Rencheng Yu

Detection of the neurotoxin BMAA within cyanobacteria isolated from freshwater in China

The cyanobacterial neurotoxin, beta-N-methylamino-l-alanine (BMAA), has been suggested as an important environmental factor for neurodegenerative disease such as amyotrophic lateral sclerosis- Parkinsonism dementia complex (ALS/PDC) in Guam. BMAA was detected within the majority of cyanobacterial isolates surveyed in both free and symbiotic cyanobacteria, living in freshwater as well as marine environments. In this study, we report two methods using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) each coupled with a different type of hydrophilic interaction liquid chromatography (HILIC) column to detect BMAA. A third method using AQC-derivatized BMAA was also used for comparison. Axenic cultures of Microcystis aeruginosa and Nostoc sp. isolated from Chinese freshwater were analyzed for both free and protein-bound BMAA at the exponential growth stage. Cultures of two strains of M. aeruginosa collected at four growth stages were also analyzed for the presence of BMAA. BMAA was detected in the Nostoc sp. at very low concentrations (<0.07pmoles on column) only when precolumn AQC derivatization was used. No BMAA was detected in the Chinese derived axenic cultures of Microcystis; detection limits for the LC-ESI-MS and LC-ESI-MS/MS without precolumn derivatization were 10ng and 2pg BMAA on column, respectively. We suggest that cyanobacteria grown under some culture conditions may be relatively free of BMAA.

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

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.

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.

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.

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.

Dynamic adsorption of diarrhetic shellfish poisoning (DSP) toxins in passive sampling relates to pore size distribution of aromatic adsorbent

Solid-phase adsorption toxin tracking (SPATT) technology was developed as an effective passive sampling method for dissolved diarrhetic shellfish poisoning (DSP) toxins in seawater. HP20 and SP700 resins have been reported as preferred adsorption substrates for lipophilic algal toxins and are recommended for use in SPATT testing. However, information on the mechanism of passive adsorption by these polymeric resins is still limited. Described herein is a study on the adsorption of OA and DTX1 toxins extracted from Prorocentrum lima algae by HP20 and SP700 resins. The pore size distribution of the adsorbents was characterized by a nitrogen adsorption method to determine the relationship between adsorption and resin porosity. The Freundlich equation constant showed that the difference in adsorption capacity for OA and DTX1 toxins was not determined by specific surface area, but by the pore size distribution in particular, with micropores playing an especially important role. Additionally, it was found that differences in affinity between OA and DTX1 for aromatic resins were as a result of polarity discrepancies due to DTX1 having an additional methyl moiety.