[Show abstract][Hide abstract] ABSTRACT: As long-lived predators that integrate exposures across multiple trophic levels, cetaceans are recognized as sentinels for the health of marine ecosystems. Their utility as sentinels requires the establishment of baseline health parameters. Because cetaceans are protected, measurements obtained with minimal disruption to free ranging animals are highly desirable. In this study we investigated the utility of skin gene expression profiling to monitor health and contaminant exposure in common bottlenose dolphins (Tursiops truncatus). Remote integument biopsies were collected in the northern Gulf of Mexico prior to the Deepwater Horizon oil spill (May 2010) and during summer and winter for two years following oil contamination (2010-2011). A bottlenose dolphin microarray was used to characterize the skin transcriptomes of 94 individuals from three populations: Barataria Bay, Louisiana, Chandeleur Sound, Louisiana, and Mississippi Sound, Mississippi/Alabama. Skin transcriptomes did not differ significantly between populations. In contrast, season had a profound effect on gene expression, with nearly one-third of all genes on the array differing in expression between winter and the warmer seasons (moderated T-test; p<0.01, fold-change≥1.5). Persistent organic pollutants (POPs) in blubber changed concurrently, reaching >two-fold higher concentrations in summer compared to winter, due to a seasonal decrease in blubber thickness and loss of stored lipid. However, global gene expression did not correlate strongly with seasonally changing contaminant concentrations, most likely because the refractory, lipid-stored metabolites are not substrates for phase I or II xenobiotic detoxification pathways. Rather, processes related to cell proliferation, motility, and differentiation dominated the differences in expression in winter and the warmer seasons. More subtle differences were seen between spring and summer (1.5% of genes differentially expressed). However, two presumed oil-exposed animals from spring presented gene expression profiles more similar to the summer animals (presumed exposed) than to other spring animals. Seasonal effects have not previously been considered in studies assessing gene expression in cetaceans, but clearly must be taken into account when applying transcriptomic analyses to investigate their contaminant exposure or health status.
PLoS ONE 06/2015; 10(6):e0130934. DOI:10.1371/journal.pone.0130934 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As top-level predators, common bottlenose dolphins (Tursiops truncatus) are particularly sensitive to chemical and biological contaminants that accumulate and biomagnify in the marine food chain.
This work investigates the potential use of microarray technology and gene expression profile analysis to screen common bottlenose dolphins for exposure to environmental contaminants through the immunological and/or endocrine perturbations associated with these agents. A dolphin microarray representing 24,418 unigene sequences was used to analyze blood samples collected from 47 dolphins during capture-release health assessments from five different US coastal locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). Organohalogen contaminants including pesticides, polychlorinated biphenyl congeners (PCBs) and polybrominated diphenyl ether congeners were determined in blubber biopsy samples from the same animals. A subset of samples (n=10, males; n=8, females) with the highest and the lowest measured values of PCBs in their blubber was used as strata to determine the differential gene expression of the exposure extremes through machine learning classification algorithms. A set of genes associated primarily with nuclear and DNA stability, cell division and apoptosis regulation, intra – and extra-cellular traffic, and immune response activation was selected by the algorithm for identifying the two exposure extremes. In order to test the hypothesis that these gene expression patterns reflect PCB exposure, we next investigated the blood transcriptomes of the remaining dolphin samples using machine-learning approaches, including K-nn and Support Vector Machines classifiers. Using the derived gene sets, the algorithms worked very well (100% success rate) at classifying dolphins according to the contaminant load accumulated in their blubber. These results suggest that gene expression profile analysis may provide a valuable means to screen for indicators of chemical exposure.
