Transcriptome profiling of blood from common bottlenose dolphins (Tursiops truncatus) in the northern Gulf of Mexico to enhance health assessment capabilities.

Following the 2010 Deepwater Horizon disaster and subsequent unusual mortality event, adverse health impacts have been reported in bottlenose dolphins in Barataria Bay, LA including impaired stress response and reproductive, pulmonary, cardiac, and immune function. These conditions were primarily diagnosed through hands-on veterinary examinations and analysis of standard diagnostic panels. In human and veterinary medicine, gene expression profiling has been used to identify molecular mechanisms underlying toxic responses and disease states. Identification of molecular markers of exposure or disease may enable earlier detection of health effects or allow for health evaluation when the use of specialized methodologies is not feasible. To date this powerful tool has not been applied to augment the veterinary data collected concurrently during dolphin health assessments. This study examined transcriptomic profiles of blood from 76 dolphins sampled in health assessments during 2013-2018 in the waters near Barataria Bay, LA and Sarasota Bay, FL. Gene expression was analyzed in conjunction with the substantial suite of health data collected using principal component analysis, differential expression testing, over-representation analysis, and weighted gene co-expression network analysis. Broadly, transcript profiles of Barataria Bay dolphins indicated a shift in immune response, cytoskeletal alterations, and mitochondrial dysfunction, most pronounced in dolphins likely exposed to Deepwater Horizon oiling. While gene expression profiles in Barataria Bay dolphins were altered compared to Sarasota Bay for all years, profiles from 2013 exhibited the greatest alteration in gene expression. Differentially expressed transcripts included genes involved in immunity, inflammation, reproductive failure, and lung or cardiac dysfunction, all of which have been documented in dolphins from Barataria Bay following the Deepwater Horizon oil spill. The genes and pathways identified in this study may, with additional research and validation, prove useful as molecular markers of exposure or disease to assist wildlife veterinarians in evaluating the health of dolphins and other cetaceans.

Modeling population effects of the Deepwater Horizon oil spill on a long-lived species.

The 2010 Deepwater Horizon (DWH) oil spill exposed common bottlenose dolphins (Tursiops truncatus) in Barataria Bay, Louisiana to heavy oiling that caused increased mortality and chronic disease and impaired reproduction in surviving dolphins. We conducted photographic surveys and veterinary assessments in the decade following the spill. We assigned a prognostic score (good, fair, guarded, poor, or grave) for each dolphin to provide a single integrated indicator of overall health, and we examined temporal trends in prognostic scores. We used expert elicitation to quantify the implications of trends for the proportion of the dolphins that would recover within their lifetime. We integrated expert elicitation, along with other new information, in a population dynamics model to predict the effects of observed health trends on demography. We compared the resulting population trajectory with that predicted under baseline (no spill) conditions. Disease conditions persisted and have recently worsened in dolphins that were presumably exposed to DWH oil: 78% of those assessed in 2018 had a guarded, poor, or grave prognosis. Dolphins born after the spill were in better health. We estimated that the population declined by 45% (95% CI 14-74) relative to baseline and will take 35 years (95% CI 18-67) to recover to 95% of baseline numbers. The sum of annual differences between baseline and injured population sizes (i.e., the lost cetacean years) was 30,993 (95% CI 6607-94,148). The population is currently at a minimum point in its recovery trajectory and is vulnerable to emerging threats, including planned ecosystem restoration efforts that are likely to be detrimental to the dolphins' survival. Our modeling framework demonstrates an approach for integrating different sources and types of data, highlights the utility of expert elicitation for indeterminable input parameters, and emphasizes the importance of considering and monitoring long-term health of long-lived species subject to environmental disasters. Article impact statement: Oil spills can have long-term consequences for the health of long-lived species; thus, effective restoration and monitoring are needed.

