustochytrid Aurantiochytrium limacinum SR21. Enzymatically prepared spruce hydrolysate was combined with a complex nitrogen source and different amounts of salts. Shake flask batch cultivations revealed that addition of extra salts was not needed for optimal growth. Upscaling to fed-batch bioreactors yielded up to 55 g/L cell dry mass and a total fatty acid content of 44% (w/w) out of which 1/3 was DHA. Fourier transform infrared spectroscopy was successfully applied as a rapid method for monitoring lipid accumulation in A. limacinum SR21. Thus, this proof-of-principle study clearly demonstrates that crude spruce hydrolysates can be directly used as a novel and sustainable resource for production of DHA.

Protist-Lactic Acid Bacteria Co-Culture as a Strategy to Bioaccumulate Polyunsaturated Fatty Acids in the Protist Aurantiochytrium sp. T66.

Thraustochytrids are aquatic unicellular protists organisms that represent an important reservoir of a wide range of bioactive compounds, such as essential polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), which are involved in the regulation of the immune system. In this study, we explore the use of co-cultures of Aurantiochytrium sp. and bacteria as a biotechnological tool capable of stimulating PUFA bioaccumulation. In particular, the co-culture of lactic acid bacteria and the protist Aurantiochytrium sp. T66 induce PUFA bioaccumulation, and the lipid profile was evaluated in cultures at different inoculation times, with two different strains of lactic acid bacteria capable of producing the tryptophan dependent auxins, and one strain of Azospirillum sp., as a reference for auxin production. Our results showed that the Lentilactobacillus kefiri K6.10 strain inoculated at 72 h gives the best PUFA content (30.89 mg g-1 biomass) measured at 144 h of culture, three times higher than the control (8.87 mg g-1 biomass). Co-culture can lead to the generation of complex biomasses with higher added value for developing aquafeed supplements.

Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy.

Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution-alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of ∼7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear-nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids.

Low dissolved oxygen supply functions as a global regulator of the growth and metabolism of Aurantiochytrium sp. PKU#Mn16 in the early stages of docosahexaenoic acid fermentation.

BACKGROUND: Thraustochytrids accumulate lipids with a high content of docosahexaenoic acid (DHA). Although their growth and DHA content are significantly affected by the dissolved oxygen (DO) supply, the role of DO on the transcriptional regulation of metabolism and accumulation of intracellular metabolites remains poorly understood. Here we investigate the effects of three different DO supply conditions (10%, 30%, and 50%) on the fed-batch culture of the Aurantiochytrium PKU#Mn16 strain to mainly reveal the differential gene expressions and metabolite profiles. RESULTS: While the supply of 10% DO significantly reduced the rates of biomass and DHA production in the early stages of fermentation, it achieved the highest amounts of biomass (56.7 g/L) and DHA (6.0 g/L) on prolonged fermentation. The transcriptome analyses of the early stage (24 h) of fermentation revealed several genes involved in the central carbon, amino acid, and fatty acid metabolism, which were significantly downregulated at a 10% DO level. The comparative metabolomics results revealed the accumulation of several long-chain fatty acids, amino acids, and other metabolites, supporting the transcriptional regulation under the influence of a low oxygen supply condition. In addition, certain genes involved in antioxidative systems were downregulated under 10% DO level, suggesting a lesser generation of reactive oxygen species that lead to oxidative damage and fatty acid oxidation. CONCLUSIONS: The findings of this study suggest that despite the slow growth and metabolism in the early stage of fermentation of Aurantiochytrium sp. PKU#Mn16, a constant supply of low dissolved oxygen can yield biomass and DHA content better than that with high oxygen supply conditions. The critical information gained in this study will help to further improve DHA production through bioprocess engineering strategies.

Enhanced docosahexaenoic acid production from cane molasses by engineered and adaptively evolved Schizochytrium sp.

