A PCR‐BASED TEST TO ASSESS THE POTENTIAL FOR MICROCYSTIN OCCURRENCE IN CHANNEL CATFISH PRODUCTION PONDS1,2

Microcystis aeruginosa is a common form of cyanobacteria (blue‐green algae) capable of forming toxic heptapeptides (microcystins) that can cause illness or death. Occasionally, blooms of cyanobacteria have caused toxic fish‐kills in catfish production ponds. We have developed a PCR test that will detect the presence of microcystin‐producing cyanobacteria. Microcystin producers are detected by the presence of the microcystin peptide synthetase B gene (an obligate enzyme in the microcystin pathway), which appears to be present only in toxin‐producing cyanobacteria. These PCR amplifications can be performed in multiplex using purified DNA from pond waters or by two‐stage amplification from native water samples. A synoptic survey of 476 channel catfish production ponds from four states in the southeastern United States revealed that 31% of the ponds have the genetic potential to produce microcystins by toxic algae.     

Sensitivity of Daphnia to toxic cyanobacteria: effects of genotype and temperature

1. We studied the effects of both acute and chronic exposure of Daphnia pulex to toxic Microcystis aeruginosa . We focused on the effects of Daphnia genotype and temperature (19 and 24 °C).
2. The study revealed variation among ten Daphnia pulex clones in survivorship under acute Microcystis exposure, measured as EC₅₀. An increase in temperature caused a clear decrease in EC₅₀, although the ranking of clones according to sensitivity remained the same at both temperatures.
3. In the chronic exposure of two of the clones, toxic Microcystis reduced survival and reproduction. The two clones differed in their responses, indicating different means of coping with toxic cyanobacteria. Toxic cyanobacteria reduced slightly more at 24 °C than 19 °C.
4. The clonal difference in sensitivity to toxic cyanobacteria at acute exposure was reversed at chronic exposure. This indicates that the results from short-term toxicity cannot be used to predict life history responses under sublethal exposure.     

Sensitivity of Daphnia to toxic cyanobacteria: effects of genotype and temperature

1. We studied the effects of both acute and chronic exposure of Daphnia pulex to toxic Microcystis aeruginosa . We focused on the effects of Daphnia genotype and temperature (19 and 24 °C).
2. The study revealed variation among ten Daphnia pulex clones in survivorship under acute Microcystis exposure, measured as EC₅₀. An increase in temperature caused a clear decrease in EC₅₀, although the ranking of clones according to sensitivity remained the same at both temperatures.
3. In the chronic exposure of two of the clones, toxic Microcystis reduced survival and reproduction. The two clones differed in their responses, indicating different means of coping with toxic cyanobacteria. Toxic cyanobacteria reduced slightly more at 24 °C than 19 °C.
4. The clonal difference in sensitivity to toxic cyanobacteria at acute exposure was reversed at chronic exposure. This indicates that the results from short-term toxicity cannot be used to predict life history responses under sublethal exposure.     

The occurrence, distribution and toxicity of cyanobacteria in the estuary of Lagoa dos Patos, Rio Grande do Sul

Several blooms of Microcystis aeruginosa have been observed in the Patos Lagoon estuary during the last fifteen years without a proper investigation of their ecological importance or possible toxicity. The present study has identified and quantified the presence of cyanobacteria in the Patos Lagoon estuary, particularly of M. aeruginosa. During this survey, identification and quantification of the main phytoplankton groups were done in relation to geographical distribution in the estuary. The presence of M. aeruginosa colonies in the estuarine region confirmed their superficial distribution throughout the estuarine waters during twelve months with a maximum of 1, 3.10(6) cells. L-1 in December, 1994 and a minimum of 1, 5.10(5) cells. L-1 in August, 1995 and also confirmed that M. aeruginosa originated from waters in the north of the estuary. The period of the highest cell and colonies densities was coincident with high chlorophyll-a levels in surface waters. Toxicity of M. aeruginosa bloom material was determined by bioassay and concentrations of hepatotoxins microcystins were identified by HPLC-DAD. M. aeruginosa blooms were considered highly toxic, presenting a 24 h-LD50 lower than 100 mg.Kg-1 b.w. and a toxin content higher than 1 microgram.mg-1 d.w. Several microcystin variants were found in the extracts with microcystin-LR predominating.     

