Shear Stress-Responsive Polymersome Nanoreactors Inspired by the Marine Bioluminescence of Dinoflagellates.

Some marine plankton called dinoflagellates emit light in response to the movement of surrounding water, resulting in a phenomenon called milky seas or sea sparkle. The underlying concept, a shear-stress induced permeabilisation of biocatalytic reaction compartments, is transferred to polymer-based nanoreactors. Amphiphilic block copolymers that carry nucleobases in their hydrophobic block are self-assembled into polymersomes. The membrane of the vesicles can be transiently switched between an impermeable and a semipermeable state by shear forces occurring in flow or during turbulent mixing of polymersome dispersions. Nucleobase pairs in the hydrophobic leaflet separate when mechanical force is applied, exposing their hydrogen bonding motifs and therefore making the membrane less hydrophobic and more permeable for water soluble compounds. This polarity switch is used to release payload of the polymersomes on demand, and to activate biocatalytic reactions in the interior of the polymersomes.

Unravelling the mechanism of pH-regulation in dinoflagellate luciferase.

Dinoflagellates are the dominant source of bioluminescence in coastal waters. The luminescence reaction involves the oxidation of luciferin by a luciferase enzyme, which only takes place at low pH. The pH-dependence has previously been linked to four conserved histidines. It has been suggested that their protonation might induce a conformational change in the enzyme, thereby allowing substrate access to the binding pocket. Yet, the precise mechanism of luciferase activation has remained elusive. Here, we use computational tools to predict the open structure of the luciferase in Lingulodinium polyedra and to decipher the nature of the opening mechanism. Through accelerated molecular dynamics simulations, we demonstrate that the closed-open conformational change likely takes place via a tilt of the pH-regulatory helix-loop-helix domain. Moreover, we propose that the molecular basis for the transition is electrostatic repulsion between histidine-cation pairs, which destabilizes the closed conformation at low pH. Finally, by simulating truncated mutants, we show that eliminating the C-terminus alters the shape of the active site, effectively inactivating the luciferase.

Paralytic Shellfish Toxins (PST)-Transforming Enzymes: A Review.

Paralytic shellfish toxins (PSTs) are a group of toxins that cause paralytic shellfish poisoning through blockage of voltage-gated sodium channels. PSTs are produced by prokaryotic freshwater cyanobacteria and eukaryotic marine dinoflagellates. Proliferation of toxic algae species can lead to harmful algal blooms, during which seafood accumulate high levels of PSTs, posing a health threat to consumers. The existence of PST-transforming enzymes was first remarked due to the divergence of PST profiles and concentrations between contaminated bivalves and toxigenic organisms. Later, several enzymes involved in PST transformation, synthesis and elimination have been identified. The knowledge of PST-transforming enzymes is necessary for understanding the processes of toxin accumulation and depuration in mollusk bivalves. Furthermore, PST-transforming enzymes facilitate the obtainment of pure analogues of toxins as in natural sources they are present in a mixture. Pure compounds are of interest for the development of drug candidates and as analytical reference materials. PST-transforming enzymes can also be employed for the development of analytical tools for toxin detection. This review summarizes the PST-transforming enzymes identified so far in living organisms from bacteria to humans, with special emphasis on bivalves, cyanobacteria and dinoflagellates, and discusses enzymes' biological functions and potential practical applications.

Transcriptomic analysis of polyketide synthases in a highly ciguatoxic dinoflagellate, Gambierdiscus polynesiensis and low toxicity Gambierdiscus pacificus, from French Polynesia.

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain ß-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.

Transcriptome survey and toxin measurements reveal evolutionary modification and loss of saxitoxin biosynthesis genes in the dinoflagellates Amphidinium carterae and Prorocentrum micans.

