RESUMO
Proteins, lipids, and carbohydrates from the harmful algal bloom (HAB)-causing organism Pyrodinium bahamense were characterized to obtain insights into the biochemical processes in this environmentally relevant dinoflagellate. Shotgun proteomics using label-free quantitation followed by proteome mapping using the P. bahamense transcriptome and translated protein databases of Marinovum algicola, Alexandrium sp., Cylindrospermopsis raciborskii, and Symbiodinium kawagutii for annotation enabled the characterization of the proteins in P. bahamense. The highest number of annotated hits were obtained from M. algicola and highlighted the contribution of microorganisms associated with P. bahamense. Proteins involved in dimethylsulfoniopropionate (DMSP) degradation such as propionyl CoA synthethase and acryloyl-CoA reductase were identified, suggesting the DMSP cleavage pathway as the preferred route in this dinoflagellate. Most of the annotated proteins were involved in amino acid biosynthesis and carbohydrate degradation and metabolism, indicating the active roles of these molecules in the vegetative stage of P. bahamense. This characterization provides baseline information on the cellular machinery and the molecular basis of the ecophysiology of P. bahamense.
Assuntos
Dinoflagellida/metabolismo , Proliferação Nociva de Algas , Compostos de Sulfônio/metabolismo , Dinoflagellida/genéticaRESUMO
Many aspects of the biology and ecology of the toxic dinoflagellate Pyrodinium bahamense var. compressum are still poorly understood. In this brief note, we present identification of its associated intracellular bacteria or endosymbionts via PCR cloning and 16s rRNA gene sequencing and their localization by confocal microscopy, a first for Pyrodinium. The most frequently observed species in the endosymbiotic microflora were from Roseobacter clade (Alphaproteobacteria, 68%) and Gilvibacter sediminis (Flavobacteriaceae, 20%). Roseobacter lineage, the most abundant taxa in this study, is known to be involved in dimethylsulfoniopropionate metabolism which is highly produced in dinoflagellates-a possible strong factor shaping the structure of the associated bacterial community.
Assuntos
Dinoflagellida/microbiologia , Roseobacter/fisiologia , RNA Ribossômico 16S/genética , Roseobacter/genética , Roseobacter/isolamento & purificação , Roseobacter/metabolismo , Compostos de Sulfônio/metabolismo , SimbioseRESUMO
In the Southeast Asian region, the Philippines and Malaysia are two of the most affected by Harmful Algal Blooms (HABs). Using long-term observations of HAB events, we determined if these are increasing in frequency and duration, and expanding across space in each country. Blooms of Paralytic Shellfish Toxin (PST)-producing species in the Philippines did increase in frequency and duration during the early to mid-1990s, but have stabilized since then. However, the number of sites affected by these blooms continue to expand though at a slower rate than in the 1990s. Furthermore, the type of HABs and causative species have diversified for both toxic blooms and fish kill events. In contrast, Malaysia showed no increasing trend in the frequency of toxic blooms over the past three decades since Pyrodinium bahamense was reported in 1976. However, similar to the Philippines, other PST producers such as Alexandrium minutum and Alexandrium tamiyavanichii have become a concern. No amnesic shellfish poisoning (ASP) has been confirmed in either Philippines or Malaysia thus far, while ciguatera fish poisoning cases are known from the Philippines and Malaysia but the causative organisms remain poorly studied. Since the 1990s and early 2000s, recognition of the distribution of other PST-producing species such as species of Alexandrium and Gymnodinium catenatum in Southeast Asia has grown, though there has been no significant expansion in the known distributions within the last decade. A major more recent problem in the two countries and for Southeast Asia in general are the frequent fish-killing algal blooms of various species such as Prorocentrum cordatum, Margalefidinium polykrikoides, Chattonella spp., and unarmored dinoflagellates (e.g., Karlodinium australe and Takayama sp.). These new sites affected and the increase in types of HABs and causative species could be attributed to various factors such as introduction through mariculture and eutrophication, and partly because of increased scientific awareness. These connections still need to be more concretely investigated. The link to the El Niño Southern Oscillation (ENSO) should also be better understood if we want to discern how climate change plays a role in these patterns of HAB occurrences.
Assuntos
Dinoflagellida , Intoxicação por Frutos do Mar , Animais , Proliferação Nociva de Algas , Malásia , FilipinasRESUMO
Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10-12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis. The complementary proteomics approach provided insights into the biochemistry of these two closely related HAB-causing organisms. The identified proteins are potential biomarkers for organismal toxicity and could be explored for environmental monitoring.