Steatitis in egrets and herons from Japan

More than 70 egrets and herons were found sick or dead at an agricultural water reservoir in Kanagawa Prefecture, Japan between September and October 2008. The birds showed weakness, lethargy, and inability to fly before death. Postmortem findings included large amounts of firm subcutaneous and cavitary fat comprised of necrotic adipose tissues with infiltrates of heterophils and macrophages. The birds were diagnosed with steatitis on the basis of the gross lesions and histopathology. Egrets with steatitis had low blood levels of vitamin E. High counts of cyanobacteria (Microcystis aeruginosa) were found in the reservoir concurrent with the outbreak of steatitis. No microcystin was detected in the reservoir water or the livers from the egrets. This is the first report of steatitis in wild birds in Japan.     

Release of heptapeptide toxin (microcystin) during the decomposition process of Microcystis aeruginosa.

The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Outbreaks of type C botulism in waterfowl in Japan

Four outbreaks of botulism in waterfowl were encountered over a five-year period of 1973 to 1977 in Japan. In all the outbreaks toxin was detected from all 12 sera, twenty-three of 24 gizzard contents from diseased or dead birds and one of three maggots. It was neutralized with Clostridium botulinum type C antitoxin serum, regardless of its origin. By using CO2 gas jet method, C. botulinum was isolated from four of 11 gizzards from diseased birds, five of 7 ones from dead birds, one of one maggot and one of one sludge sample, that is, eleven of 20 specimens in total. All 20 strains were identical with C. botulinum type C in biological properties. Most of the isolates showed a toxin titer ranging from 1,000 to 200,000 LD50 for mice. Four of them were identified as type C by mouse neutralization tests with antitoxin sera. The toxic suspensions of a strain 1-15 were administered orally to Chinese spot-billed ducks, which died when more than 200,000 LD50 mouse toxin was administered. Environmental conditions for occurrences of waterfowl botulism were discussed.     

Determination of oligopeptide diversity within a natural population of Microcystis spp. (cyanobacteria) by typing single colonies by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a natural Microcystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa (Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.     

Distribution of Microcystins in a Lake Foodweb: No Evidence for Biomagnification

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.     

Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes

Abstract Toxic strains of Microcystis aeruginosa produce cyclic heptatoxins (microcystins) that are believed to be synthesized non-ribosomally by peptide synthetases. We analysed toxin-producing and non-toxic strains of M. aeruginosa with respect to the presence of DNA sequences potentially encoding peptide synthetases. Hybridizations of genomic DNA of various M. aeruginosa strains with PCR-amplificated fragments possessing homologies to adenylate-forming domains of peptide synthetase genes provided first evidence for the existence of corresponding genes in cyanobacteria. Furthermore we isolated and sequenced from genomic libraries overlapping fragments of M. aeruginosa DNA with a total length of 2982 bp showing significant homology to genes encoding peptide synthetases and hybridizing exclusively with DNA from toxic strains. Our results indicate that both toxic and non-toxic strains of M. aeruginosa possess genes coding for peptide synthetases and that hepatotoxin-producing and non-toxic strains differ in their content of genes for specific peptide synthetases.     