Marine environmental research 09/2014; 100. DOI:10.1016/j.marenvres.2014.03.007 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ciguatoxins (CTXs) are potent neurotoxins responsible for the food-borne illness known as ciguatera that occurs after consumption of contaminated fish. Benthic dinoflagellates of the genus Gambierdiscus spp. are known as the main producers of CTXs. CTXs are polycyclic polyethers, presumed to be synthesized by polyketide synthase (PKS) complexes; however, the mechanisms of CTX biosynthesis remain unresolved. Here, we investigated a de novo transcriptome assembly of Gambierdiscus polynesiensis TB-92 clone, a highly toxic producer of Pacific ciguatoxins, and focused on the identification of PKS transcripts. A cDNA library generated using a spliced leader (SL) priming approach, which specifically targets the dinoflagellate nuclear transcriptome, was sequenced by Roche 454. This strategy produced 1,221,335 raw reads, assembled into 16,336 unique contigs. Contigs were subjected to BLAST search, annotated with Gene Ontology (GO) terms and enriched with enzyme codes (EC) from Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Thirty-three PKS-related sequences were thus identified. Twenty-two contigs encoded single domain β-ketoacyl synthases (KS) with sequence similarity to Type I PKSs, as reported in other dinoflagellates. A conserved motif previously observed near the 5′ end of dinoflagellate KS domain transcripts was present in G. polynesiensis, and distinguished two groups of KS domain sequences. Ketoreductase (KR), acyltransferase (AT), and acyl carrier protein (ACP) domains were also found on single domain containing transcripts. KEGG pathway mapping placed three of the KS sequences containing the PKS conserved domain (cd00833) in the fatty acid biosynthesis pathway. No contigs were found encoding the conserved domains typically found in elongating ketosynthase domains of fatty acid synthases (cd00832, Type I or cd00834, Type II). Contigs mapping to other parts of the fatty acid biosynthesis pathway similarly encoded individual domains, suggesting that fatty acid synthesis takes place in multiprotein complexes. Other than the three KS domains, none of the sequences mapping to the fatty acid biosynthesis pathway overlapped with those annotated as PKSs. These data lend support to the idea that PKSs may contribute to both polyketide and fatty acid synthesis in dinoflagellates. This dataset provides important background to future research in order to understand the complex mechanism of toxin production in this dinoflagellate.
[Show abstract][Hide abstract] ABSTRACT: The observation of caspase-like activity during cell death has provided a new framework for understanding the evolutionary and ecological contexts of programmed cell death in phytoplankton. However, additional roles for this caspase-like activity, the enzymes responsible, and the targets of this enzyme activity in phytoplankton remain largely undefined. In the present study, the role of caspase-like activity in aging and ROS-mediated cell death were investigated and death programs both dependent on and independent of caspase-like activity were observed in the toxic dinoflagellate, Karenia brevis. The dual use of in situ caspase 3/7 and TUNEL staining identified previously undescribed death-associated morphotypes in K. brevis. In silico motif analysis identified several enzymes with predicted caspase-like activity in the K. brevis transcriptome, although bona fide caspases are absent. Lastly, computational prediction of downstream caspase substrates, using sequence context and predicted secondary structure, identified proteins involved in a wide range of biological processes including regulation of protein turnover, cell cycle progression, lipid metabolism, coenzyme metabolism, apoptotic and autophagic death. To confirm the computational predictions, a short peptide was designed around the predicated caspase cleavage site in a predicted novel K. brevis caspase 3/7-like target, S-adenosylmethionine synthetase (KbAdoMetS). Cleavage of the peptide substrate with recombinant caspase 3 enzyme was determined by MALDI-TOF MS, confirming that KbAdoMetS is indeed a bona fide caspase substrate. These data identify the involvement of caspase-like activity in both aging and cell death in K. brevis and identify novel executioner enzymes and downstream targets that may be important for bloom termination.
[Show abstract][Hide abstract] ABSTRACT: Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [3H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10−10) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.
Journal of Phycology 12/2013; 49(6):1118–1127. DOI:10.1111/jpy.12120 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the roles of differential mRNA stability and de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of increases in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.
PLoS ONE 06/2013; 8(6):e66347. DOI:10.1371/journal.pone.0066347 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the global proliferation of toxic harmful algal bloom species, there is a need to identify the environmental and biological factors that regulate toxin production. One such species, Karenia brevis, forms nearly annual blooms that threaten coastal regions throughout the Gulf of Mexico. This dinoflagellate produces brevetoxins, which are potent neurotoxins that cause neurotoxic shellfish poisoning and respiratory illness in humans, as well as massive fish kills. A recent publication reported that a rapid decrease in salinity increased cellular toxin quotas in K. brevis and hypothesized that brevetoxins serve a role in osmoregulation. This finding implied that salinity shifts could significantly alter the toxic effects of blooms. We repeated the original experiments separately in three different laboratories and found no evidence for increased brevetoxin production in response to low-salinity stress in any of the eight K. brevis strains we tested, including three used in the original study. Thus, we find no support for an osmoregulatory function of brevetoxins. The original publication also stated that there was no known cellular function for brevetoxins. However, there is increasing evidence that brevetoxins promote survival of the dinoflagellates by deterring grazing by zooplankton. Whether they have other as-yet-unidentified cellular functions is currently unknown.
Proceedings of the National Academy of Sciences 06/2013; 110(25). DOI:10.1073/pnas.1217716110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.
