Differential allelopathic effects of mangrove plants Kandelia obovata and Aegiceras corniculatum on harmful algal species: Potential applications in algal bloom control.

This study examined effects of mangrove plants Kandelia obovata and Aegiceras corniculatum on harmful algal species. While A. corniculatum leaf extract had no inhibitory effect, K. obovata leaf extract significantly inhibited the growth of two harmful algal species Alexandrium tamarense and Karenia mikimotoi. The inhibitory effect was concentration-dependent, with over 90 % inhibition at the highest concentration. Morphological changes and cell size reduction were observed in both microalgae. Excessive production of reactive oxygen species and damage to algal photosynthetic system were found. The allelopathic effect of K. obovata on K. mikimotoi with low-concentration repeated exposure was more effective than high-concentration single exposure. The EC50 of K. obovata (0.33 g L-1) was lower than reported values on other coastal plants. Higher inhibitory effects of K. obovata were found on naked algal species than the armoured ones. These findings suggest potential applications of K. obovata leaf extract in controlling harmful algal blooms.

Revealing the algicidal characteristics of Maribacter dokdonensis: An investigation into bacterial strain P4 isolated from Karenia mikimotoi bloom water.

Harmful algal blooms (HABs) are a global environmental concern, causing significant economic losses in fisheries and posing risks to human health. Algicidal bacteria have been suggested as a potential solution to control HABs, but their algicidal efficacy is influenced by various factors. This study aimed to characterize a novel algicidal bacterium, Maribacter dokdonensis (P4), isolated from a Karenia mikimotoi (Hong Kong strain, KMHK) HAB and assess the impact of P4 and KMHK's doses, growth phase, and algicidal mode and the axenicity of KMHK on P4's algicidal effect. Our results demonstrated that the algicidal effect of P4 was dose-dependent, with the highest efficacy at a dose of 25% v/v. The study also determined that P4's algicidal effect was indirect, with the P4 culture and the supernatant, but not the bacterial cells, showing significant effects. The algicidal efficacy was higher when both P4 and KMHK were in the stationary phase. Furthermore, the P4 culture at the log phase could effectively kill KMHK cells at the stationary phase, with higher algicidal efficacy in the bacterial culture than that of the supernatant alone. Interestingly, P4's algicidal efficacy was significantly higher when co-culturing with xenic KMHK (~90% efficacy at day 1) than that with the axenic KMHK (~50% efficacy at day 1), suggesting the presence of other bacteria could regulate P4's algicidal effect. The bacterial strain P4 also exhibited remarkable algicidal efficacy on four other dinoflagellate species, particularly the armored species. These results provide valuable insights into the algicidal effect of M. dokdonensis on K. mikimotoi and on their interactions.

Intra- and inter-habitat variation in sediment heavy metals, antibiotics and ecological risks in Mai Po RAMSAR, China.

Distribution of heavy metals (HMs) and antibiotics (ABs) in surface sediments of three habitats: mudflat, mangrove and gei wai (inter-tidal shrimp ponds), at Mai Po RAMSAR were determined with inductively coupled plasma and liquid chromatograph tandem - mass spectrometry, respectively. Eight HMs (Cr, As, Pb, Cd, Mn, Ni, Cu and Zn), and ten ABs (tetracyclines, quinolones, macrolides and sulphonamides) were detected in all habitats, with relatively lower concentration in gei wai. Ecological risk assessment based on PNEC revealed that HMs posed a higher ecological risk to microorganisms than ABs. All metals except Mn were above their respective threshold effect levels according to sediment quality guidelines, indicating their potential toxicity to benthos. The enrichment factor and geo-accumulation index on background values suggested sediments were moderately polluted by Zn, Cu and Cd, possibly from anthropogenic inputs. This study implies that HMs pollution must be prevented through proper regulation of agricultural and industrial discharge.

Upregulation of Peridinin-Chlorophyll A-Binding Protein in a Toxic Strain of Prorocentrum hoffmannianum under Normal and Phosphate-Depleted Conditions.