In the present study, we characterized the potential toxin genes for polyketide synthase (PKS) and saxitoxin (STX) biosynthesis using the transcriptomes of two non-STX producing dinoflagellates Amphidinium carterae and Prorocentrum micans. RNA sequencing revealed 94 and 166 PKS contigs in A. carterae and P. micans, respectively. We first detected type III PKS, which was closely related to bacteria. In addition, dozens of homologs of 20 STX biosynthesis genes were identified. Interestingly, the core STX-synthesizing genes sxtA and sxtB were only found in P. micans, whereas sxtD was detected in A. carterae alone. Bioinformatic analysis showed that the first two core genes (sxtA and sxtG) had a low sequence similarity (37.0-67.6%) and different domain organization compared to those of other toxigenic dinoflagellates, such as Alexandrium pacificum. These might result in the breakdown of the initial reactions in STX production and ultimately the loss of the ability to synthesize the toxins in both dinoflagellates. Our findings suggest that toxin-related PKS and sxt genes are commonly found in non-STX producing dinoflagellates. In addition to their involvement in the synthesis of toxins, our result indicates that genes may also have other molecular metabolic functions.

New Perspectives Related to the Bioluminescent System in Dinoflagellates: Pyrocystis lunula, a Case Study.

Pyrocystis lunula is considered a model organism due to its bioluminescence capacity linked to circadian rhythms. The mechanisms underlying the bioluminescent phenomenon have been well characterized in dinoflagellates; however, there are still some aspects that remain an enigma. Such is the case of the presence and diversity of the luciferin-binding protein (LBP), as well as the synthesis process of luciferin. Here we carry out a review of the literature in relation to the molecular players responsible for bioluminescence in dinoflagellates, with particular interest in P. lunula. We also carried out a phylogenetic analysis of the conservation of protein sequence, structure and evolutionary pattern of these key players. The basic structure of the luciferase (LCF) is quite conserved among the sequences reported to date for dinoflagellate species, but not in the case of the LBP, which has proven to be more variable in terms of sequence and structure. In the case of luciferin, its synthesis has been shown to be complex process with more than one metabolic pathway involved. The glutathione S-transferase (GST) and the P630 or blue compound, seem to be involved in this process. In the same way, various hypotheses regarding the role of bioluminescence in dinoflagellates are exposed.

Alkaline phosphatase activities and regulation in three harmful Prorocentrum species from the coastal waters of the East China Sea.

Harmful blooms of Prorocentrum donghaiense occur annually in the phosphorus-scarce coastal waters of the East China Sea (ECS). The enzymatic activities of alkaline phosphatase (AP) and its regulation by external phosphorus were studied during a P. donghaiense bloom in this area. The AP characteristics of P. donghaiense was further compared with Prorocentrum minimum and Prorocentrum micans in monocultures with both bulk and single-cell enzyme-labeled fluorescence AP assays. Concentrations of dissolved inorganic phosphorus (DIP) varied between 0.04 and 0.73 µmol l-1, with more than half recording stations registering concentrations below 0.10 µmol l-1. Concentrations of dissolved organic phosphorus (DOP) were comparable or even higher than those of DIP. P. donghaiense suffered phosphorus stress and expressed abundant AP, especially when DIP was lower than 0.10 µmol l-1. The AP activities showed a negative correlation with DIP but a positive correlation with DOP. The AP activities were also regulated by internal phosphorus pool. The sharp increase in AP activities was observed until cellular phosphorus was exhausted. Most AP of P. donghaiense was located on the cell surface and some were released into the water with time. Compared with P. minimum and P. micans, P. donghaiense showed a higher AP affinity for organic phosphorus substrates, a more efficient and energy-saving AP expression quantity as a response to phosphorus deficiency. The unique AP characteristic of P. donghaiense suggests that it benefits from the efficient utilization of DOP, and outcompete other species in the phosphorus-scarce ECS.

Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8.

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH4)2SO4 fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography-coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31-32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles.

Origin and roles of a novel copper-zinc superoxide dismutase (CuZnSOD) gene from the harmful dinoflagellate Prorocentrum minimum.

