Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many ...slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes.
Stomatopod crustaceans, or mantis shrimp, are renowned for their complex visual systems. Their array of 16 types of photoreceptors provides complex color reception, as well as linear and circular ...polarization sensitivity 1–6. The least-understood components of their retina are the UV receptors, of which there are up to six distinct, narrowly tuned spectral types 4. Here we show that in the stomatopod species Neogonodactylus oerstedii, this set of receptors is based on only two visual pigments. Surprisingly, five of the six UV receptor types contain the same visual pigment. The various UV receptors are spectrally tuned by a novel set of four short- and long-pass UV-specific optical filters in the overlying crystalline cones. These filters are composed of various mycosporine-like amino acid (MAA) pigments. Commonly referred to as “nature’s sunscreens,” MAAs are usually employed for UV photoprotection 7, 8, but mantis shrimp uniquely incorporate them into powerful spectral tuning filters, extending and diversifying their preeminently elaborate photoreceptive arsenal.
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•Mantis shrimp have only two UV visual pigments in their retinas•Four types of UV optical filters are found in specific facets of the compound eye•The four UV filters and two visual pigments combine to tune six receptor types•The filters are composed of pigments akin to biological sunscreens
Bok et al. describe a unique system of UV spectral tuning in the eyes of the mantis shrimp Neogonodactylus oerstedii. Specifically, two visual pigments in conjunction with four optical filters, composed of biological sunscreen pigments, produce six spectral types of UV photoreceptors.
The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species ...have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.
Marine microbes have received growing attention as sources of bioactive metabolites and offer a unique opportunity to both increase the number of marine natural products in clinical trials as well as ...expedite their development. This review focuses specifically on those molecules currently in the clinical pipeline that are established or highly likely to be produced by bacteria based on expanding circumstantial evidence. We also include an example of how compounds from harmful algal blooms may yield both tools for measuring environmental change as well as leads for pharmaceutical development. An example of the karlotoxin class of compounds isolated from the dinoflagellate Karlodinium veneficum reveals a significant environmental impact in the form of massive fish kills, but also provides opportunities to construct new molecules for the control of cancer and serum cholesterol assisted by tools associated with rational drug design.
•Karlodinium veneficum coincided with a fish kill in the Swan River Estuary, Western Australia.•Diatoms declined as K. veneficum numbers increased in the two months preceding the bloom.•A karlotoxin ...congener was isolated and characterized from the K. veneficum bloom.•Isolated karlotoxins from this study were shown to be ichthyotoxic in laboratory assays.•Observations in this study suggest synergistic effects of karlotoxins and hypoxia in fish kills.
Ichthyotoxic Karlodinium veneficum has become a persistent problem in the eutrophic Swan River Estuary (SRE) near Perth, Western Australia. Karlotoxin (KmTx) concentrations and K. veneficum were sampled from March to July 2005, spanning a bloom confirmed by microscopy and genetics (ITS sequence), and a fish kill coincident with end of the bloom. The objective of this study was to investigate K. veneficum cell and toxin dynamics, and water quality conditions, leading up to the bloom and fish kill in this estuarine system. Abundance of K. veneficum increased as diatom abundance decreased over a 3-month period (Jan–Mar) preceding the bloom. Low freshwater flow to the SRE characterized the bloom initiation period, while elevated seasonal flows altered water quality and preceded the end of the bloom and fish kill. The bloom of K. veneficum was localized over a bottom layer of hypoxic water in a stratified water column. Low nitrate levels, DIN:DIP (mol) near unity, and particulate C:N:P of K. veneficum-rich water samples were consistent with nitrogen limitation of phytoplankton. A KmTx 2 congener was present in the concentration range 0–1052ngKmTxmL−1, levels that were sufficient to kill larval fish in the laboratory within 4h. A KmTx cell quota of 2.8pgKmTxcell−1 was estimated for the bloom, which is moderately high for the species. Gill histopathology of fish from this fish kill showed signs of damage similar to those caused by KmTx in the lab. Results from this study suggest that conditions in the SRE, including elevated K. veneficum abundance and KmTx cell quotas, as well as hypoxia in the upper SRE, likely contribute to seasonal fish kills observed in this system.
