Alkaline phosphatase activities and regulation in three harmful Prorocentrum species from the coastal waters of the East China Sea.

Harmful blooms of Prorocentrum donghaiense occur annually in the phosphorus-scarce coastal waters of the East China Sea (ECS). The enzymatic activities of alkaline phosphatase (AP) and its regulation by external phosphorus were studied during a P. donghaiense bloom in this area. The AP characteristics of P. donghaiense was further compared with Prorocentrum minimum and Prorocentrum micans in monocultures with both bulk and single-cell enzyme-labeled fluorescence AP assays. Concentrations of dissolved inorganic phosphorus (DIP) varied between 0.04 and 0.73 µmol l-1, with more than half recording stations registering concentrations below 0.10 µmol l-1. Concentrations of dissolved organic phosphorus (DOP) were comparable or even higher than those of DIP. P. donghaiense suffered phosphorus stress and expressed abundant AP, especially when DIP was lower than 0.10 µmol l-1. The AP activities showed a negative correlation with DIP but a positive correlation with DOP. The AP activities were also regulated by internal phosphorus pool. The sharp increase in AP activities was observed until cellular phosphorus was exhausted. Most AP of P. donghaiense was located on the cell surface and some were released into the water with time. Compared with P. minimum and P. micans, P. donghaiense showed a higher AP affinity for organic phosphorus substrates, a more efficient and energy-saving AP expression quantity as a response to phosphorus deficiency. The unique AP characteristic of P. donghaiense suggests that it benefits from the efficient utilization of DOP, and outcompete other species in the phosphorus-scarce ECS.

Emerging harmful algal bloom species over the last four decades in China.

The first recorded micro-algae bloom in Chinese coastal waters dates back to 1933 and was caused by a mixture of Noctiluca scintillans and Skeletonema costatum sensu lato along the Zhejiang coast (the East China Sea). While well-documented harmful algal blooms (HABs) appeared to be extremely scarce from the 1950s to 1990, both the frequency and intensity have been reportedly increasing since 1990. Among them, the fish-killing HABs, mainly caused by Karenia mikimotoi, Karlodinium digitatum, Karlodinium veneficum, Margalefidinium polykrikoides, and Heterocapsa spp., have intensified. Karenia mikimotoi was responsible for at least two extremely serious events in the Pearl River Estuary in 1998 and the Taiwan Strait (in the East China Sea) in 2012, which appeared to be associated with abnormal climate conditions and excessive nutrients loading. Other major toxic algal blooms have been caused by the species responsible for paralytic shellfish poisoning (including Alexandrium catenella, Alexandrium pacificum, Gymnodinium catenatum) and diarrhetic shellfish poisoning (including Dinophysis spp., and a couple of benthic dinoflagellates). Consequent closures of shellfish farms have resulted in enormous economic losses, while consumption of contaminated shellfish has led to occasional human mortality in the Bohai Sea and the East China Sea. Expansions of these HABs species along the coastline of China have occurred over the last four decades and, due to the projected global changes in the climate and marine environments and other anthropological activities, there is potential for the emergence of new types of HABs in China in the future. This literature review aimed to present an updated overview of HABs species over the last four decades in China.

Bioavailability of Organic Phosphorus Compounds to the Harmful Dinoflagellate Karenia mikimotoi.

Karenia mikimotoi is one of the most well-known harmful bloom species in temperate coastal waters. The present study investigated the characteristics of alkaline phosphatase (APase) and phosphodiesterase (PDEase) activities in hydrolysis of two phosphomonoesters (adenosine triphosphate (ATP) and ribulose 5-phosphate (R5P)) and a phosphodiester (cyclic adenosine monophosphate (cAMP)) in K. mikimotoi and compared its growth and physiological responses to the different forms of phosphorus substrates. K. mikimotoi produced comparable quantities of APase and PDEase to hydrolyze the organic phosphorus substrates, although hydrolysis of the phosphomonoesters was much faster than that of the phosphodiester. The growth of K. mikimotoi on organic phosphorus substrates was comparable to or better than that on inorganic phosphate. The difference in particulate organic nutrients (carbon, nitrogen, and phosphorus) and hemolytic activity supported different rates of hydrolysis-assimilation of the various organic phosphorus substrates by K. mikimotoi. The hemolytic activities of K. mikimotoi in the presence of organic phosphorus substrates were several times those in the presence of inorganic phosphate during the exponential phase. This suggested the potential important role of organic phosphorus in K. mikimotoi blooms.