El derrame de petróleo Deepwater Horizon (DWH) en 2010 expuso gravemente a este hidrocarburo a los delfines (Tursiops truncatus) de la Bahía Barataria, Luisiana, causando un incremento en la mortalidad y en las enfermedades crónicas, y deteriorando la reproducción de los delfines sobrevivientes. Realizamos censos fotográficos y evaluaciones veterinarias durante la década posterior al derrame. Asignamos un puntaje pronóstico (bueno, favorable, moderado, malo, o grave) a cada delfín para proporcionar un indicador integrado único de la salud en general. También examinamos las tendencias temporales de estos puntajes. Usamos información de expertos para cuantificar las implicaciones de las tendencias para la proporción de delfines que se recuperaría dentro de su periodo de vida. Integramos esta información, junto con información nueva, a un modelo de dinámica poblacional para predecir los efectos sobre la demografía de las tendencias observadas en la salud. Comparamos la trayectoria poblacional resultante con aquella pronosticada bajo condiciones de línea base (sin derrame). Las condiciones de enfermedad persistieron y recientemente han empeorado en los delfines que supuestamente estuvieron expuestos al petróleo de DWH: 78% de aquellos evaluados en 2018 tuvieron un pronóstico moderado, malo o grave. Los delfines que nacieron después del derrame contaron con mejor salud. Estimamos que la población declinó en un 45% (95% CI 14-74) relativo a la línea base y tardará 35 años (95% CI 18-67) en recuperar el 95% de los números de línea base. La suma de las diferencias anuales entre el tamaño poblacional de línea base y el dañado (es decir, los años cetáceos perdidos) fue de 30,993 (95% CI 6,607-94,148). La población actualmente está en un punto mínimo de su trayectoria de recuperación y es vulnerable a las amenazas emergentes, incluyendo los esfuerzos de restauración ambiental planeada que probablemente sean nocivos para la supervivencia de los delfines. Nuestro marco de modelado demuestra una estrategia para la integración de diferentes fuentes y tipos de datos, resalta la utilidad de la información de expertos para los parámetros de aportación indeterminable, y enfatiza la importancia de la consideración y el monitoreo de la salud a largo plazo de las especies longevas sujetas a los desastres ambientales. Modelado de los Efectos Poblacionales del Derrame de Petróleo Deepwater Horizon sobre Especies Longevas.

Biochemical and hematological biomarkers of reproductive failure in bottlenose dolphins Tursiops truncatus.

The physiological demands of pregnancy inevitably result in alterations in both biochemical and hematological parameters as fetal development occurs. The shifts observed in successful pregnancy in bottlenose dolphins Tursiops truncatus to support both fetal physiological needs and maternal basal requirements have been established according to each trimester. Detecting aberrations in blood-based biomarkers could help facilitate diagnosis of gestational abnormalities, improve our understanding of factors influencing reproductive outcomes and aid in prediction of reproductive failure. This study retrospectively analyzed 263 blood samples from 15 bottlenose dolphins in 21 failed pregnancies over 28 yr (1989-2017). Most samples remained within normal pregnancy reference ranges; however, significant shifts were observed between trimesters. Hematological alterations, compared to successful pregnancy reference ranges from previously published data, were consistent across failed pregnancies and included an increased prevalence of elevated 2nd and 3rd trimester neutrophils, elevated 2nd trimester monocytes and decreased 3rd trimester eosinophils. In addition, low hematocrit and low red blood cells were more prevalent in the 2nd trimester. Biochemical shifts included an increased prevalence of elevated creatine phosphokinase in the 3rd trimester outside of the normal reference ranges. Across failed pregnancies, calcium and iron were decreased in the 3rd trimester. Significantly decreased progesterone in the 3rd trimester was a negative prognostic indicator of pregnancy outcome with decreasing 3rd trimester progesterone associated with failed pregnancy. This study demonstrates the use of blood-based biomarkers as possible predictors of pregnancy outcome in bottlenose dolphins.

Age determination of common bottlenose dolphins (Tursiops truncatus) using dental radiography pulp:tooth area ratio measurements.

Age is an important parameter to better understand wildlife populations, and is especially relevant for interpreting data for fecundity, health, and survival assessments. Estimating ages for marine mammals presents a particular challenge due to the environment they inhabit: accessibility is limited and, when temporarily restrained for assessment, the window of opportunity for data collection is relatively short. For wild dolphins, researchers have described a variety of age-determination techniques, but the gold-standard relies upon photo-identification to establish individual observational life histories from birth. However, there are few populations with such long-term data sets, therefore alternative techniques for age estimation are required for individual animals without a known birth period. While there are a variety of methods to estimate ages, each involves some combination of drawbacks, including a lack of precision across all ages, weeks-to-months of analysis time, logistical concerns for field applications, and/or novel techniques still in early development and validation. Here, we describe a non-invasive field technique to determine the age of small cetaceans using periapical dental radiography and subsequent measurement of pulp:tooth area ratios. The technique has been successfully applied for bottlenose dolphins briefly restrained during capture-release heath assessments in various locations in the Gulf of Mexico. Based on our comparisons of dental radiography data to life history ages, the pulp:tooth area ratio method can reliably provide same-day estimates for ages of dolphins up to about 10 years old.

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.

Pregnancy profiles in the common bottlenose dolphin (Tursiops truncatus): Clinical biochemical and hematological variations during healthy gestation and a successful outcome.