PLoS ONE 08/2012; 7(8):e42974. DOI:10.1371/journal.pone.0042974 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Harmful algal blooms (HABs) aff ect aquatic ecosys-tems around the world, adversely aff ecting marine animal and human health, coastal ecosystem integrity, and economies that depend on coastal resources. Shellfi sh poisoning events involving humans who had ingested bivalves contaminated with HAB toxins primarily drove early scientifi c and social interest in HABs. More recently, research eff orts have shown that HABs are oft en temporally and spatially correlated with the occurrence of acute morbidity or mortality of marine animals (Landsberg et al. 2005), and to date at least four classes of algal toxins have been associated with such events. Although fi sh, seabirds, and many other groups of marine wildlife are aff ected, these mortality events frequently involve marine mammals, and as such this chapter will focus primarily on the latt er. In addition, since marine mammals are impor-tant sentinel species that act as barometers of ocean health and demonstrate the link between ocean and human health, the importance placed on these species in this context is warranted (Aguirre and Tabor 2004 ; Tabor and Aguirre 2004 ; Wells et al. 2004 ; Bossart 2006). Th e frequency of associated marine mammal mortality events and HABs appears to have increased in recent years. Th is may be a refl ection of several of factors, including (1) the increased scientifi c and popular att ention given to marine mammal mortality events, (2) an increase in resources and observer eff ort dedicated to detection and study of HAB spe-cies in coastal waters, (3) an improved ability to detect algal toxins in marine mammal tissues and fl uids, and (4) an apparent increase in both the frequency and the geographic distribution of HABs that has been documented over the past quarter-century (Van Dolah 2000 ; Hallegraeff 2003 ; Sellner et al. 2003). Exposure of marine mammals to algal toxins occurs via food-web transfer or directly through respiratory exposure. Th e susceptibility of marine mammals to algal toxins is therefore dependent not only upon the occurrence of toxin-producing algae within their habitat but, in the case of food-web transfer, on the co-occurrence of prey species at the time of a HAB to serve as vectors to higher trophic levels. Th us, management of the impacts of algal toxins on marine mammals requires an understanding of the causes and consequences of harmful algal blooms, the rea-sons for their apparent increase, and their dependence on large-scale oceanographic and climate changes as well as their local and regional infl uences.
New Directions in Conservation Medicine: Applied Cases of Ecological Health, 05/2012: chapter 26: pages 374-389; Oxford University Press.
[Show abstract][Hide abstract] ABSTRACT: A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic e shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with (3)H saxitoxin (STX) diHCl for binding to voltage-gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (microg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1-93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r(2)) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r(2) of 0.92. When samples were sorted according to increasing toxin concentration (microg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 microg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.
Journal of AOAC International 05/2012; 95(3):795-812. DOI:10.5740/jaoacint.CS2011_27 · 1.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The toxic dinoflagellate, Karenia brevis, forms dense blooms in the Gulf of Mexico that persist for many months in coastal waters, where they can cause extensive marine animal mortalities and human health impacts. The mechanisms that enable cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. To gain an understanding of processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out to identify changes in the global transcriptome that accompany the entry and maintenance of stationary phase up to the onset of cell death. The transcriptome of K. brevis was assayed using a custom 10,263 feature oligonucleotide microarray from mid-logarithmic growth to the onset of culture demise. A total of 2958 (29%) features were differentially expressed, with the mid-stationary phase timepoint demonstrating peak changes in expression. Gene ontology enrichment analyses identified a significant shift in transcripts involved in energy acquisition, ribosome biogenesis, gene expression, stress adaptation, calcium signaling, and putative brevetoxin biosynthesis. The extensive remodeling of the transcriptome observed in the transition into a quiescent non-dividing phase appears to be indicative of a global shift in the metabolic and signaling requirements and provides the basis from which to understand the process of chronological aging in a dinoflagellate.
[Show abstract][Hide abstract] ABSTRACT: Conservation biologists face many challenges in assessing health, immune status and infectious diseases in protected species. These challenges include unpredictable sample populations, diverse genetic and environmental backgrounds of the animals, as well as the practical, legal and ethical issues involved in experimentation. The use of whole genome scale transcriptomics with animal samples obtained in a minimally invasive manner is an approach that shows promise for health assessment. In this study we assessed the utility of a microarray to identify changes in gene expression predictive of health status by interrogating blood samples from California sea lions (Zalophus californianus) in rehabilitation. A custom microarray was developed from the commercially available dog microarray (Canis familiaris) by selecting probes that demonstrated reliable cross-hybridization with RNA in sea lion blood. This custom microarray was used for the analysis of RNA from 73 sea lion blood samples, from animals with a broad spectrum of health changes. Both traditional classifying techniques and newer artificial neural network approaches correctly classified sea lions with respect to health status, primarily distinguishing between leptospirosis infection and domoic acid exposure. Real time PCR validation for a small set of genes, followed by sequencing, showed good correlation with array results and high identity (96-98%) between the dog and sea lion sequences. This approach to health status classification shows promise for disease identification in a clinical setting, and assessment of health status of wildlife.