Exploration of resting cysts (stages) and their relevance for possibly HABs-causing species in China.

The studies on the species diversity, distribution, environmental implications, and molecular basis of resting cysts (stages) of dinoflagellates and a few species of other groups conducted in China during the last three decades are reviewed. The major achievements are summarized as the following five aspects: 1) The continual efforts in detecting the species diversity of resting cysts (spores) in dinoflagellates and other classes using either morphological or molecular approaches, or both, in the four seas of China, which led to identifications of 106 species of dinoflagellate resting cysts and 4 species of resting stages from other groups of microalgae, with a total of 64 species of dinoflagellate cysts and the resting stage of the brown tide-causing Aureococcus anophagefferens being unequivocally identified via molecular approaches from the sediments of Chinese coastal waters; 2) The well-known toxic and HABs-causing dinoflagellates Karenia mikimotoi, Karlodinium veneficum, Akashiwo sanguinea and the pelagophyte A. anophagefferens were proven to be resting cyst (stage) producers via laboratory studies on their life cycles and field detections of resting cysts (resting stage cells). And, via germination experiment and subsequent characterization of vegetative cells, numerous dinoflagellate species that had never been described or found to form cysts were discovered and characterized; 3) The distributions of the resting cysts of Alexandrium catenella, A. pacificum, Gymnodinium catenatum, K. mikimotoi, K. veneficum and Azadinium poporum and the resting stage cells of A. anophagefferens were morphologically and molecularly mapped in all four seas of China, with A. anophagefferens proven to have been present in the Bohai Sea for at least 1,500 years; 4) Obtaining important insights into the 'indicator' values of the dinoflagellate cyst assemblages in sediment cores for tracking eutrophication, environmental pollution and other anthropological influences in coastal waters; 5) Studies on the cyst-pertinent processes and genetic basis (transcriptomics together with physiological and chemical measurements) of resting cyst dormancy not only revealed the regulating patterns of some environmental factors in cyst formation and germination, but also identified many characteristically active or inactive metabolic pathways, differentially expressed genes, and the possibly vital regulating function of the phytohormone abscisic acid and a group of molecular chaperones in resting cysts. We also identified seven issues and three themes that should be addressed and explored by Chinese scientists working in the area in the future.

Alkaline phosphatase activity during a phosphate replete dinoflagellate bloom caused by Prorocentrum obtusidens.

Prorocentrum obtusidens Schiller (formerly P. donghaiense Lu), a harmful algal species common in the East China Sea (ECS), often thrives with the depletion of phosphate. Three cruises in the spring of 2013 sampled an entire P. obtusidens bloom process to investigate the dynamics of alkaline phosphatase activity (APA) and phosphorus (P) status of the bloom species using both bulk and cell-specific assays. Unlike previous studies, the bloom of P. obtusidens occurred in a phosphate replete environment. Very high APA, with an average of 76.62 ± 90.24 nmol L-1 h-1, was observed during the early-bloom phase, a value comparable to that in low phosphate environments. The alkaline phosphatase (AP) hydrolytic kinetics also suggested a more efficient AP system with a lower half-saturation constant (Ks), but higher maximum potential hydrolytic velocity (Vmax) in this period. The APA decreased significantly with an average of 24.98 ± 30.98 nmol L-1 h-1 when the bloom reached its peak. The lack of a correlation between dissolved inorganic phosphate (DIP) or dissolved organic phosphate (DOP) concentration and APA suggested that the APA was regulated by the internal P growth demand, rather than the external P availability during the phosphate replete P. obtusidens bloom. These findings facilitate an understanding of the P. obtusidens acclimation strategy with respect to P variations in terms of AP expression during blooms in the ECS.

Comparative uptake and assimilation of nitrate, ammonium, and urea by dinoflagellate Karenia mikimotoi and diatom Skeletonema costatum s.l. in the coastal waters of the East China Sea.

The nitrogen uptake kinetics and physiological growth of Karenia mikimotoi and Skeletonema costatum sensu lato grown on different N substrates and concentrations were compared in the laboratory. In the presence of three N substrates, both species preferred to take up NH4+. K. mikimotoi and S. costatum s.l. showed the highest substrate affinities for urea and NO3-, respectively. Both species grew well on three N substrates, and the growth parameters were comparable among the different N substrates. However, K. mikimotoi assimilated urea more efficiently than it assimilated either NO3- or NH4+. Different with S. costatum s.l., K. mikimotoi grew slowly and steady and the physiological and growth activities in N-depleted conditions were higher than those in N-replete conditions. Our results suggested that K. mikimotoi shows a greater readiness for uptake and assimilation of urea, and that this species is more competitive in an N-depleted environment when compared with S. costatum s.l.