The physiological demands of pregnancy inevitably result in changes of both biochemical and hematological parameters as the fetus develops. Alterations in blood parameters have been observed to shift according to both trimester and species, to support fetal physiological needs and maternal basal requirements. Establishing normal reference ranges for each stage in gestation is important to facilitate diagnosis of underlying health concerns and prevent over-diagnosing abnormalities. Despite bottlenose dolphins (Tursiops truncatus) being one of the most highly studied cetaceans, the blood profile changes occurring as a result of pregnancy have not been previously described. A retrospective analysis was performed from blood samples obtained from 42 successful pregnancies from 20 bottlenose dolphins in a managed population over 30 years. Samples were compared to non-pregnant states and among trimesters of pregnancy. Blood profile fluctuations occurred throughout gestation, however significant alterations predominantly occurred between the 2nd and 3rd trimester. Hematological changes from the 2nd to the 3rd trimester included a decrease in lymphocytes, decrease in platelet count, and hemoconcentration with increased hematocrit and hemoglobin. Biochemical changes in the 3rd trimester included significant reductions in ALKP (alkaline phosphatase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase) with significant increases observed in albumin, globulins, total protein, cholesterol, triglycerides and CO2. It's important to note that despite significant shifts occurring between the 2nd and 3rd trimester, there was no significant change in platelets, hematocrit, hemoglobin, lymphocytes or CO2 between non-pregnant and 3rd trimester blood samples. The normal reference ranges for each trimester established herein, will enable future identification of abnormalities occurring during pregnancy and help improve our understanding of factors potentially influencing a failed or successful pregnancy outcome.

Remote blubber sampling paired with liquid chromatography tandem mass spectrometry for steroidal endocrinology in free-ranging bottlenose dolphins (Tursiops truncatus).

Liquid chromatography tandem mass spectrometry allows for the measurement of steroid hormone suites in the blubber of marine mammals. By combining this technology with minimally invasive techniques such as remote biopsy, endocrine profiles can be assessed, allowing for studies of hormonal profile variation over time. In this study, we explored associations among different steroidogenic pathways and seasonal differences in blubber hormone profiles of free-ranging common bottlenose dolphins along the coast of South Carolina, USA. Male dolphins experience a peak in testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone in the spring, likely related to an upregulation of the androgen steroidogenic pathway during mating season. We also observed increased cortisol concentrations during summer compared to winter. Among females, there was an increase in androstenedione with elevated progesterone concentrations indicative of pregnancy, highlighting another potential endocrine marker for pregnancy in free-ranging dolphins. This work emphasizes the importance of selecting the appropriate season for studies on endocrine status to effectively uncover physiological variation or disruption in free-ranging cetaceans.

Skin Transcriptomes of common bottlenose dolphins (Tursiops truncatus) from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts.

Common bottlenose dolphins serve as sentinels for the health of their coastal environments as they are susceptible to health impacts from anthropogenic inputs through both direct exposure and food web magnification. Remote biopsy samples have been widely used to reveal contaminant burdens in free-ranging bottlenose dolphins, but do not address the health consequences of this exposure. To gain insight into whether remote biopsies can also identify health impacts associated with contaminant burdens, we employed RNA sequencing (RNA-seq) to interrogate the transcriptomes of remote skin biopsies from 116 bottlenose dolphins from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts. Gene expression was analyzed using principal component analysis, differential expression testing, and gene co-expression networks, and the results correlated to season, location, and contaminant burden. Season had a significant impact, with over 60% of genes differentially expressed between spring/summer and winter months. Geographic location exhibited lesser effects on the transcriptome, with 23.5% of genes differentially expressed between the northern Gulf of Mexico and the southeastern U.S. Atlantic locations. Despite a large overlap between the seasonal and geographical gene sets, the pathways altered in the observed gene expression profiles were somewhat distinct. Co-regulated gene modules and differential expression analysis both identified epidermal development and cellular architecture pathways to be expressed at lower levels in animals from the northern Gulf of Mexico. Although contaminant burdens measured were not significantly different between regions, some correlation with contaminant loads in individuals was observed among co-expressed gene modules, but these did not include classical detoxification pathways. Instead, this study identified other, possibly downstream pathways, including those involved in cellular architecture, immune response, and oxidative stress, that may prove to be contaminant responsive markers in bottlenose dolphin skin.

Both modular and single-domain Type I polyketide synthases are expressed in the brevetoxin-producing dinoflagellate, Karenia brevis (Dinophyceae).

