Mingjiang Zhou

A Modified HPLC Method for Analysis of PSP Toxins in Algae and Shellfish from China

SummaryImprovements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins reponsible for paralytic shellfish poisoning (PSP). The PSP toxin cotent of two strains ofAlexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

Abstract To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l−1 ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W>NW>N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l−1 ammonia-N reduced the immunocompetence of P. japonicus and may have decreased the virulence of WSSV.

A Recent Shellfish Toxin Investigation in China

A shellfish toxin investigation along the Chinese coast has recently been conducted using both HPLC and mouse assay methods. The results showed that DSP was widely distributed in different shellfish species in China. 26 out of 89 samples had DTX1 (dinophysistoxin-1) or OA (okadaic acid) but the DSP content in most shellfish samples did not reach the regulatory limit for human consumption adopted in many countries (20 mu g/100 g soft tissue). PSP was also found in 5 out of 96 samples along the coast. One sample, Chlamys nobilis from Hong Kong contained high levels of PSP (320 mu g STX equivalent/100 g soft tissue), compared to the regulatory limit (80 mu g STX equivalent/100 g soft tissue). After the recent outbreak of red tide in Hong Kong waters, three further shellfish samples were collected within 40 days to investigate the impacts of this event, It was shown that high levels of PSP continued to exist in Hong Kong waters. This report provides the first report of DSP and PSP distribution along the Chinese coast

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.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects. Likewise, exposure of eggs to standard STX toxin, as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above; no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae. The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2)mouse unit (MU) per gram of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus.

Mariculture: significant and expanding cause of coastal nutrient enrichment

Mariculture (marine aquaculture) generates nutrient waste either through the excretion by the reared organisms, or through direct enrichment by, or remineralization of, externally applied feed inputs. Importantly, the waste from fish or shellfish cannot easily be managed, as most is in dissolved form and released directly to the aquatic environment. The release of dissolved and particulate nutrients by intensive mariculture results in increasing nutrient loads (finfish and crustaceans), and changes in nutrient stoichiometry (all mariculture types). Based on different scenarios, we project that nutrients from mariculture will increase up to six fold by 2050 with exceedance of the nutrient assimilative capacity in parts of the world where mariculture growth is already rapid. Increasing nutrient loads and altered nutrient forms (increased availability of reduced relative to oxidized forms of nitrogen) and/or stoichiometric proportions (altered nitrogen:phosphorus ratios) may promote an increase in harmful algal blooms (HABs) either directly or via stimulation of algae on which mixotrophic HABs may feed. HABs can kill or intoxicate the mariculture product with severe economic losses, and can increase risks to human health.

Effects of the dinoflagellate Alexandrium tamarense on early development of the scallop Argopecten irradians concentricus

The effects of Alexandrium tamarense (strain ATHK) on early development of the bay scallop Argopecten irradians concentricus were studied under laboratory conditions. The algal culture was verified by HPLC to produce paralytic shellfish poisoning (PSP) at a level of 37.48 fmol/cell. Survival of the scallop larvae was not affected when they were grown with A. tamarense at concentrations of 500-10,000 cells/ml for 48 h. However, the activity of D-shape larvae was inhibited after 48-h exposure to A. tamarense at the algal cell density of 10,000 cells/ml. Scallop growth was inhibited significantly by A. tantarense during a 14-day exposure starting at the eye-spot larval stage. The size of juvenile scallops in the group of 10,000 cells/ml was only about 32% of that of the controls, although no obvious effect of A. tamarense was found on the rate of larval metamorphosis. All juvenile scallops survived in algal concentrations of 600-2400 cells/ml, however, attachment rates were significantly lower than control values after a 5-h exposure to A. tamarense at concentrations >600 cells/ml, while they were not obviously reduced after only 1 h of exposure. At concentrations >600 cells/ml, the climbing ability of juveniles was clearly reduced by exposure to A. tamarense after only 1 h. The climbing rate and height were only 55% and 45%, respectively, of those of the controls, when exposed to A. tantarense at a concentration of 600 cells/ml. The results indicated that A. tamarense blooms may have detrimental impacts on shellfish at early life stages, therefore, special attention should be paid to the toxic algal blooms in shellfish breeding area

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection.

Abstract The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis of l-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F. chinensis increased slightly from 0 to 12h postchallenge, indicated by the variations of l-citrulline (from 11.15±0.10 to 12.08±0.64μM) and total nitrite/nitrate concentrations (from 10.45±0.65 to 12.67±0.52μM). Then it decreased sharply till the end of the experiment (84h postchallenge), the concentrations of l-citrulline and total nitrite/nitrate at 84h were 1.58±0.24 and 2.69±0.70μM, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72h postchallenge, the concentrations of l-citrulline and total nitrite/nitrate increased from 7.82±0.77 at 0h to 10.79±0.50μM at 72h, and from 8.98±0.43 at 0h to 11.20±0.37μM at 72h, respectively. Then it decreased till the end of the experiment (216h postchallenge), and the concentrations of l-citrulline and total nitrite/nitrate at 216h were 5.66±0.27 and 4.68±0.16μM, respectively. More importantly, an apparent increase of LPS-stimulated NOS activity was observed in M. japonicus at 48h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66h, much more quickly than M. japonicus, whose accumulative mortality reached 100% after 240h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F. chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F. chinensis and M. japonicus to WSSV infection.

Pigment characterization for the 2011 bloom in Qinhuangdao implicated “brown tide” events in China

A large-scale bloom occurred from May to June in 2011 in sea area near Qinhuangdao of the Bohai Sea, leading to huge damage of the scallop culture industry. Similar blooms have been observed in this region for three years. The causative species of the bloom, which dominated the phytoplankton community with the maximum cell density around 109 cell/L, could not be identified with morphological features due to the small cell size (∼2 m m). A pigment analytical method was then adopted to analyze the pigment profile of the phytoplankton samples collected from the blooming sea area. It was found that pico-sized (<2 m m), nano-sized (2–20 m m), and bulk phytoplankton samples had similar pigment profile, representing the pigment signature of the bloom-causative species. The major pigments detected included 19-butanoyloxyfucoxanthin (But-fuco), fucoxanthin (Fuco), diadinoxanthin (Diad) and chlorophyll a (Chl a), and high content of But-fuco was the most significant characteristics of the phytoplankton samples. Based on the pigment composition and content, the bloom-causative species could be tentatively identified as pelagophyte, “type 8” group of haptophyte, or silicoflagellate. Some unique features of the bloom, such as the extremely high cell density, small-sized and But-fuco containing cells, occurring in early summer, and the feeding-cessation effects on scallops, suggest it be a “brown tide” event similar to those reported in the east coast of the United States of America. The recurrent “brown tide” events and their dramatic impacts on the shellfish mariculture industry in Qinhuangdao need close attention in the coming years.