Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.

Genetic diversity: Geographical distribution and toxin profiles of Microcystis strains (Cyanobacteria) in China

Twenty strains of Microcystis Kutz were isolated from different freshwater bodies in China to analyze the diversity, geographical distribution and toxin profiles. Based on whole-cell polymerase chain reaction of cpcBA-IGS nucleotide sequence, the derived neighbor-joining (NJ) and maximum parsimony (MP) trees indicate that these strains of Microcystis can be divided into four clusters. The strains from south, middle and north region of China formed distinct lineages, suggesting high diversity and a geographical distribution from south to north locations. Moreover, the results being indicating high variable genotypes of the strains of the Microcystis strains from the same lake show that there is high diversity of Microcystis within a water bloom population. Comparing the results of the present study with those reported for compared with 43 strains of Microcystis from other locations, also reveals Chinese strains have high similarity with those from regions in the North Hemispherical. This suggests that the Microcystis strains in the world might have a geographical distribution. Analysis of 30 strains using the primers MCF/TER and TOX2P/TOX2M showed that there was no correlation between the gene of cpcBA-IGS and the presence of mcy. Toxic strains were founded to be predominant in different water bodies throughout China.

Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 mu M NaNO3) Were supplemented with 793.8 mu M NaNO3 at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 mu M NaH2PO4) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense. (c) 2005 Elsevier Ltd. All rights reserved.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins

Phytoplanktivorous bighead carp were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 200 and 500 MC-LReq. mu g kg(-1) bw, and the changes in extractable MCs in liver and in the ultrastructure of hepatocytes were studied at 1, 3, 12, 24 and 48 h after injection. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS, respectively. MC concentration in the liver reached the maxima at 12 It (2.89 mu g MCs g(-1) dry weight at the lower dose) or at 3 h (5.43 mu g MCs g(-1) dry weight at the higher dose) post-injection, followed by sharp declines afterwards, whereas the ultrastructural changes of hepatocytes in both dose groups suggest progressive increases in severity toward the directions of apoptosis and necrosis from I to 24 h, respectively. There were two new findings in fish: widening of intercellular spaces was among the early ultrastructural changes induced by MCs and ultrastructural recovery of hepatocytes was evident at 48 h post-injection in both dose groups. Both the present and previous studies suggest that with in vivo or in vitro exposure to microcystins, hepatocyte damage in fish tends to proceed toward the direction of apoptosis at lower MC concentrations but toward the direction of necrosis at high MC concentrations. The temporal dynamics of MCs in the liver suggest that bighead carp may have a mechanism to degrade or bind MC-LR actively after it enters the blood system. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of nitrogen forms on the production of cyanobacterial toxin microcystin-LR by an isolated Microcystis aeruginosa

A cyanobacterial strain, which produced high content of microcystin-LR (MC-LR) but no rnicrocystin-RR (MC-RR), was isolated from the hypertrophic Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of nitrogen containing chemicals and trace elements on the growth and the production of MC-LR by this strain were Studied. In the presence of bicine, compared with urea and ammonium, nitrate greatly promoted the growth and the production of MC-LR. However, leucine and arginine, which were the constitutional components in the molecular structure of MC-LR or RR, inhibited the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the growth of M. aeruginosa DC-1, but the production of MC-LR was apparently enhanced. Under all conditions studied here, only MC-LR but no RR was detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of nitrogen, rather than the usually concerned the total nitrogen, Lind trace elements played important roles in the production of MC toxins during cyanobacterial blooms.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

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. (C) 1999 Elsevier Science Ltd, All rights reserved.

Impact of germanium on the growth of Pseudonitzschia pungens f. multiseries and the production of algal toxin

The impacts of germanic acid Ge(OH)(4) on the growth of Psuedonitzschia pungens f. multiseries and on the production of the algal toxin, domoic acid, were studied. The results showed that germanic acid in the range of concentrations could inhibit the growth of the algal cells and the inhibition was enhancing with the concentrations of germanic acid increasing. Germanic acid also could inhibit the production of the algal toxin, domoic acid in cells and the inhibition reached up to 100% at Ge/Si = 35. Based on the results, the mechanism was discussed.

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. (C) 2008 Elsevier Ltd. All rights reserved.