Dinoflagellates are prolific producers of polyketide compounds, many of which are potent toxins with adverse impacts on human and marine animal health. To identify polyketide synthase (PKS) genes in the brevetoxin-producing dinoflagellate, Karenia brevis, we assembled a transcriptome from 595 million Illumina reads, sampled under different growth conditions. The assembly included 125,687 transcripts greater than 300 nt in length, with over half having >100× coverage. We found 121 transcripts encoding Type I ketosynthase (KS) domains, of which 99 encoded single KS domains, while 22 contained multiple KS domains arranged in 1-3 protein modules. Phylogenetic analysis placed all single domain and a majority of multidomain KSs within a monophyletic clade of protist PKSs. In contrast with the highly amplified single-domain KSs, only eight single-domain ketoreductase transcripts were found in the assembly, suggesting that they are more evolutionarily conserved. The multidomain PKSs were dominated by trans-acyltransferase architectures, which were recently shown to be prevalent in other algal protists. Karenia brevis also expressed several hybrid nonribosomal peptide synthetase (NRPS)/PKS sequences, including a burA-like sequence previously reported in a wide variety of dinoflagellates. This contrasts with a similarly deep transcriptome of Gambierdiscus polynesiensis, which lacked NRPS/PKS other than the burA-like transcript, and may reflect the presence of amide-containing polyketides in K. brevis and their absence from G. polynesiensis. In concert with other recent transcriptome analyses, this study provides evidence for both single domain and multidomain PKSs in the synthesis of polyketide compounds in dinoflagellates.

De novo transcriptome assembly and RNA-Seq expression analysis in blood from beluga whales of Bristol Bay, AK.

Assessing the health of marine mammal sentinel species is crucial to understanding the impacts of environmental perturbations on marine ecosystems and human health. In Arctic regions, beluga whales, Delphinapterus leucas, are upper level predators that may serve as a sentinel species, potentially forecasting impacts on human health. While gene expression profiling from blood transcriptomes has widely been used to assess health status and environmental exposures in human and veterinary medicine, its use in wildlife has been limited due to the lack of available genomes and baseline data. To this end we constructed the first beluga whale blood transcriptome de novo from samples collected during annual health assessments of the healthy Bristol Bay, AK stock during 2012-2014 to establish baseline information on the content and variation of the beluga whale blood transcriptome. The Trinity transcriptome assembly from beluga was comprised of 91,325 transcripts that represented a wide array of cellular functions and processes and was extremely similar in content to the blood transcriptome of another cetacean, the bottlenose dolphin. Expression of hemoglobin transcripts was much lower in beluga (25.6% of TPM, transcripts per million) than has been observed in many other mammals. A T12A amino acid substitution in the HBB sequence of beluga whales, but not bottlenose dolphins, was identified and may play a role in low temperature adaptation. The beluga blood transcriptome was extremely stable between sex and year, with no apparent clustering of samples by principle components analysis and <4% of genes differentially expressed (EBseq, FDR<0.05). While the impacts of season, sexual maturity, disease, and geography on the beluga blood transcriptome must be established, the presence of transcripts involved in stress, detoxification, and immune functions indicate that blood gene expression analyses may provide information on health status and exposure. This study provides a wealth of transcriptomic data on beluga whales and provides a sizeable pool of preliminary data for comparison with other studies in beluga whale.

Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations. Graphical abstract The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.

RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins.

BACKGROUND: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome. RESULTS: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters. CONCLUSIONS: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Global analysis of mRNA half-lives and de novo transcription in a dinoflagellate, Karenia brevis.

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.

Osmotic stress does not trigger brevetoxin production in the dinoflagellate Karenia brevis.

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.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

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. [(3) H]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.

Transcriptome remodeling associated with chronological aging in the dinoflagellate, Karenia brevis.

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.

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Liver genomic responses to ciguatoxin: evidence for activation of phase I and phase II detoxification pathways following an acute hypothermic response in mice.

Ciguatoxins (CTX) are polyether neurotoxins that target voltage-gated sodium channels and are responsible for ciguatera, the most common fish-borne food poisoning in humans. This study characterizes the global transcriptional response of mouse liver to a symptomatic dose (0.26 ng/g) of the highly potent Pacific ciguatoxin-1 (P-CTX-1). At 1 h post-exposure 2.4% of features on a 44K whole genome array were differentially expressed (p < or = 0.0001), increasing to 5.2% at 4 h and decreasing to 1.4% by 24 h post-CTX exposure. Data were filtered (/fold change/ > or = 1.5 and p < or = 0.0001 in at least one time point) and a trend set of 1550 genes were used for further analysis. Early gene expression was likely influenced prominently by an acute 4 degrees C decline in core body temperature by 1 h, which resolved by 8 h following exposure. An initial downregulation of 32 different solute carriers, many involved in sodium transport, was observed. Differential gene expression in pathways involving eicosanoid biosynthesis and cholesterol homeostasis was also noted. Cytochrome P450s (Cyps) were of particular interest due to their role in xenobiotic metabolism. Twenty-seven genes, mostly members of Cyp2 and Cyp4 families, showed significant changes in expression. Many Cyps underwent an initial downregulation at 1 h but were quickly and strongly upregulated at 4 and 24 h post-exposure. In addition to Cyps, increases in several glutathione S-transferases were observed, an indication that both phase I and phase II metabolic reactions are involved in the hepatic response to CTX in mice.