[Show abstract][Hide abstract] ABSTRACT: The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' trans-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of K. brevis is responsive to nitrogen and phosphorus and is informative of nutrient status.
Microarray analysis of N-depleted K. brevis cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes.
Microarray analysis provided transcriptomic evidence for N- but not P-limitation in K. brevis. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.
[Show abstract][Hide abstract] ABSTRACT: Karenia brevis is a toxic dinoflagellate responsible for red tides in the Gulf of Mexico. The molecular mechanisms controlling its cell cycle are important to bloom formation because blooms develop through vegetative cell division. This study identifies a suite of conserved S-phase genes in K. brevis-proliferating cell nuclear antigen (PCNA), ribonucleotide reductase 2, replication factor C, and replication protein A-and characterizes their expression at the mRNA and protein level over the cell cycle. In higher eukaryotes, the expression of these genes is controlled by transcription, activated at S-phase entry by the E2F transcription factor, which ensures their timely availability for DNA synthesis. In the dinoflagellate, these transcripts possess a 5'-transspliced leader sequence, which suggests they may be under post-transcriptional control as demonstrated in trypanosomes. Using quantitative polymerase chain reaction (qPCR), we confirmed that their transcript levels are unchanged over the cell cycle. However, their proteins are maximally expressed during S-phase. This suggests their cell-cycle-dependent expression may be achieved at the level of translation and/or stability. Proliferating cell nuclear antigen further undergoes an increase in size of ∼9 kDa that dominates during S-phase. This coincides with a change in its distribution, with prominent staining of chromatin-bound PCNA occurring during S-phase. We hypothesize that the change in the observed size of PCNA is due to post-translational modification. Together, these studies demonstrate post-transcriptional regulation of S-phase genes in K. brevis. Differential expression of these S-phase proteins may be useful in the development of biomarkers to assess bloom growth status in the field.
[Show abstract][Hide abstract] ABSTRACT: The marine neurotoxin domoic acid (DA) is a rigid analogue of the neurotransmitter glutamate and a potent agonist of kainate subtype glutamate receptors. Persistent activation of these receptor subtypes results in rapid excitotoxicity, calcium-dependent cell death, and neuronal degeneration in regions of the brain where glutamatergic pathways are concentrated. Previous work has shown that DA promotes the expression of inflammatory genes in the brain, such as cyclooxygenase 2 (COX2). To investigate the impact of inflammation on the development of neurodegeneration, and ultimately survival following DA administration, we used selective (L745337, Merck) and non-selective (acetylsalicylic acid (ASA)) COX inhibitors in DA exposed mice. Adult male ICR mice were given a regime of either ASA or L23547 both before and after a single LD50 dose of DA. Mice were observed immediately after toxin introduction and then sacrificed at 2 days post exposure. Our lower dose of L23547 increased survival and was most effective at decreasing neuronal degeneration in the CA1 and CA3 regions of the hippocampus, areas especially sensitive to DA excitotoxicity. This study shows that COX2 plays a role in DA induced neurodegeneration and death, and that inhibitors may be of value for treatment in human and wildlife DA exposure.
[Show abstract][Hide abstract] ABSTRACT: Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data.
A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results.
Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.