Assuntos
Dinoflagellida/metabolismo , Proteômica/métodos , Dinoflagellida/genética , Regulação da Expressão Gênica , Proliferação Nociva de Algas , Toxinas Marinhas/toxicidade , Especificidade da EspécieRESUMO
Bivalve molluscs represent an important food source within the Philippines, but the health of seafood consumers is compromised through the accumulation of harmful algal toxins in edible shellfish tissues. In order to assess the dynamics of toxin risk in shellfish, this study investigated the uptake, depuration, assimilation, and analogue changes of paralytic shellfish toxins in Perna viridis. Tank experiments were conducted where mussels were fed with the toxic dinoflagellate Alexandrium minutum. Water and shellfish were sampled over a six day period to determine toxin concentrations in the shellfish meat and water, as well as algal cell densities. The maximum summed toxin concentration determined was 367 µg STX eq./100 g shellfish tissue, more than six times higher than the regulatory action limit in the Philippines. Several uptake and depuration cycles were observed during the study, with the first observed within the first 24 h coinciding with high algal cell densities. Toxin burdens were assessed within different parts of the shellfish tissue, with the highest levels quantified in the mantle during the first 18 h period but shifting towards the gut thereafter. A comparison of toxin profile data evidenced the conversion of GTX1,4 in the source algae to the less potent GTX2,3 in the shellfish tissue. Overall, the study illustrated the temporal variability in Perna viridis toxin concentrations during a modelled algal bloom event, and the accumulation of toxin from the water even after toxic algae were removed.
Assuntos
Bivalves/metabolismo , Toxinas Marinhas/metabolismo , Frutos do Mar/análise , Animais , Transporte Biológico , Proliferação Nociva de Algas , Toxinas Marinhas/farmacocinéticaRESUMO
The coastal waters of Bolinao, Pangasinan, Philippines experienced environmental changes over a 10-year period (1995-2005), the most significant effect of which was the major fish kill event in 2002 that coincided with the first reported Philippine bloom of a dinoflagellate Prorocentrum minimum. Days before the bloom, dissolved oxygen was < 2.0 mg/l in the waters that were stratified. These conditions may be linked to the uncontrolled proliferation of fish pens and cages to more than double the allowable limit of 544 units for Bolinao waters. Mariculture activities release organic matter from unconsumed feed and fecal material that accumulate in the water and sediments. In over 10 years, water quality conditions have become eutrophic with ammonia increasing by 56%, nitrite by 35%, nitrate by 90%, and phosphate by 67%. The addition of more fish pens and cages placed additional stress to this poorly flushed, shallow area that affected water quality due to changes in the water residence time.
Assuntos
Eutrofização , Pesqueiros , Peixes/fisiologia , Animais , Diatomáceas/fisiologia , Dinoflagellida/fisiologia , Oxigênio/análise , Oceano Pacífico , Filipinas , Plâncton/classificação , Plâncton/fisiologia , Densidade Demográfica , Água do Mar/químicaRESUMO
The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.
Assuntos
Carbono/química , Ecossistema , Fertilizantes/análise , Ureia/química , Ureia/farmacologia , Efeito Estufa , Oceanos e MaresRESUMO
Morphological and phylogenetic analysis showed that the Gambierdiscus isolate from Bolinao, Philippines belongs to the species of G. carpenteri. It was morphologically more similar to the Merimbula strain than the subtropical Florida Keys strain. Growth and toxin production were also investigated at varying levels of temperature, salinity, and irradiance. Gambierdiscus are known to grow favorably in a low light environment. However, this study showed high growth rates of G. carpenteri even at high irradiance levels. Generally, cells produced more toxins at lower treatment levels. Highest cellular toxin content recorded was 7.48⯱â¯0.49â¯pgâ¯Pbtxâ¯eq/cell at culture conditions of 25⯰C, 100⯵molâ¯photonsâ¯m-2â¯s-1, and salinity of 26. Growth rate and toxin production data suggest that cells produced more toxins during the slowest growth at certain range of treatments. This information gives insight into how changes in environmental conditions may affect toxin production and growth of G. carpenteri.
