Ingestion of microcystins by Daphnia: Intestinal uptake and toxic effects

We investigated the intestinal uptake and adverse effects of microcystins ingested with Microcystis on Daphnia galeata. The gut structure, blood microcystin concentration, appearance, and movements of Daphnia fed Microcystis PCC 7806 or a microcystin-deficient PCC 7806 mutant were monitored over time. Microcystins were rapidly taken up from the digestive cavity into the blood. This process apparently required a preceding disruption of the gut epithelium by an as-yet-unknown Microcystis factor. Once microcystins entered the blood, they affected the neuromuscular communication or another life function that influences major muscle systems. Consequently, the beat rates of the thoracic legs, mandibles, and second antennae as well as the activity of the foregut decreased, whereas the midgut muscles were stimulated. Finally, the animals exhibited symptoms of exhaustion and died. The present results suggest that an ingestion of between 10.2 ng and 18.3 ng of microcystin per 1 mg of Daphnia body fresh weight is sufficient to kill D. galeata within 2 d. Many freshwater cyanobacteria share the ability to synthesize bioactive compounds that may affect other organisms. In particular, the bloom-forming taxa, which are members of the orders Nostocales and Chroococcales, including Microcystis, Anabaena, Aphanizomenon, and Planktothrix spp., are rich sources of potent enzyme inhibitors, cellular disrupters, and compounds with a diverse range of other biological activities (Carmichael 1992; Codd 1995). Some of these metabolites present serious threats to animal and human health. Prominent examples are the microcystins, which frequently are produced by Microcystis sp. and have the potency to induce various harmful effects, such as an inhibition of protein phosphatases (e.g., MacKintosh et al. 1990). Microcystins usually are cell-bound. Although Microcystis cells may exhibit a limited excretion of microcystins 1 Corresponding author ([email protected]). Acknowledgments The authors express their gratitude to N. Willumsen for his laboratory assistance. The authors also thank J. Weckesser, W. Lampert, and M. Henning for providing stock cultures of Microcystis, Scenedesmus, and Daphnia. This study was supported by grants FMRX-CT97-0097 and FMRX-CT98-0246 from the European Community to K.C. and T.B., respectively. I.N. was supported by grant Praxis XXI/BD/21757/99 from Fundação para a Ciência e Tecnologia. (Kaebernick et al. 2000), their internal microcystin content is often high. Microcystins therefore may harm animals that ingest Microcystis. Special interest has been directed to the effects on Daphnia sp. (e.g., Jungmann and Benndorf 1994; Reinikainen 1994), which constitute key elements of freshwater food webs (Riemann and Christoffersen 1993). Daphnia are also grazers of natural Microcystis populations (Thompson et al. 1982; Schoenberg and Carlson 1984) and, thus, may ingest considerable amounts of microcystins in nature. This may result in various adverse effects. As recently shown by Rohrlack et al. (2001) and Kaebernick et al. (2001), microcystins taken in together with Microcystis cells can rapidly kill five common Daphnia sp. The basis of this finding has been experiments with genetically engineered and spontaneous Microcystis mutants for microcystin production. In addition, Rohrlack et al. (2001) have presented a correlation between the microcystin ingestion rate and the survival time of various Daphnia sp. originating from different regions and types of waters. According to their work, differences in survival among Daphnia sp. exposed to Microcystis are caused by variation in feeding activity rather than by differences in susceptibility to foodassociated microcystins. In fact, those authors demonstrated that all five tested Daphnia sp. shared virtually the same sensitivity to microcystins ingested with Microcystis cells. 441 Effects of microcystins on Daphnia Another conclusion from the correlation between microcystin ingestion rate and survival time was that even low rates of microcystin ingestion, as may occur when Daphnia feed on a natural mixture of Microcystis and other types of phytoplankton, can result in lethal effects. Moreover, those authors found colony-forming Microcystis strains to be capable of poisoning daphnids and, therefore, concluded that natural, microcystin-producing Microcystis populations may well be toxic to Daphnia. In contrast to the work by Rohrlack et al. (2001), other studies, including one by DeMott (1999), have found Daphnia sp. and clones to differ in their response to microcystin-producing Microcystis. An explanation for this deviation in results may be that Rohrlack et al. (2001) used pure Microcystis cultures in their experiments, whereas DeMott (1999) exposed Daphnia to a mixture of Microcystis