[Show abstract][Hide abstract] ABSTRACT: The dinoflagellate Karenia brevis (C. C. Davis) Gert Hansen et Moestrup produces a suite of brevetoxins, potent neurotoxins that have adverse effects on marine animal and human health. Brevetoxins are polyketides proposed to be synthesized by polyketide synthases (PKSs), and genes for type I PKSs have been predicted by PCR and transcript analysis. However, the full-length transcripts in K. brevis predict an unusual protein structure for type I PKS in that individual transcripts encode for single catalytic domains. In this study, we developed peptide antibodies to in silico translated transcripts for two PKS proteins to characterize their expression and localization. Immunoreactive proteins identified by Western blotting at 40 kDa (KR domain) and 100 kDa (KS domain) are consistent with the sizes predicted by the full-length transcripts. Immunolocalization and Western blot analysis indicate that these PKSs are associated with the chloroplasts. In order to establish evidence for a role in brevetoxin biosynthesis, PKS transcript and protein levels were examined in a “nontoxic”K. brevis substrain and its parental toxic isolate, K. brevis Wilson. DNA microarray analysis of the global transcript profiles in the “nontoxic” isolate showed that ∼7% of transcripts were differentially expressed, including photosystem genes; however, no difference was observed in PKS transcript abundance. By contrast, KS domain proteins were 55%–70% less abundant in “nontoxic”K. brevis cultures compared to toxic cultures. This finding suggests that K. brevis PKS expression is regulated posttranscriptionally, like many other processes in dinoflagellates. Further, the decrease in PKS protein abundance in the “nontoxic” cultures provides correlative evidence for their involvement in brevetoxin biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: A single-laboratory validation (SLV) study was conducted for the microplate receptor binding assay (RBA) for paralytic shellfish poisoning (PSP) toxins in shellfish. The basis of the assay is the competition between [3H]saxitoxin (STX) and STX in a standard or sample for binding to the voltage dependent sodium channel. A calibration curve is generated by the addition of 0.01-1000 nM STX, which results in the concentration dependent decrease in [3H]STX-receptor complexes formed and serves to quantify STX in unknown samples. This study established the LOQ, linearity, recovery, accuracy, and precision of the assay for determining PSP toxicity in shellfish extracts, as performed by a single analyst on multiple days. The standard curve obtained on 5 independent days resulted in a half-maximal inhibition (IC50) of 2.3 nM STX +/- 0.3 (RSD = 10.8%) with a slope of 0.96 +/- 0.06 (RSD = 6.3%) and a dynamic range of 1.2-10.0 nM. The LOQ was 5.3 microg STX equivalents/100 g shellfish. Linearity, established by quantification of three levels of purified STX (1.5, 3, and 6 nM), yielded an r2 of 0.97. Recovery from mussels spiked with three levels (40, 80, and 120 microg STX/100 g) averaged 121%. Repeatability (RSD(r)), determined on six naturally contaminated shellfish samples on 5 independent days, was 17.7%. A method comparison with the AOAC mouse bioassay yielded r2 = 0.98 (slope = 1.29) in the SLV study. The effects of the extraction method on RBA-based toxicity values were assessed on shellfish extracted for PSP toxins using the AOAC mouse bioassay method (0.1 M HCI) compared to that for the precolumn oxidation HPLC method (0.1% acetic acid). The two extraction methods showed linear correlation (r2 = 0.99), with the HCl extraction method yielding slightly higher toxicity values (slope = 1.23). A similar relationship was observed between HPLC quantification of the HCI- and acetic acid-extracted samples (r2 = 0.98, slope 1.19). The RBA also had excellent linear correlation with HPLC analyses (r2 = 0.98 for HCl, r2 = 0.99 for acetic acid), but gave somewhat higher values than HPLC using either extraction method (slope = 1.39 for HCl extracts, slope = 1.32 for acetic acid). Overall, the excellent linear correlations with the both mouse bioassay and HPLC method and sufficient interassay repeatability suggest that the RBA can be effective as a high throughput screen for estimating PSP toxicity in shellfish.
Journal of AOAC International 11/2009; 92(6):1705-13. · 1.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Avian vacuolar myelinopathy (AVM) is a neurological disease affecting bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), waterfowl, and other birds in the southeastern United States. The cause of the disease is unknown, but is thought to be a naturally produced toxin. AVM is associated with aquatic macrophytes, most frequently hydrilla (Hydrilla verticillata), and researchers have linked the disease to an epiphytic cyanobacterial species associated with the macrophytes. The goal of this study was to develop an extraction protocol for separating the putative toxin from a hydrilla-cyanobacterial matrix. Hydrilla samples were collected from an AVM-affected reservoir (J. Strom Thurmond Lake, SC) and confirmed to contain the etiologic agent by mallard (Anas platyrhynchos) bioassay. These samples were then extracted using a solvent series of increasing polarity: hexanes, acetone, and methanol. Control hydrilla samples from a reference reservoir with no history of AVM (Lake Marion, SC) were extracted in parallel. Resulting extracts were administered to mallards by oral gavage. Our findings indicate that the methanol extracts of hydrilla collected from the AVM-affected site induced the disease in laboratory mallards. This study provides the first data documenting for an "extractable" AVM-inducing agent.