Some strains of the dinoflagellate species Prorocentrum hoffmannianum show contrasting ability to produce diarrhetic shellfish poisoning (DSP) toxins. We previously compared the okadaic acid (OA) production level between a highly toxic strain (CCMP2804) and a non-toxic strain (CCMP683) of P. hoffmannianum and revealed that the cellular concentration of OA in CCMP2804 would increase significantly under the depletion of phosphate. To understand the molecular mechanisms, here, we compared and analyzed the proteome changes of both strains growing under normal condition and at phosphate depletion using two-dimensional gel electrophoresis (2-DE). There were 41 and 33 differential protein spots observed under normal condition and phosphate depletion, respectively, of which most were upregulated in CCMP2804 and 22 were common to both conditions. Due to the lack of matched peptide mass fingerprints in the database, de novo peptide sequencing was applied to identify the differentially expressed proteins. Of those upregulated spots in CCMP2804, nearly 60% were identified as peridinin-chlorophyll a-binding protein (PCP), an important light-harvesting protein for photosynthesis in dinoflagellates. We postulated that the high expression of PCP encourages the production of DSP toxins by enhancing the yields of raw materials such as acetate, glycolate and glycine. Other possible mechanisms of toxicity related to PCP might be through triggering the transcription of non-ribosomal peptide synthetase/polyketide synthase genes and the transportation of dinophysistoxin-4 from chloroplast to vacuoles.

Relationship between phytoplankton community and water parameters in planted fringing mangrove area in South China.

Reforestation has been commonly adopted to increase the mangrove areas lost due to developments. A diverse phytoplankton community is critical to mangrove ecosystem functions; however, its compositions in planted mangrove habitats have seldom been reported. The present study, based on the temporal and spatial variations of phytoplankton community and water parameters, evaluated their relationships in a 20-year old artificially planted fringing mangrove in South China. Thirty-one phytoplankton taxa were identified from tidal water collected from three sites along a planted mangrove shoreline: within, and at the edge of mangroves, and at bare shoreline without mangroves. In all three sites, Bacillariophyta (diatom) was the most abundant phylum, dominated by Navicula and Nitzschia at 9.82-83.76% and 2.57-33.97%, respectively. The overall diversity ranged between 0.41 and 1.94. The temporal variations of phytoplankton and water parameters were more obvious than site differences. Higher phytoplankton diversity was found in summer, with increased green algae and cyanobacteria abundance accompanied by riverine discharge. There was site-specific dominance in some phyla between June and January, noticeably Chlorophyta, Cyanophyta, Dinophyta, and Euglenophyta. A potential toxic dinoflagellate Peridinium sp. was identified during fall and winter, serving as a warning to probable harmful bloom events. Multivariate statistical analyses revealed the abundance of major phytoplankton taxa significantly related to water physiochemical parameters, especially nitrate, ammonium, total phosphorous and orthophosphate. The present study suggests that mangrove vegetation may influence water quality and shape phytoplankton composition, but temporal differences were more significant. Compared with the protected natural mangroves in the National Nature Reserve nearby, this planted fringing mangrove habitat not only had lower nutrients, especially total nitrogen in tidal water, but also had different dominant phytoplankton species and lower species richness. The 20-year old artificially planted mangroves may still not serve the same ecological function as the protected natural mangrove forest.

Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi.

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi's ichthyotoxicity.

Establish axenic cultures of armored and unarmored marine dinoflagellate species using density separation, antibacterial treatments and stepwise dilution selection.

Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.

How mangrove plants affect microplastic distribution in sediments of coastal wetlands: Case study in Shenzhen Bay, South China.

Microplastic pollution is common in marine and coastal ecosystems, especially in mangrove wetlands. However, factors affecting the distribution of microplastics, such as plants, have not been sufficiently studied. We investigated the effect of different plant species on the distribution of sediment microplastics in two Nature Reserves in South China, viz. Futian Mangrove and Mai Po Mangrove. In Futian Mangrove, the abundance of total microplastics among three monospecific mangrove stands dominated by Sonneratia caseolaris, Kandelia obovata, and Sonneratia apetala was similar. The abundance of microplastics in the mudflat was similar to that in the forest interior, except for the fact that more fiber was found in the mudflat than in the interior of Sonneratia apetala. This suggested that the dense pneumatophores at the fringe prevented fibers from entering the mangrove forest. The significant positive dependence (p < 0.05) between the density of Sonneratia pneumatophores and the abundance of fibers highlighted the importance of pneumatophores. The abundance of total microplastics, predominantly in the form of fibers, in sediments at the forest fringe (2835 ± 713 items/kg d.w. and 2070 ± 224 items/kg d.w. in Futian and Mai Po, respectively) was higher than that in the forest interior and mudflat. There was no difference between the two latter locations in both mangroves, which demonstrated the significance of the fringe effect. This paper reports for the first time that the spatial distribution of microplastics in mangrove sediments was affected by plant species, which provides useful information for environmental processes of microplastics in coastal wetlands.