Inhibition of the growth of Alexandrium tamarense by algicidal substances in Chinese fir (Cunninghamia lanceolata).

The wood sawdust from Chinese fir (Cunninghamia lanceolata) exhibited stronger inhibition on the growth of Alexandrium tamarense than those from alder (Alnus cremastogyne), pine (Pinus massoniana), birch (Betula alnoides) and sapele (Entandrophragma cylindricum). The water extract, acetone-water extract and essential oil from fir sawdust were all shown to inhibit the growth of A. tamarense. The inhibition of fir essential oil was the strongest among all the above wood sources while the half effective concentration was only 0.65 mg/L. These results suggested that the fir essential oil may play an important role in the algicidal effect of Chinese fir.

Ocean urea fertilization for carbon credits poses high ecological risks.

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.

[Sodium dichlorinated isocyanuric acid and trichloroisocyanuric acid for removing cells of Phaeochystis globosa].

Removal and control of Phaeochystis globosa cells by sodium dichlorinated isocyanuric acid and tri-chloroisocyanuric acid were studied. Removal efficiency of the two algaecides was contrasted and removal mechanism was also discussed. The results suggest both of the algaecides have excellent ability to remove and control Phaeochystis globosa cells. Hypochlorous acid is released slowly by the two algaecides and maintains it's stabilization. The effective concentration of available chlorine is 4.5 mg/L. The removal efficiency is more than 90% within 24 hours at the available chlorine's dosage of 5.0 mg/L. Trichloroisocyanuric acid could remove Phaeochystis globosa cells more effectively than sodium dichlorinated isocyanuric acid when concentration of available chlorine was more than 5.0 mg/L. The conclusion was identified by t test of available chlorine and removal efficiency.

[Distribution of dinoflagellate resting cysts in surface sediments from the Changjiang River estuary].

In order to understand the potential of algal blooms, surface sediments were collected from 12 stations in the Changjiang River estuary with longitude from 122 degrees to 123.5 degrees E and latitude from 29 degrees to 32 degrees N from April to May 2002 to investigate the distribution of dinoflagellate resting cysts, 29 different cyst morphotypes representing 19 genera and 6 groups were identified. Among them, there were 11 autotrophic species and 18 heterotrophic ones. The number of species observed in each sample was low, and varied from 10 to 21. Cyst concentrations varied from 11.7 to 587 cysts per gram dry weight. Cyst concentrations and species diversity were higher in offshore areas, and increased from the west to the east, and from the north to the south within the studied area. Cysts of Alexandrium distributed widely in the Changjiang River estuary, with the maximum concentration of 40.4 cysts per gram dry weight. Cysts of harmful algal bloom causative species such as Alexandrium, Gymnodinium catenatum, Lingulodinium polyedra, Scrippsiellla trochoidea, Polykrikos kofoidii and P. schwartzii were also observed in this survey.

[What the Prorocentrum species should be?--a review on identification of a Prorocentrum species from the East China Sea].

In the past decade, the Prorocentrum bloom was the most frequently occurred bloom in the East China Sea. It occurred every one year or two years, and covered huge areas. There were arguments on the identification of Prorocentrum bloom causative species. Some scientists thought the species being P. dentatum Stein, but the others thought that it was a new species, P. donghaiense Lu. For achieving the proper classification of the species, an "International Workshop on HAB Causative Species--Taxonomic View on a species of Prorocentrum in the East China Sea" was held in Shenzhen, China in November, 2002. By reviewing as much as available references, and by comparing the Prorocentrum specimen from the East China Sea with those from Japan, Korea and USA, scientists participated in this Workshop agreed that: 1) the specimens from Japanese and Korean waters, and from CCMP, USA were the same species; 2) the features of anterior projection and cell surface marking were similar to those of originally described P. dentatum Stein; 3) the major difference is the cell size. The specimens from the East China Sea were mostly less than 20 microns long, whereas the size of the original described P. deatatum was larger than 50 microns; 4) the minor difference is the shape of the cell end. The original described P. deatatum was with a pointed end, whereas the cells from the East China Sea were mostly with a rounded end. There was no final agreement for the species identification at the Workshop. By summing all views of scientists from the Workshop, the authors suggested that the bloom causative species of Prorocentrum in the East China Sea, as well as so-called species of P. dentatum from other East Asian waters, should be the same species. They are all P. donghaiense Lu.

