Martin L. Saker

The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus

Redclaw crayfish, Cherax quadricarinatus harvested from an aquaculture pond infested by a bloom of the cyanobacterium Cylindrospermopsis raciborskii (order: Nostocales), were shown to accumulate the toxic alkaloid cylindrospermopsin. Pond water samples collected during the bloom contained 589 microg l(-1) of the toxin (93% in the cyanobacterial cells, 7% in the water). Crayfish from the pond contained cylindrospermopsin at concentrations of 4.3 microg g freeze dried hepatopancreas tissue and 0.9 microg g freeze dried muscle tissue. Trichomes of C. raciborskii were observed in gut contents of crayfish harvested during the cyanobacterial bloom, indicating that the most likely mechanism for accumulation of the toxin was by ingestion of cyanobacterial cells. Crayfish subjected to an extract of harvested bloom material under laboratory conditions for a period of 14 days were also found to accumulate cylindrospermopsin, indicating that this toxin is also absorbed into the tissues by direct uptake of the toxin in solution.

Varied Diazotrophies, Morphologies, and Toxicities of Genetically Similar Isolates of Cylindrospermopsis raciborskii(Nostocales, Cyanophyceae) from Northern Australia

ABSTRACT The potentially toxic freshwater cyanobacteriumCylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO3−, NH4+, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH4+ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH4+. Cultures supplied with NO3− were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH4+ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO3− < NH4+). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications forCylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.

The effect of temperature on growth and cylindrospermopsin content of seven isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from water bodies in northern Australia

Abstract This study investigates the effect of temperature (range, 20–35°C) on the growth and cylindrospermopsin (CYL) content of seven isolates of Cylindrospermopsis raciborskii grown in batch culture. The concentration of CYL, measured late in the exponential growth phase for cultures grown at 25°C, ranged from below detection (< 2 × 10−5% freeze-dried weight) for isolates from Lake Julius (20°08′S; 139°44′E) and an aquaculture pond in Townsville (19°16′S; 146°49′E) up to 0.46% for an isolate from Solomon Dam, Palm Island (18°45′S; 146°35′E). Maximum growth rates for all isolates occurred at temperatures between 25 and 30°C. Only one isolate failed to grow at 20°C. For isolates containing undetectable concentrations of CYL, the production of this compound could not be induced by an increase in temperature. For strains producing detectable concentrations, there was a strong negative correlation between CYL content and temperature. While all the isolates grew well at 35°C, none produced any detectable amount of CYL. When cultures of isolates that normally produce CYL were transferred from 35°C to a lower temperature, production of CYL was restored. In the cultured isolates, less than 10% of the total CYL was released from exponentially growing cells. In one CYL-producing isolate, extracellular CYL accounted for c. 50% of the total in stationary phase (20 day old) cultures.

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world‐wide. This species is of concern from a water‐quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic vari‐ation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium‐specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between‐country variation was greater than within‐country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

First report and toxicological assessment of the cyanobacterium Cylindrospermopsis raciborskii from Portuguese freshwaters

The freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in freshwaters worldwide. This species is a concern from a water quality perspective due to its known ability to produce a potent hepatotoxic alkaloid cylindrospermopsin, which has been implicated in outbreaks of human sickness and cattle mortality. C. raciborskii strains isolated from Brazil have also been found to produce the highly toxic paralytic shellfish poisons (PSPs). This article reports the toxicity of four strains of C. raciborskii taken from three reservoirs and one river in Portugal, as well as the occurrence of this species in other water bodies used for potable and recreational purposes. All four strains grown in pure culture in the laboratory were found to be toxic in the mouse bioassay at 8-24h after intraperitoneal administration of single doses ranging from 1337 to 1572 mgkg(-1) Histological examination indicated that liver damage was the primary lesion; in addition, there was inflammation in the intestine. HPLC/MS tests for the presence of cylindrospermopsin, microcystins, and PSP toxins were negative. The available evidence suggests that another toxin may be present. This constitutes the first report of toxic C. raciborskii in Europe and draws attention to the need for increased monitoring of this cyanobacterium in water bodies used for potable and recreational purposes.

Cattle mortality attributed to the toxic cyanobacterium Cylindrospermopsis raciborskii in an outback region of North Queensland

Three cows and ten calves were found dead near a farm dam on a cattle property at McKinlay in northwest Queensland, Australia. At that time, the dam contained an algal bloom which was identified as a monoculture of the cyanobacterium C. raciborskii. Histological examination of the liver of a calf carcass showed signs consistent with poisoning caused by hepatotoxin. The hepatotoxic alkaloid cylindrospermopsin was detected in material harvested from the dam (4.1×10−15 g cell−1) and in a pure culture of an isolate from the bloom (4.4×10−15 g cell−1). An extract of this material was lethal to mice after 24 h at an intraperitoneal concentration of 153 mg kg−1. This appears to be the first report of animal poisonings attributed to the cyanobacterium C. raciborskii. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 179–182, 1999

TWO MORPHOLOGICAL FORMS OF CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) ISOLATED FROM SOLOMON DAM, PALM ISLAND, QUEENSLAND

Cylindrospermopsis raciborskii Woloszynska is a prominent constituent of a number of water bodies in northern Australia. In Solomon Dam, this species occurs as two distinct morphological forms, one with straight trichomes and one with coiled trichomes. Isolates of the two forms have been grown in pure culture and have been shown to maintain their respective characteristic form over successive generations. Both forms were similar with respect to cylindrospermopsin content expressed as a percentage of freeze‐dried culture material, ranging from 0.14% to 0.20%, depending on the N source provided in the medium. A morphological comparison between natural populations of the two forms showed significant differences in vegetative, heterocyst, and akinete cell dimensions. These characteristics are the primary taxonomic criteria at the species level in this genus. In culture, the coiled form grew slightly faster than the straight form over the range of conditions investigated in this study. The coiled form was better suited to growth under low‐light conditions. These clear and consistent morphological and physiological differences contrast with the 99.8% similarity between the two forms in their 16S rRNA gene nucleotide sequences. It is concluded that although taxonomic separation of the two forms at the species level might not be warranted, the two strains investigated are clearly distinct morphotypes, and it is recommended that they be so recorded in monitoring programs.

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.

Occurrence of blooms of the cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju in a north Queensland domestic water supply

This paper describes seasonally recurring blooms of the potentially toxic cyanobacterium Cylindrospermopsis raciborskii in relation to some limnological characteristics of Lake Julius, a large man-made water impoundment in Australia’s semi-arid tropics. These blooms have occurred each year since 1991, with subsurface concentrations of >50 000 cells mL–1. Periods of greater cyanobacterial abundance are characterized by reduced rates of vertical mixing of the water column, reduced mixed:euphotic depth ratios and high epilimnetic temperatures (>25˚C). Surface scums were not observed and, in general, this species displays a fairly uniform distribution throughout the euphotic zone and below. An isolate of C. raciborskii taken from Lake Julius during a bloom in November 1995 and grown in pure culture produced no symptoms of poisoning when tested by mouse bioassay, and absence of detectable concentrations of the hepatotoxin cylindrospermopsin was confirmed by HPLC/MS-MS. Low concentrations of cylindrospermopsin (~1–2 g L–1) were detected in the lake during blooms of C. raciborskii.

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

Aims:  The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2‐month period.