Antibiotic-Resistant Bacteria in Hydroponic Lettuce in Retail: A Comparative Survey.

Hydroponic produce is gaining popularity due to its suitability for urban agriculture. The general public also considers that hydroponic produce is free from microbiological contamination. In this study, we compared the frequency and abundance of tetracycline-resistant and sulphadiazine-resistant bacteria and the minimal inhibitory concentration (MIC) of these isolates in conventional, organic, and hydroponic lettuce sold in retail. We also determined the frequency of samples carrying tetB, tetX, sul1, sul2, and int1 genes by PCR and further quantified the copy number of tetX, sul1, and int1 genes in samples positive for these genes using qPCR. As expected, the number of resistant bacteria and the MICs of these isolates were lowest in hydroponic lettuce and highest in organic lettuce. All tested resistant genes, except int1, were detected in samples of all three production methods, but no significant difference was observed between the three groups in the frequency of samples carrying the resistance genes examined or in their copy number. To the best of our knowledge, it is the first study directly reporting the existence of antibiotic-resistant bacteria and resistance genes in hydroponic vegetables sold in retail. The result highlights that the risk of antibiotic-resistant bacteria contamination in hydroponic produce should be further investigated.

Citizen science: An alternative way for water monitoring in Hong Kong.

Nowadays, citizen science has become increasingly popular, especially in Western countries. In Hong Kong, citizen science projects are mostly used for public education, while utilizing citizen scientists in published scientific research is very rare. On the other hand, with the increasing threats to global water security, Hong Kong requires new adaptation and strategy in facing the impairment of local freshwater systems. However, unfortunately, the number of full-scale urban river research appears to be declining. In this regard, citizen science can offer an alternative option as one of the new integrated water management strategies in Hong Kong. In this study, the water quality of seven rivers and streams in Hong Kong was studied monthly for two years by a group of citizen scientists. The main goal is to examine the reliability of data collected by citizen scientists by comparing it with the official data from the Environmental Protection Department of Hong Kong (EPD). Results show that the water temperature and conductivity data acquired by the citizen scientists were highly comparable to the official data. Also, moderate to strong correlations in water pH, turbidity, and dissolved oxygen levels were found between citizen scientists and official data. Since the citizen science data remained as high as 70% of relevance to the official data, we believe that this may serve as a supplement to the lacking official or professional water quality monitoring data in Hong Kong. Even though the use of volunteer data in water quality monitoring unavoidably exists with errors and bias, this study demonstrates a successful outcome of utilizing citizen science programme in urban river monitoring in Hong Kong.

Production of Paralytic Shellfish Toxins (PSTs) in Toxic Alexandrium catenella is Intertwined with Photosynthesis and Energy Production.

To investigate the mechanism for the production of paralytic shellfish toxins (PST) in toxic dinoflagellates, with a 2D-gel based approach, we had made two sets of proteomic comparisons: (a) between a toxic Alexandrium catenella (AC-T) and a phylogenetically closely related non-toxic strain (AC-N), (b) between toxic AC-T grown in a medium with 10% normal amount of phosphate (AC-T-10%P) known to induce higher toxicity and AC-T grown in normal medium. We found that photosynthesis and energy production related proteins were up-regulated in AC-T when compared to AC-N. However, the same group of proteins was down-regulated in AC-T-10%P when compared to normal AC-T. Examining the relationship of photosynthesis and toxin content of AC-T upon continuous photoperiod experiment revealed that while growth and associated toxin content increased after 8 days of continuous light, toxin content maintained constant when cells were shifted from continuous light to continuous dark for 3 days. This emphasized the cruciality of light availability on toxin biosynthesis in AC-T, while another light-independent mechanism may be responsible for higher toxicity in AC-T-10%P compared to normal AC-T. Taken all together, it is believed that the interplay between "illumination", "photosynthesis", "phosphate availability", and "toxin production" is much more complicated than what we had previously anticipated.

Production of high-quality two-dimensional gel electrophoresis profile for marine medaka samples by using Trizol-based protein extraction approaches.

BACKGROUND: Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. METHODS: Three Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches. RESULTS: Any of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins. CONCLUSIONS: Trizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile.

Optimization of protein extraction and two-dimensional gel electrophoresis profiles for the identification of Cordyceps sinensis and other similar species.