Diversity of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> isolates (Chroococcales,Cyanobacteria) from East-African water bodies

With exception of South Africa, very little is known about the presence and abundance of toxic cyanobacteria and cyanobacterial blooms on the African continent. The close proximity between society and nature, and the use of the sparse water resources as drinking water in large parts of Africa, lead to the recognition that more knowledge on toxic cyanobacterial blooms is of major importance. The bloom forming cyanobacterium Microcystis aeruginosa is known to produce cyclic heptatoxins (microcystins) which can be toxic to humans. In this study the morphological, genetic, and chemical characters of 24 strains of M. aeruginosa from several water bodies in Kenya and Uganda, some of them used as drinking water sources, were examined. The M. aeruginosa strains possessed different levels of diversity depending on characterisation method. Four morphotypes were identified based on the traditional morphological approach, 10 genotypes by DNA sequence comparison of the PC-IGS and ITS1 rDNA regions, and 10 chemotypes based on MALDI-TOF-MS oligopeptide analysis. Only 4 of the 24 isolated strains from East Africa were found to produce microcystins, while oligopeptides belonging to the aeruginosin and cyanopeptolin class were detected in most strains.     

Responses of Daphnia pulex populations to toxic cyanobacteria

1. We studied the effects of toxic cyanobacteria, Microcystis aeruginosa , on Daphnia pulex populations. The experiment was performed at room temperature in laboratory microcosms to which we added toxic Microcystis in two pulses. Green alga, Scenedesmus obtusiusculus, was provided in two concentrations.
2. Microcystis exposure resulted in a decreased population density of Daphnia. The proportion of adolescents was higher in the Microcystis treatment than in the control, while the proportion of newborns did not differ significantly from the control. This indicates delayed maturation of Daphnia exposed to Microcystis . We found no significant impacts of cyanobacteria, food level or clonal origin on population variability after correcting for differences in population size.
3. Juveniles of the two clones studied showed different sensitivities to toxic Microcystis in the acute test. However, in the population experiment we did not find clonal differences between Microcystis and control treatments in the relative proportion of juveniles.
4. The number of ephippia produced per adult was highest in the treatments with Microcystis. This indicates that cyanobacterial toxins may be one of several factors inducing ephippia formation, acting directly or through inhibition of feeding.     

A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia

Microcystis aeruginosa strain MRC is unique in its' possession of the mcyA-J gene cluster, which encodes microcystin synthetase, but its' inability to produce microcystins. M. aeruginosa strain MRD is genetically identical to MRC at numerous genomic loci examined, but produces a variety of microcystins, mainly with the amino acid tyrosine in the molecule. Zooplankton studies with Daphnia galeata and D. pulicaria , using the mutant (MRC) and its' wild type (MRD), showed for the first time that microcystins other than microcystin-LR can be responsible for the poisoning of Daphnia by Microcystis . Regardless of microcystin content, both Daphnia exhibited significantly reduced ingestion rates when fed with either strain of M. aeruginosa compared with the green alga Scenedesmus acutus . A disruption of the molting process in both Daphnia spp. was noted when these species were fed with MRC cells. Such symptoms on Daphnia have not been previously reported for cyanobacteria and may point to a bioactive compound, other than microcystin, which inhibits the hardening of protein–chitin complexes in Daphnia .     

阴离子表面活性剂LAS对产毒和无毒微囊藻生长及产毒特性的影响

文章研究了低浓度范围内不同浓度梯度的阴离子表面活性剂直链烷基苯磺酸盐(LAS)对产毒微囊藻(Microcystis aeruginosa, FACHB905)和无毒微囊藻(Microcystis wesenbergii, FACHB908)生长、光合特性、种间竞争及毒素合成的影响。结果表明,在0.05—5.0 mg/L LAS浓度梯度处理下,产毒微囊藻的生物量、产毒基因mcyD表达量和每细胞MCs含量均在培养12d后显著增加。产毒微囊藻胞内和胞外MCs含量在各LAS浓度处理后分别为0.069、0.052、0.061、0.038和0.037 fg/fg Chl.a及107.1、103.7、127.1、99.6和113.7 ng/L。即使在0.5 mg/L低浓度LAS处理条件下,上述3个参数也分别比对照组显著增加了24.2%、12.4倍和10.4%。浓度为0—0.2 mg/L LAS对无毒微囊藻的生物量无明显影响,而较高浓度的LAS(0.5—5.0 mg/L)明显抑制了无毒微囊藻的生长。在两株微囊藻混合培养时, 0.2—1.0 mg/L LAS处理组的产毒铜绿微囊藻mcy D的表达对LAS...     