Cane molasses (CM) is a sugar-rich agro-industrial byproduct. The purpose of this study is to synthesize docosahexaenoic acid (DHA) in Schizochytrium sp. by using CM. The single factor analysis showed that sucrose utilization was the main factor limiting the utilization of CM. Therefore, the endogenous sucrose hydrolase (SH) was overexpressed in Schizochytrium sp., which enhanced the sucrose utilization rate 2.57-fold compared to the wild type. Furthermore, adaptive laboratory evolution was used to further improve sucrose utilization from CM. Comparative proteomics and RT-qPCR were used out to analyze the metabolic differences of evolved strain grown on CM and glucose, respectively. Finally, a constant flow rate CM feeding strategy was implemented, whereby the DHA titer and lipid yield of the final strain OSH-end reached 25.26 g/L and 0.229 g/g sugar, respectively. This study demonstrated the CM is a cost-effective carbon source for industrial DHA fermentation.

Revealing holistic metabolic responses associated with lipid and docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1.

Aurantiochytrium sp. SW1, a marine thraustochytrid, has been regarded as a potential candidate as a docosahexaenoic acid (DHA) producer. Even though the genomics of Aurantiochytrium sp. are available, the metabolic responses at a systems level are largely unknown. Therefore, this study aimed to investigate the global metabolic responses to DHA production in Aurantiochytrium sp. through transcriptome and genome-scale network-driven analysis. Of a total of 13,505 genes, 2527 differentially expressed genes (DEGs) were identified in Aurantiochytrium sp., unravelling the transcriptional regulations behinds lipid and DHA accumulation. The highest number of DEG were found for pairwise comparison between growth phase and lipid accumulating phase where a total of 1435 genes were down-regulated with 869 genes being up-regulated. These uncovered several metabolic pathways that contributing in DHA and lipid accumulation including amino acid and acetate metabolism which involve in the generation of crucial precursors. Upon applying network-driven analysis, hydrogen sulphide was found as potential reporter metabolite that could be associated with the genes related to acetyl-CoA synthesis for DHA production. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific cultivation phases during DHA overproduction in Aurantiochytrium sp. SW1.

Rapid measurement of brown tide algae using Zernike moments and ensemble learning based on excitation-emission matrix fluorescence.

Accurate real-time prediction of microalgae density has great practical significance for taking countermeasures before the advent of Harmful algal blooms (HABs), and the non-destructive and sensitive property of excitation-emission matrix fluorescence (EEMF) spectroscopy makes it applicable to online monitoring and control. In this study, an efficient image preprocessing algorithm based on Zernike moments (ZMs) was proposed to extract compelling features from EEM intensities images. The determination of the highest order of ZMs considered both reconstruction error and computational cost, then the optimal subset of preliminarily extracted 36 ZMs was screened via the BorutaShap algorithm. Aureococcus anophagefferens concentration prediction models were developed by combining BorutaShap and ensemble learning models (random forest (RF), gradient boosting decision tree (GBDT), and XGBoost). The experimental results show that BorutaShap_GBDT preserved the superior subset of ZMs, and the integration of BorutaShap_GBDT and XGBoost achieved the highest prediction accuracy. This research provides a new and promising strategy for rapidly measuring microalgae cell density.

The First Identification of a Narnavirus in Bigyra, a Marine Protist.

Current information on the diversity and evolution of eukaryotic RNA viruses is biased towards host lineages, such as animals, plants, and fungi. Although protists represent the majority of eukaryotic diversity, our understanding of the protist RNA virosphere is still limited. To reveal untapped RNA viral diversity, we screened RNA viruses from 30 marine protist isolates and identified a novel RNA virus named Haloplacidia narnavirus 1 (HpNV1). A phylogenetic ana-lysis revealed that HpNV1 is a new member of the family Narnaviridae. The present study filled a gap in the distribution of narnaviruses and implies their wide distribution in Stramenopiles.

Taxonomic composition, community structure and molecular novelty of microeukaryotes in a temperate oligomesotrophic lake as revealed by metabarcoding.