Assuntos
Dinoflagellida/classificação , Dinoflagellida/crescimento & desenvolvimento , Toxinas Marinhas/metabolismo , Dinoflagellida/metabolismo , Ecossistema , Filipinas , Filogenia , Salinidade , Temperatura , Clima TropicalRESUMO
In contrast to temperate Harmful Algal Blooms (HABs), knowledge on the mechanisms driving tropical HABs are less well studied. The interaction of a seasonal temperature window, cysts (for certain species) and large-scale transport are some of the key processes in temperate HABs. In the Philippines, HABs occur not along long open coastlines, but in embayments that are highly influenced by run-off and stratification. These embayments are typically also the sites of cultured or wild harvest shellfish and other aquaculture activities. Sorsogon Bay in the northeastern Philippines has experienced prolonged shellfish-harvesting bans due to blooms by Pyrodinium bahamense var. compressum severely affecting the fisheries industry in this area, as well as leading to Paralytic Shellfish Poisoning illnesses and fatalities. A novel integrated model was developed that mechanistically captures the interactions between hydrodynamic conditions, nutrients, the life history (cells and cysts) of Pyrodinium, as well as the cultured shellfish within the bay and their ensuing toxicities due to ingestion of toxic Pyrodinium cells and cysts. This is the second model developed for HABs in the Philippines, and the first to integrate different components of Pyrodinium bloom dynamics. The model is modularly composed of a watershed nutrient and diffusion model, a 3D hydrodynamic model, a Pyrodinium population model and a shellfish toxin model. It was able to capture the observed temporal variations of Pyrodinium and shellfish toxicity. It was also able to represent some aspects of the spatial distribution in Sorsogon Bay though there were discrepancies. To explore the dynamics of blooms, the linkages between the bloom and decline of the Pyrodinium population with shellfish toxicity as affected by temperature, salinity and nutrients were investigated. Comparisons with field results showed the seasonality of blooms in Sorsogon Bay is driven by increased rainfall. The timing of these conditions is important in facilitating Pyrodinium excystment and reproduction. Model results showed as well the potential significance of shellfish grazing and dinoflagellate cell mortality in influencing the decline of the bloom, and toxicity levels. This approach is promising in helping to understand mechanisms for HABs more holistically, and the model can be further improved to provide more precise quantitative information.
Assuntos
Dinoflagellida/fisiologia , Estuários , Proliferação Nociva de Algas , Modelos Teóricos , Ecossistema , Hidrodinâmica , Toxinas Marinhas , Nitrogênio/análise , Temperatura , Água/química , Movimentos da ÁguaRESUMO
The growth and toxin production of a Philippine Pyrodinium bahamense isolate in nutrient replete batch cultures were investigated under conditions affected by varying salinity, temperature and combined effects of salinity and temperature. Early exponential growth stage was reached after 7 days with a cell division rate of 0.26 div day(-1). The toxin content reached a peak of 298 fmol cell-1 at mid exponential phase and rapidly declined to 54 fmol cell-1 as it approached the death phase. Only three sets of toxins composed of STX, dcSTX and B1 were detected in which STX made up to 85-98 mol%toxincell-1. P. bahamense was able to grow in salinities and temperatures ranging from 26 per thousand to 36 per thousand and 23 to 36 degrees C, respectively. The optimum growth under varying salinity and temperature conditions was observed at 36 per thousand and 25 degrees C. Toxin content reached a peak of 376 fmol cell-1 at 25 degrees C and was lower (80-116 fmol cell-1) at higher temperatures (32-35 degrees C). Combined effects of salinity and temperature showed that P. bahamense was not able to grow at low salinity and temperature (i.e. below 26 per thousand-28 degrees C). Optimum growth was observed in higher salinities at all temperature conditions.
Assuntos
Dinoflagellida/crescimento & desenvolvimento , Dinoflagellida/patogenicidade , Toxinas Marinhas/biossíntese , Cloreto de Sódio/farmacologia , Animais , Meios de Cultura , Toxinas Marinhas/análise , TemperaturaRESUMO
From 1995 up to the present, fish pens proliferated in the municipal waters of Bolinao, northern Philippines. Since then, fish kills and phytoplankton blooms have been recurrent. Have fishpens altered the phytoplankton community composition and production of these waters? The phytoplankton community in Cape Bolinao, Lingayen Gulf is typical of a tropical coastal area where diatoms alternate with dinoflagellates during the dry and wet seasons. In the nutrient-rich fish pens, phytoplankton in this study showed a lower diatom/dinoflagellate ratio and unusually high phytoplankton counts of 10(4) cells/l and even as high as 10(5) cells/l. Correlations between physico-chemical parameters, phytoplankton production and community composition were made in 2001. This paper tried to explain the occurrence of a Cylindrotheca closterium bloom (10(5) cells/l), during the dry season of the same year and a Prorocentrum minimum bloom (4.7 x 10(5) cells/l), which accompanied a massive fish kill during January 2002.
Assuntos
Aquicultura , Eutrofização , Fitoplâncton/crescimento & desenvolvimento , Animais , Dinoflagellida/crescimento & desenvolvimento , Monitoramento Ambiental , Peixes , Mortalidade , Filipinas , Clima TropicalRESUMO
The number of areas affected by toxic harmful algal bloom (HAB) in the Philippines has been increasing since its first recorded occurrence in 1983. Thus far, HAB has been reported in about 20 areas in the Philippines including major fishery production areas. The HAB-causing organism (Pyrodinium bahamense var. compressum) produces a cyst during its life cycle. Pyrodinium cysts which are deposited in the sediment column may play a role in initiating a toxic bloom. 210Pb-derived sedimentation rate studies in the two important fishing grounds of Manila Bay and Malampaya Sound, Palawan have shown that Pyrodinium cysts may have been present in the sediment even before the first recorded toxic algal bloom in these areas. High sedimentation rates (approximately 1 cm/year) have been observed in the northern and western parts of Manila Bay. The results indicate that the sedimentation processes occurring in these bays would require subsurface cyst concentration analysis in evaluating the potential of an area to act as seed bed.