Genotoxicity of microcystic cyanobacteria extract of a water source in China.

The water pollution of toxic cyanobacteria (blue-green algae) is a worldwide problem and worsens with industrialization. Microcystins are potent cyclic heptapeptidic hepatotoxins produced mainly by Microcystis aeruginosa, and their hepatotoxicity has been well-documented. In contrast, information on the genotoxic effects of microcystins is relatively scarce. In our present study, the genotoxicity of microcystic cyanobacteria extract (MCE) of a water source in China was studied using Salmonella typhimurium assay (Ames test), comet assay (Single cell gel electrophoresis) and mouse micronucleus test. Results from Ames test indicated that MCE had strong mutagenicity regardless of the presence of S9. Moreover, MCE was able to induce DNA damage in primary cultured rat hepatocytes examined by comet assay. In addition, MCE also enhanced bone marrow micronucleated polychromatic erythrocytes in mice. The analysis of HPLC showed that the main component of MCE was microcystin-LR. The understanding of the potent genotoxicity of MCE will help to establish the possible link between water cyanobacteria contamination and high risk of primary liver cancer found in some endemic areas.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Testicular toxicity of cyanobacterial biomass in Japanese quails

Previous studies demonstrated the toxic effects of cyanobacteria in Japanese quails (Coturnix coturnix japonica) in various experimental set-ups including acute, sub-chronic and reproduction toxicity, co-exposures with toxic metals and the Newcastle vaccination. This study aimed to assess the testicular toxicity of a complex cyanobacterial biomass administered to Japanese quails in the feed for eight weeks (daily dose of 61.62 μg microcystins pro toto including 26.54 μg MC-RR, 7.62 μg MC-YR and 27.39 μg MC-LR). There was no mortality in the controls or the biomass-exposed birds. However, males exposed to cyanobacteria in the feed showed moderate to marked atrophy of the seminiferous tubular epithelium with only sparse numbers of the developmental stages of spermatozoa and Sertoli cells. Biomass-exposed birds had elevated catalase activities but decreased glutathione peroxidase activities and surprisingly lower levels of lipid peroxides in the testes. The other biochemical parameters studied (i.e., glutathione level and glutathione reductase, glutathione-S-transferase and superoxide dismutase activities) showed no differences. The cell defensive system protecting testicular tissue from the damage associated with toxic effects of the complex cyanobacterial biomass seemed to be insufficient and partly depleted after the chronic exposure of male birds to this biomass.     

Grazing rates on toxic and non-toxic strains of cyanobacteria by Hypophthalmichthys molitrix and Oreochromis niloticus

The tilapia Oreochromis niloticus and the silver carp Hypophthalmichthys molitrix were exposed to toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa in order to determine if cells of the toxic strain were ingested and, if not, by what mechanism they were excluded. Enumeration of cyanobacterial particles before and after exposure to fish showed that there were no significant differences (P<0.05) at the end of the trial between the toxic treatment and the control consisting of toxic M. aeruginosa with no fish. Fish exposed to the non-toxic strain increased opercular beat rate, elevating the volumes of water and food material passed over the gills whereas those that were held in the toxic strain did not. Of the cyanobacterial toxins (microcystins) presented to the fish, most were in the cyanobacterial cells, toxin levels in the water being below the level of detectability (<250 ng l⁻¹), The ability of the fish to differentiate between toxic and non-toxic cyanobacterial strains may thus be determined by very low levels of extracellular microcystins or/and other features which distinguish toxic from non-toxic M. aeruginosa strains, such as cell surface components.