Microbial eukaryotes are diverse and ecologically important organisms, yet sampling constraints have hindered the understanding of their distribution and diversity in freshwater ecosystems. Metabarcoding has provided a powerful complement to traditional limnological studies, revealing an unprecedented diversity of protists in freshwater environments. Here, we aim to expand our knowledge of the ecology and diversity of protists in lacustrine ecosystems by targeting the V4 hypervariable region of the 18S rRNA gene in water column, sediment and biofilm samples collected from Sanabria Lake (Spain) and surrounding freshwater ecosystems. Sanabria is a temperate lake, which are relatively understudied by metabarcoding in comparison to alpine and polar lakes. The phylogenetic diversity of microbial eukaryotes detected in Sanabria spans all currently recognized eukaryotic supergroups, with Stramenopiles being the most abundant and diverse supergroup in all sampling sites. Parasitic microeukaryotes account for 21% of the total protist ASVs identified in our study and were dominated by Chytridiomycota, both in terms of richness and abundance, in all sampling sites. Sediments, biofilms and water column samples harbour distinct microbial communities. Phylogenetic placement of poorly assigned and abundant ASVs indicates molecular novelty inside Rhodophyta, Bigyra, early-branching Nucletmycea and Apusomonadida. In addition, we report the first freshwater incidence of the previously exclusively marine genera Abeoforma and Sphaeroforma. Our results contribute to a deeper understanding of microeukaryotic communities in freshwater ecosystems, and provide the first molecular reference for future biomonitoring surveys in Sanabria Lake.

Solid-state fermentation-based enzyme-assisted extraction of eicosapentaenoic acid-rich oil from Nannochloropsis sp.

Enzymatic treatment of microalgal biomass is a promising approach for extraction of microalgal lipid, but high cost of commercially sourcing enzyme is a major drawback in industrial implementation. Present study involves extraction of eicosapentaenoic acid-rich oil from Nannochloropsis sp. biomass using low cost cellulolytic enzymes produced from Trichoderma reesei in a solid-state fermentation bioreactor. Maximum total fatty acid recovery of 369.4 ± 4.6 mg/g dry weight (total fatty acid yield of 77%) was achieved in 12 h from the enzymatically treated microalgal cells, of which the eicosapentaenoic acid content was 11%. Sugar release of 1.70 ± 0.05 g/L was obtained post enzymatic treatment at 50 °C. The enzyme was reused thrice for cell wall disruption without compromising on total fatty acid yield. Additionally, high protein content of 47% in the defatted biomass could be explored as a potential aquafeed, thus enhancing the overall economics and sustainability of the process.

Horizontal Gene Transfer and Fusion Spread Carotenogenesis Among Diverse Heterotrophic Protists.

Thraustochytrids (phylum: Labyrinthulomycota) are nonphotosynthetic marine protists. Some thraustochytrids have crtIBY, a trifunctional fusion gene encoding a protein capable of ß-carotene biosynthesis from geranylgeranyl pyrophosphate. Here we show that crtIBY is essential in, and encodes the sole pathway for, carotenoid biosynthesis in the thraustochytrid Aurantiochytrium limacinum ATCC MYA-1381. We explore the evolutionary origins of CrtIBY and discover that the closest related protein domains are present in a small but diverse group of other heterotrophic protists, including the apusomonad Thecamonas trahens and the dinoflagellates Oxyrrhis marina and Noctiluca scintillans. Each organism within this cluster also contains one or more ß-carotene 15-15' oxygenase genes (blh and rpe65), suggesting that the acquisition of ß-carotene biosynthesis genes may have been related to the production of retinal. Our findings support a novel origin of eukaryotic (apo)carotenoid biosynthesis by horizontal gene transfer from Actinobacteria, Bacteroidetes, and/or Archaea. This reveals a remarkable case of parallel evolution of eukaryotic (apo)carotenogenesis in divergent protistan lineages by repeated gene transfers.