Toxins produced by the harmful algal bloom (HAB) forming, mixotrophic dinoflagellate Karlodinium veneficum have long been associated with fish kills. To date, the perceived ecological role for toxins ...has been relief from grazing pressures. Here, we demonstrate that karlotoxins also serve as a predation instrument. Using high-speed holographic microscopy, we measure the swimming behavior of several toxic and nontoxic strains of K. veneficum and their prey, Storeatula major, within dense suspensions. The selected strains produce toxins with varying potency and dosages, including a nontoxic one. Results clearly show that mixing the prey with the predatory, toxic strains causes prey immobilization at rates that are consistent with the karlotoxins' potency and dosage. Even prey cells that continue swimming slow down after exposure to toxic predators. The swimming characteristics of predators vary substantially in pure suspensions, as quantified by their velocity, radii of helical trajectories, and direction of helical rotation. When mixed with prey, all toxic strains that are involved in predation slow down. Furthermore, they substantially reduced their predominantly vertical migration, presumably to remain in the vicinity of their prey. Conversely, the nontoxic control strain does not alter its swimming and does not affect prey behavior. In separate experiments, we show that exposing prey to exogenous toxins also causes prey immobilization at rates consistent with potency. Clearly, the toxic predatory strains use karlotoxins as a means of stunning their prey, before ingesting it. These findings add a substantiated critical understanding for why some HAB species produce such complex toxin molecules.
Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact ...mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.
After publication of karlotoxin 2 (KmTx2; 1), the harmful algal bloom dinoflagellate Karlodinium sp. was collected and scrutinized to identify additional biologically active complex polyketides. The ...structure of 1 was validated and revised at C49 using computational NMR tools including J‐based configurational analysis and chemical‐shift calculations. The characterization of two new compounds KmTx8 (2) and KmTx9 (3) was achieved through overlaid 2D HSQC NMR techniques, while the relative configurations were determined by comparison to 1 and computational chemical‐shift calculations. The detailed evaluation of 2 using the NCI‐60 cell lines, NMR binding studies, and an assessment of the literature supports a mode of action (MoA) for targeting cancer‐cell membranes, especially of cytostatic tumors. This MoA is uniquely different from that of current agents employed in the control of cancers for which 2 shows sensitivity.
In bloom: Harmful algal bloom toxins derived from the dinoflagellate Karlodinium sp. produce an interesting suite of complex polyketides which have been shown to have hemolytic, cytotoxic and ichthyotoxic activity. Better understanding the mechanism of action for this class of compounds could lead to the development of new agents for targeting cancer‐cell membranes.
Dietary insufficiencies have been well documented to decrease growth rates and survival (and therefore overall production) in fish aquaculture. By contrast, the effects of dietary insufficiencies on ...the sensory biology of cultured fish remains largely unstudied. Diets based solely on plant protein sources could have advantages over fish-based diets because of the cost and ecological effects of the latter, but plant proteins lack the amino acid taurine. Adequate levels of taurine are, however, necessary for the development of a fully functional visual system in mammals. As part of ongoing studies to determine the suitability of plant-based diets, we investigated the effects of normal and reduced taurine dietary levels on retinal anatomy and function in European sea bass (Dicentrarchus labrax). We could not demonstrate any effects of dietary taurine level on retinal anatomy, nor the functional properties of luminous sensitivity and temporal resolution (measured as flicker fusion frequency). We did, however, find an effect on spectral sensitivity. The peak of spectral sensitivity of individuals fed a 5% taurine diet was rightward shifted (i.e., towards longer wavelengths) relative to that of fish fed a 0% or 1.5% taurine diet. This difference in in spectral sensitivity was due to a relatively lower level of middle wavelength pigment (maximum absorbance .500 nm) in fish fed a 5% taurine diet. Changes in spectral sensitivity resulting from diets containing different taurine levels are unlikely to be detrimental to fish destined for market, but could be in fishes that are being reared for stock enhancement programs.
This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and ...Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7–40% from log phase growth to late log – early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20–31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60–90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC–MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.
► Gambierdiscus species exhibit significant hemolytic activity (HA). ► HA was caused by maitotoxins and differs significantly among species. ► HA varied geographically and with growth phase. ► Intraspecies isolates obtained from the same region exhibited similar HA. ► HA (maitotoxin) was constitutively produced and under strong genetic control.
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