Biochemical Mapping of Pyrodinium bahamense Unveils Molecular Underpinnings behind Organismal Processes.

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.

Over 30 years of HABs in the Philippines and Malaysia: What have we learned?

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.

Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics.

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.

Paralytic Shellfish Toxin Uptake, Assimilation, Depuration, and Transformation in the Southeast Asian Green-Lipped Mussel (Perna viridis).

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.

Insights into the dynamics of harmful algal blooms in a tropical estuary through an integrated hydrodynamic-Pyrodinium-shellfish model.

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.

Potential DMSP-degrading Roseobacter clade dominates endosymbiotic microflora of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro.

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.

Thalassic biogas production from sea wrack biomass using different microbial seeds: cow manure, marine sediment and sea wrack-associated microflora.

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.

Paralytic shellfish toxin concentration and cell density changes in Pyrodinium bahamense -Noctiluca scintillans feeding experiments.

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.

Eutrophic waters, algal bloom and fish kill in fish farming areas in Bolinao, Pangasinan, Philippines.

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.

Growth response and toxin concentration of cultured Pyrodinium bahamense var. compressum to varying salinity and temperature conditions.

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.

Bacterial endosymbionts of Pyrodinium bahamense var. compressum.

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.

The community composition and production of phytoplankton in fish pens of Cape Bolinao, Pangasinan: a field study.

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.

Application of 210Pb-derived sedimentation rates and dinoflagellate cyst analyses in understanding Pyrodinium bahamense harmful algal blooms in Manila Bay and Malampaya Sound, Philippines.

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.