Superoxide dismutase (SOD) acts as the first line of defence against reactive oxygen species (ROS) within cells. In this study, we characterized a novel SOD gene (PmCuZnSOD) from the dinoflagellate Prorocentrum minimum, and examined its structural features, putative origin and gene expression. The SOD cDNA is 895 bp in length, containing dinoflagellate splice-leader (dinoSL) sequence, 714-bp ORF (237 aa), and poly (A) tail. In addition, PmCuZnSOD is coded on the dinoflagellate nuclear genome without introns and in a non-tandem repeat manner; however, the encoded protein is probably localized in chloroplasts. Phylogenetic analysis indicated that it might be acquired from cyanobacteria via horizontal gene transfer (HGT) and then the gene possibly relocated from the chloroplast to the nuclear genome. Excess copper dramatically increased the PmCuZnSOD transcripts and SOD activity in cells, caused by ROS generation and decrease of photosynthetic efficiency in the treated cells. These suggest that CuZnSOD may function to defend against oxidative stress for the survival of the dinoflagellate.

Modelling dimethylsulfide diffusion in the algal external boundary layer: implications for mutualistic and signalling roles.

Dimethylsulfide (DMS), a dominant organic sulfur species in the surface ocean, may act as a signalling molecule and contribute to mutualistic interactions between bacteria and marine algae. These proposed functions depend on the DMS concentration in the vicinity of microorganisms. Here, we modelled the DMS enrichment at the surface of DMS-releasing marine algal cells as a function of DMS production rate, algal cell radius and turbulence. Our results show that the DMS concentration at the surface of unstressed phytoplankton with low DMS production rates can be enriched by <1 nM, whereas for mechanically stressed algae with high activities of the enzyme DMSP-lyase (a coccolithophore and a dinoflagellate) DMS cell surface enrichments can reach ~10 nM, and could potentially reach µM levels in large cells. These DMS enrichments are much higher than the median DMS concentration in the surface ocean (1.9 nM), and thus may attract and support the growth of bacteria living in the phycosphere. The bacteria in turn may provide photoactive iron chelators (siderophores) that enhance algal iron uptake and provide algal growth factors such as auxins and vitamins. The present study highlights new insights on the extent and impact of microscale DMS enrichments at algal surfaces, thereby contributing to our understanding of the potential chemoattractant and mutualistic roles of DMS in marine microorganisms.

Recurrent acquisition of cytosine methyltransferases into eukaryotic retrotransposons.

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Fates of Evolutionarily Distinct, Plastid-type Glyceraldehyde 3-phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates.

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome-one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte-derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid-type glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte-derived tertiary plastids: "gapC1h" acquired from the haptophyte endosymbiont and "gapC1p" inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid-type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT-derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.

DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton.

Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1-3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton 4 , and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified 5 . However, eukaryotic phytoplankton probably produce most of Earth's DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.

CpMCA, a novel metacaspase gene from the harmful dinoflagellate Cochlodinium polykrikoides and its expression during cell death.

Metacaspases (MCAs) are cysteine proteases that share sequence homology with caspases, and may play roles in programmed cell death (PCD). In the present study, we identified a novel MCA gene (CpMCA) from the red tide dinoflagellate Cochlodinium polykrikoides, and examined its molecular characteristics and gene expression in response to algicide-induced cell death. CpMCA cDNA is 1164 bp in length, containing a dinoflagellate spliced leader sequence (dinoSL), an 879-bp open reading frame (ORF), which codes for a 293-aa protein, and a poly (A) tail. Multi-sequence comparison indicated that CpMCA belongs to type I MCA, but it has a different structure at the N-terminal. Phylogenetic analysis showed that C. polykrikoides may have acquired the MCA gene from bacteria by means of horizontal gene transfer (HGT). In addition, expressions of CpMCA significantly increased following exposure to the common algicides copper sulfate and oxidizing chlorine, which trigger cell death in dinoflagellates, suggesting that CpMCA may be involved in cell death.

Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase.

The bioluminescence reaction in dinoflagellates involves the oxidation of an open-chain tetrapyrrole by the enzyme dinoflagellate luciferase (LCF). The activity of LCF is tightly regulated by pH, where the enzyme is essentially inactive at pH ∼8 and optimally active at pH ∼6. Little is known about the mechanism of LCF or the structure of the active form of the enzyme, although it has been proposed that several intramolecularly conserved histidine residues in the N-terminal region are important for the pH regulation mechanism. Here, constant pH accelerated molecular dynamics was employed to gain insight into the conformational activation of LCF induced by acidification.

