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.

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.

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.

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.

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.