Assuntos
Dinoflagellida/crescimento & desenvolvimento , Eutrofização , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiologia , Animais , Dinoflagellida/química , Monitoramento Ambiental/métodos , Pesqueiros , Radioisótopos de Chumbo/análise , Oocistos , FilipinasRESUMO
Sea wrack (dislodged sea grasses and seaweeds) was used in biogas production. Fresh water scarcity in island communities where sea wrack could accumulate led to seawater utilization as liquid substrate. Three microbial seeds cow manure (CM), marine sediment (MS), and sea wrack-associated microflora (SWA) were explored for biogas production. The average biogas produced were 2172±156 mL (MS), 1223±308 mL (SWA) and 551±126 mL (CM). Though methane potential (396.9 mL(CH4) g(-1) volatile solid) computed from sea wrack proximate values was comparable to other feedstocks, highest methane yield was low (MS=94.33 mL(CH4) g(-1) VS). Among the microbial seeds, MS proved the best microbial source in utilizing sea wrack biomass and seawater. However, salinity (MS=42) observed exceeded average seawater salinity (34). Hence, methanogenic activity could have been inhibited. This is the first report on sea wrack biomass utilization for thalassic biogas production.
Assuntos
Bactérias/metabolismo , Biocombustíveis/microbiologia , Biomassa , Sedimentos Geológicos/microbiologia , Esterco/microbiologia , Alga Marinha/metabolismo , Alga Marinha/microbiologia , Animais , Bovinos , Fermentação , Metano/biossínteseRESUMO
For the first time the potential of Noctiluca scintillans, a non-toxic mixotrophic dinoflagellate, in bioconverting and/or excreting saxitoxin has been illustrated, thus contributing to the limited knowledge on the aspects of toxin pathways in the food chain/web and predator-prey preferences. Noctiluca growth rate increased with higher Pyrodinium concentration but the ratio of Noctiluca to Pyrodinium should at least be 1:250 cells per mL. Noctiluca fed with Pyrodinium alone was found to decrease in number suggesting that the nutrients from this prey were insufficient. This was confirmed by the improved cell density of Noctiluca upon addition of 0.01% casitone to the Pyrodinium-fed Noctiluca. The alternative prey (Gymnodinium sanguineum) slowed down the grazing impact of Noctiluca on Pyrodinium. Noctiluca depleted Gymnodinium earlier than Pyrodinium showing preference over a prey with less saxitoxin. After the feeding experiments, total saxitoxin levels decreased to 72% in the Noctiluca-Pyrodinium setup whereas no saxitoxin was detected in the Noctiluca culture fed with Pyrodinium and G. sanguineum. It is possible that Gymnodinium can provide some nutrients needed to make Noctiluca more efficient in bioconverting saxitoxin.
Assuntos
Dinoflagellida/metabolismo , Proliferação Nociva de Algas , Saxitoxina/metabolismo , Intoxicação por Frutos do Mar , Animais , Caseínas/administração & dosagem , Contagem de Células , Dinoflagellida/efeitos dos fármacos , Dinoflagellida/crescimento & desenvolvimento , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Cadeia Alimentar , Saxitoxina/análiseRESUMO
The study presents evidence in support of the bacterial theory associated with the toxicity of Pyrodinium bahamense var. compressum. Bacterial endosymbionts from Philippine P. bahamense var. compressum strain Pbc MZRVA 042595 were isolated and identified via 16S rDNA sequence analysis. Taxonomic diversity of the identified culturable intracellular microbiota associated with Philippine P. bahamense var. compressum was established to be limited to the Phyla Proteobacteria, Actinobacteria, and Firmicutes. Major endosymbionts identified included Moraxella spp., Erythrobacter spp., and Bacillus spp., whereas Pseudomonas putida, Micrococcus spp., and Dietzia maris were identified as minor isolates. All identified strains except D. maris, P. putida, and Micrococcus spp. were shown to contain either saxitoxin or neo saxitoxin or both at levels < or =73 ng/10(7) bacterial cells based on high-performance liquid chromatography analysis. Paralytic shellfish poisoning-like physiologic reactions in test animals used in the mouse assay were recorded for the endosymbionts except for P. putida. The study is the first to elucidate the possible contribution of bacterial endosymbionts in the toxicity of P. bahamense var. compressum isolated in the Philippines.