De novo transcriptome of the cosmopolitan dinoflagellate Amphidinium carterae to identify enzymes with biotechnological potential.

Dinoflagellates are phytoplanktonic organisms found in both freshwater and marine habitats. They are often studied because related to harmful algal blooms but they are also known to produce bioactive compounds for the treatment of human pathologies. The aim of this study was to sequence the full transcriptome of the dinoflagellate Amphidinium carterae in both nitrogen-starved and -replete culturing conditions (1) to evaluate the response to nitrogen starvation at the transcriptional level, (2) to look for possible polyketide synthases (PKSs) in the studied clone (genes that may be involved in the synthesis of bioactive compounds), (3) if present, to evaluate if nutrient starvation can influence PKS expression, (4) to look for other possible enzymes of biotechnological interest and (5) to test strain cytotoxicity on human cell lines. Results showed an increase in nitrogen metabolism and stress response in nitrogen-starved cells and confirmed the presence of a type I ß-ketosynthase. In addition, L-asparaginase (used for the treatment of Leukemia and for acrylamide reduction in food industries) and cellulase (useful for biofuel production and other industrial applications) have been identified for the first time in this species, giving new insights into possible biotechnological applications of dinoflagellates.

Screening and transcriptomic analysis of Crypthecodinium cohnii mutants with high growth and lipid content using the acetyl-CoA carboxylase inhibitor sethoxydim.

The heterotrophic microalga Crypthecodinium cohnii is well known for its lipid accumulation, with a high proportion of docosahexaenoic acid (DHA). In this study, we report a novel screening approach to obtain mutants of C. cohnii with high growth and lipid content using the acetyl-CoA carboxylase (ACCase) inhibitor sethoxydim. C. cohnii mutants were generated using atmospheric and room-temperature plasma (ARTP) and then screened for two rounds in media supplemented with sethoxydim. These efforts led to the identification of mutant M-1-2, which had 24.32% higher growth and 7.05% higher lipid content than the wild type, demonstrating the effectiveness of the sethoxydim-based screening. Consistently, the M-1-2 mutant displayed a 16.15% increase in ACCase enzymatic activity and 1.53-fold upregulation of its ACCase-encoding gene based on comparative ACCase activity analysis and transcriptomic analysis, respectively. In addition, transcriptomic analysis showed that transcripts involved in fatty acid biosynthesis, energy, central carbohydrate, and amino acid metabolism were upregulated in the mutant compared to the wild type.

Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates.

Warm preconditioning protects against acute heat-induced respiratory dysfunction and delays bleaching in a symbiotic sea anemone.

Preconditioning to non-stressful warming can protect some symbiotic cnidarians against the high temperature-induced collapse of their mutualistic endosymbiosis with photosynthetic dinoflagellates (Symbiodinium spp.), a process known as bleaching. Here, we sought to determine whether such preconditioning is underpinned by differential regulation of aerobic respiration. We quantified in vivo metabolism and mitochondrial respiratory enzyme activity in the naturally symbiotic sea anemone Exaiptasia pallida preconditioned to 30°C for >7 weeks as well as anemones kept at 26°C. Preconditioning resulted in increased Symbiodinium photosynthetic activity and holobiont (host+symbiont) respiration rates. Biomass-normalised activities of host respiratory enzymes [citrate synthase and the mitochondrial electron transport chain (mETC) complexes I and IV] were higher in preconditioned animals, suggesting that increased holobiont respiration may have been due to host mitochondrial biogenesis and/or enlargement. Subsequent acute heating of preconditioned and 'thermally naive' animals to 33°C induced dramatic increases in host mETC complex I and Symbiodinium mETC complex II activities only in thermally naive E. pallida These changes were not reflected in the activities of other respiratory enzymes. Furthermore, bleaching in preconditioned E. pallida (defined as the significant loss of symbionts) was delayed by several days relative to the thermally naive group. These findings suggest that changes to mitochondrial biogenesis and/or function in symbiotic cnidarians during warm preconditioning might play a protective role during periods of exposure to stressful heating.