[Species composition and ecological distribution of planktonic diatoms in the Changjiang River estuary during spring].

53 water samples were collected from 28 stations in typical regions where redtide frequently occurred in the Changjiang River estuary during Spring, 2002. In total, 80 diatom species and varieties belonging to 31 genera were identified, among which, genera with high species biodiversity included Coscinodiscus which contained 17 species and Pleurosigma which contained 8 species and varieties. Quantitative analysis of diatom cell density showed that Pseudo-nitzschia delicatissma (3.48 x 10(3) cells.L-1, 28.54%), Melosira sulcata (1.43 x 10(3) cells.L-1, 16.98%) and Pseudo-nitzschia pungens (0.71 x 10(3) cells.L-1, 9.85%) were dominant species, which occurred at almost all stations. Higher cell density of Pseudo-nitzschia delicatissma and Pseudo-nitzschia pungens occurred at stations along 123 degrees E, while that of Melosira sulcata occurred at stations around Changjiang River at 31-32 degrees N. The total diatom cell density ranged from 0.43 x 10(3)-23.3 x 10(3) cells.L-1, with average of 4.61 x 10(3) cells.L-1. At station DD15 located at 123 degrees E and 30.5 degrees N, the highest diatom cell density was found, which was 1.85 x 10(4) cells.L-1 at surface water layer, and 2.33 x 10(4) cells.L-1 at middle water layer (30 m) respectively. Horizontally, the diatom cell density showed scattered distribution, while vertically, it was commonly higher at surface water layer than that at middle water layer.

[Redescription of Prorocentrum donghaiense Lu and comparison with relevant Prorocentrum species].

In this paper, the detailed morphology of Prorocentrum donghaiense was studied. Taxonomic comparison was made between P. donghaiense and other relevant Prorocentrum species. The results suggested that distinct differences existed among them with respect to their conservative characteristics of morphology and cell sizes. This kind of discrepancy was far beyond that of individual variations within same species due to environmental factors. Therefore, the high biomass bloom-forming species in the East China Sea was not real Prorocentrum "dentatum" Stein, but P. donghaiense Lu that was re-described in the present paper. P. donghaiense appeared in the East China Sea, and P. "dentatum" occurred in Japanese and Korean waters should be the same species.

[Temporal dynamics of phytoplankton and nutrients during red tides].

The relationships between temporal dynamics of phytoplankton density and nutrients (NO3-, NH4+, PO4(3-), SiO3(2-), Fe) contents in the water body at Aotou waters of Daya Bay during red tide were comprehensively analysed based on the fixed position investigation of red tide in the summer of 2000 and the several years investigation data by using grey linear regression model. The relationships between phytoplankton cell density and chlorophyll a content were also analysed. The results showed that the predicted values were well consistent with the measurement values, and their correlation coefficients were between 0.51-0.83. Red tide might break out if chlorophyll a contents in water body were larger than 5.8 micrograms.dm-3. The study could provide a simple effective method to forecast red tide by sampling water samples to analyse chlorophyll a concentration, or using SeaWiFS data, and then, to calculating phytoplankton density. In addition, the primary productivity of the water areas was controlled by phosphorus.

[Povidone-iodine and isothiozolone for removing red tide algae Phaeoecystis globosa].

Studies on the removal and control of red tide algae Phaeoecystis globosa by povidone-iodine and isothiozolone algaecide showed that Phaeoecystis globosa could be killed and controlled by povidone-iodine and isothiozolone. The effective concentration of povidone-iodine was 30 mg.L-1, and that of isothiozolone was 0.30 mg.L-1. Using povidone-iodine and isothiozolone together could improve the effect, and the ideal composite ratio of povidone-iodine and isothiozolone was 1.0:0.15.

[Spectrophotometer detection of benzalkonium bromide concentration in seawater].

Benzalkonium bromide is a high-efficiency algaecide. Its concentration in seawater was measured by the method of spectrophotometer. The results indicated that the deposition appeared if the concentration of benzalkonium bromide in seawater was more than 50 mg.L-1, and affected the detection of benzalkonium bromide. But, there was a good linear relationship between concentration and absorbance when the concentration was lower than 50 mg.L-1 (R2 = 0.9996). Therefore, spectrophotometer could be used to detect benzalkonium bromide in seawater.