Given that Chinese materia medica (CMM) is expensive and rare, people take tremendous risk to adulterate and falsify Cordyceps sinensis with counterfeit species with similar morphological features. It is thus essential to develop new methods to identify the authenticity of Cordyceps sinensis. It is hypothesized in this study that Cordyceps sinensis possesses certain protein biomarkers distinct from its counterfeits, which can be identified by proteomic technologies for authentication purposes. This is the first study that aims to optimize the conditions for extracting proteins from Cordyceps sinensis, a hybrid of fungal-animal CMM, and to compare the two-dimensional gel electrophoresis (2-DE) profiles between different Cordyceps species. Two different protein extraction buffer systems, namely, phenol/sodium dodecyl sulfate (SDS) buffer or lysis buffer, were evaluated, where the preparation using lysis buffer yielded better protein content. The results also showed that extraction with lysis buffer without pre- or post-washing of samples was the most effective protocol, with over 220% of protein yield and 819 protein spots detected on a 2-DE gel. Moreover, the results demonstrated that Cordyceps sinensis possesses protein biomarkers distinct from its counterfeits, and these biomarkers are not source- or origin-dependent, strongly supporting the feasibility of using identified biomarkers as indicators for authentication of Cordyceps species. The findings of this study warrant further investigations on the structural identification of protein biomarkers of Cordyceps species.

Evaluation of the Use of TRIzol-Based Protein Extraction Approach for Gel-Based Proteomic Analysis of Dried Seafood Products and Chinese Tonic Foods.

Although the emergence of gel-free approaches has greatly enhanced proteomic studies, two-dimensional gel electrophoresis (2-DE) remains one of the most widely used proteomic techniques for its high resolving power, relatively low cost, robustness, and high resolution. Preparation of high-quality protein samples remains the key in high-quality 2-DE for proteomic analysis. Samples with high endogenous levels of interfering molecules, such as salts, nucleic acids, lipids, and polysaccharides, would yield a low-quality 2-DE gel and hinder the analysis. Recently, a TRIzol-based protein extraction method has gained prominence and has attracted attention due to its promising performance in high-quality 2-DE. The authors evaluate the use of this approach for four valuable dried food products, namely two dried seafood products (abalone slices and whelk slices) and two traditional Chinese tonic foods (ganoderma and caterpillar fungus). The results indicate that 2-DE gels obtained through the TRIzol-based method are of high-quality and are comparable to those obtained through the trichloroacetic acid⁻acetone method in terms of spot number, spot intensity, and resolution. The TRIzol-based method is generally applicable to dried food samples and is simple and fast, which greatly streamlines the protein extraction procedure. Additionally, it enables the concurrent extraction and analysis of RNA, DNA, and protein from the same sample.

The Mechanism of Diarrhetic Shellfish Poisoning Toxin Production in Prorocentrum spp.: Physiological and Molecular Perspectives.

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disorder caused by the consumption of seafood contaminated with okadaic acid (OA) and dinophysistoxins (DTXs). OA and DTXs are potent inhibitors of protein phosphatases 2A, 1B, and 2B, which may promote cancer in the human digestive system. Their expression in dinoflagellates is strongly affected by nutritional and environmental factors. Studies have indicated that the level of these biotoxins is inversely associated with the growth of dinoflagellates at low concentrations of nitrogen or phosphorus, or at extreme temperature. However, the presence of leucine or glycerophosphate enhances both growth and cellular toxin level. Moreover, the presence of ammonia and incubation in continuous darkness do not favor the toxin production. Currently, studies on the mechanism of this biotoxin production are scant. Full genome sequencing of dinoflagellates is challenging because of the massive genomic size; however, current advanced molecular and omics technologies may provide valuable insight into the biotoxin production mechanism and novel research perspectives on microalgae. This review presents a comprehensive analysis on the effects of various nutritional and physical factors on the OA and DTX production in the DSP toxin-producing Prorocentrum spp. Moreover, the applications of the current molecular technologies in the study on the mechanism of DSP toxin production are discussed.

Quantification of phytoplankton bloom dynamics by citizen scientists in urban and peri-urban environments.

Freshwater ecosystems are severely threatened by urban development and agricultural intensification. Increased occurrence of algal blooms is a main issue, and the identification of local dynamics and drivers is hampered by a lack of field data. In this study, data from 13 cities (250 water bodies) were used to examine the capacity of trained community members to assess elevated phytoplankton densities in urban and peri-urban freshwater ecosystems. Coincident nutrient concentrations and land use observations were used to examine possible drivers of algal blooms. Measurements made by participants showed a good relationship to standard laboratory measurements of phytoplankton density, in particular in pond and lake ecosystems. Links between high phytoplankton density and nutrients (mainly phosphate) were observed. Microscale observations of pollution sources and catchment scale estimates of land cover both influenced the occurrence of algal blooms. The acquisition of environmental data by committed and trained community members represents a major opportunity to support agency monitoring programmes and to complement field campaigns in the study of catchment dynamics.

Toxicity and estrogenic endocrine disrupting activity of phthalates and their mixtures.

Phthalates, widely used in flexible plastics and consumer products, have become ubiquitous contaminants worldwide. This study evaluated the acute toxicity and estrogenic endocrine disrupting activity of butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), bis(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate (DIDP), diisononyl phthalate (DINP), di-n-octyl phthalate (DNOP) and their mixtures. Using a 72 h zebrafish embryo toxicity test, the LC50 values of BBP, DBP and a mixture of the six phthalates were found to be 0.72, 0.63 and 0.50 ppm, respectively. The other four phthalates did not cause more than 50% exposed embryo mortality even at their highest soluble concentrations. The typical toxicity symptoms caused by phthalates were death, tail curvature, necrosis, cardio edema and no touch response. Using an estrogen-responsive ChgH-EGFP transgenic medaka (Oryzias melastigma) eleutheroembryos based 24 h test, BBP demonstrated estrogenic activity, DBP, DEHP, DINP and the mixture of the six phthalates exhibited enhanced-estrogenic activity and DIDP and DNOP showed no enhanced- or anti-estrogenic activity. These findings highlighted the developmental toxicity of BBP and DBP, and the estrogenic endocrine disrupting activity of BBP, DBP, DEHP and DINP on intact organisms, indicating that the widespread use of these phthalates may cause potential health risks to human beings.

Effective growth of dinoflagellate Prorocentrum minimum by cultivating the cells using municipal wastewater as nutrient source.

Several studies have been conducted worldwide in order to develop a more economical method for mass algal cultivation so that more cost-effective biomass production can be accessed. One of the directions is to reduce production costs by using wastewater as a nutrient source in algal cell cultivation. The growth ability of a red-tide causative dinoflagellate species, Prorocentrum minimum, in various concentrations of local urban wastewater was examined in this study. The highest exponential growth rate and maximum cell density (MCD) were achieved when autoclaved 10% wastewater was used for cell cultivation, although the cells could survive in 0.01-100% wastewater. Both growth rate and MCD of the cells in wastewater were found to be substantially higher than that in optimized L1 culture medium. This research highlights the potential of using wastewater as a cost-effective approach for mass cultivation of dinoflagellate cells with consequent production of valuable microalgal biomass.

Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage.

Nitrate (N) and phosphate (P) are believed to be two of the most important nutrients for the growth and toxin production for Alexandrium species. The study of the growth and toxicity characteristics of the Alexandrium spp. under the change of N and P can help us to understand the dynamics of algal bloom and toxification events in natural environments. A strain of Alexandrium tamarense (designated as Kci) was successfully isolated from the Drake Passage in 2001 and the clonal culture has been kept in our laboratory (Ho et al., 2003, 2012). In order to extend our understanding on the growth physiology and toxicity of this A. tamarense strain, growth and cellular toxin content were examined in unialgal batch cultures under different concentrations of N and P. The effects of variable N, P concentrations on growth, cellular toxicity (fg STXeq. cell(-1)), and toxin composition (% molar) were determined in both exponential and stationary growth phases. The toxin profile, determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), was found to be remained relatively stable and was consistently dominated by the N-sulfocarbamoyl C-toxins (>90%) under different conditions and growth phases. There were also trace amounts of other carbamate gonyautoxins consistently expressed. The cellular toxicity varied under different N and P concentrations, as well as different growth stages. A positive correlation was observed between cellular toxicity and N concentrations, but the toxicity was enhanced when P was depleted. Both cell densities and growth rate of the cells were severely suppressed under N- or P-depletion. However, the biovolume of the cells tended to be larger at N- or P-depleted cultures. Results from the present study provide valuable insight for the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage.

Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles.

Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01) was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX). Based on the Microbial Identification (MIDI) and 16S-23S rDNA internal transcribed spacer (ITS) phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF). Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.