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Appl Environ Microbiol. 2004 August; 70(8): 5047–5050.
PMCID: PMC492328

Cyanobacterial Protease Inhibitor Microviridin J Causes a Lethal Molting Disruption in Daphnia pulicaria

Abstract

Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.

Many cyanobacteria including Microcystis, Planktothrix, and Anabaena species share the ability to produce potentially toxic metabolites. In nature, such compounds threaten aquatic animals that feed on cyanobacterial cells. Of particular interest are the effects on the microcrustaceans Daphnia spp., which are among the grazers of planktonic cyanobacteria. Daphnids constitute a key group of organisms in freshwater ecosystems, and disturbance of these animals may well have effects throughout the aquatic community (7). Recently, Kaebernick et al. (3) have noted a lethal molting disruption in Daphnia spp. when fed with cells of the Microcystis strain UWOCC MRC. Such symptoms have not been previously described for cyanobacteria and point to the occurrence of a novel toxin in UWOCC MRC. More recently, Rohrlack et al. (8) have suspected microviridin J, a newly discovered protease inhibitor produced by Microcystis strain UWOCC MRC, of causing the lethal molting disruption observed by Kaebernick et al. (3). This hypothesis is tested here by investigating (i) whether purified microviridin J has an effect on daphnid molting; (ii) whether another major producer of microviridin J, the Microcystis strain UWOCC CBS, also disrupts molting; and (iii) whether significant changes in cellular microviridin J content affect the ability of Microcystis to cause molting problems in Daphnia spp.

Microcystis aeruginosa UWOCC CBS (University of Wisconsin Culture Collection, Oshkosh) was grown in Z8 medium (5) as nonaxenic, semicontinuous cultures (see reference 3 for a detailed protocol) that were kept at a constant temperature of 20 ± 1°C and under continuous light. Pilot experiments suggested a pronounced effect of light on microviridin J production in Microcystis. UWOCC CBS was therefore cultured at two light intensities, 11 and 25 μmol of photons m−2 s−1, to obtain cells containing different amounts of microviridin J. Scenedesmus acutus, originating from the Max Planck Institute for Limnology (Plön, Germany), was grown under conditions similar to those for Microcystis except for the light intensity (32 μmol of photons m−2 s−1). Microcystis and Scenedesmus were harvested by means of centrifugation for 10 min at 1,000 × g.

The cellular microviridin J content of Microcystis strain UWOCC CBS was determined as described in the work of Rohrlack et al. (8). Briefly, a culture volume corresponding to a cyanobacterial biovolume of 3 mm3 was filtered onto a 25-mm-diameter GF/C filter that was then transferred into a 1.5-ml microcentrifuge vial and lyophilized. After extraction with 50% (vol/vol) methanol the samples were analyzed by reversed-phase high-performance liquid chromatography. Student's t test was applied to compare means of the cellular microviridin J contents of Microcystis cultures (95% level of significance).

Daphnia pulicaria (clone culture kindly provided by W. Lampert) was cultured in a synthetic zooplankton medium (4) with S. acutus as the sole food. All other conditions and procedures were identical to those described previously (3). Individuals used in the experiments were 5 to 6 days old and originated from the same “mother culture.”

Experiments on the effect of cell-bound microviridin J on Daphnia were carried out in 300-ml glass bottles at 20 ± 1°C and a mean constant light intensity of 5 μmol of photons m−2 s−1. At the beginning of an experiment each bottle received 300 ml of a suspension of UWOCC CBS that was originally grown at either 11 or 25 μmol of photons m−2 s−1 and 10 Daphnia organisms. The food suspensions were prepared with synthetic zooplankton medium (4) and had a biovolume concentration of 10 mm3 liter−1. In addition to M. aeruginosa suspensions, nonfood controls were run to evaluate the effect of starvation. Individuals showing molting disruption (see below for a detailed description of symptoms) or that were dead were counted every 12 h. For that, all individuals were inspected microscopically (magnification, ×100). Food suspensions, or medium in the case of nonfood controls, and bottles were changed daily. The whole procedure was repeated three times for all food types and the nonfood controls, and experiments were terminated after 10 days. The effects of Microcystis treatments and starvation on daphnid molting success and survival were compared using the log-rank test (95% level of significance).

The effect of dissolved microviridin J was studied in 3-ml polystyrene vials kept at 20 ± 1°C under a constant light intensity of 5 μmol of photons m−2 s−1. At the beginning of an experiment, each vial contained 3 ml of a 40-mm3 liter−1 Scenedesmus suspension together with a specified amount of microviridin J (0, 0.75, 2, 4.5, 6.75, and 12 mg liter−1) that had been purified from UWOCC CBS as previously described (8). In addition, three Daphnia organisms were added to each vial. Every 12 h, all individuals were inspected microscopically for signs of molting disruption (see below for detailed description of symptoms) and subsequently transferred into freshly prepared Scenedesmus suspensions containing the respective amounts of microviridin J. The entire procedure was repeated three times, and experiments were terminated after 4 days.

UWOCC CBS cultures grown at a light intensity of 25 μmol of photons m−2 s−1 had a mean cellular microviridin J content of 1.05 ± 0.06 μg mm−3 (mean value ± standard deviation; n = 7). Significantly higher amounts (1.57 ± 0.13 μg mm−3, mean value ± standard deviation; n = 7) were extracted from cells adapted to 11 μmol of photons m−2 s−1.

Cells of UWOCC CBS had the same disruptive effect on daphnid molting as previously described for Microcystis strain UWOCC MRC (3). Almost all Daphnia individuals fed with UWOCC CBS were unable to shed the old integument although a new integument had been produced. Parts of the old integument remained attached to or completely warped the second antenna, preventing the antenna's branches from unfolding in a normal way. Other appendages such as the filter legs suffered the same fate. Most animals were also unable to shed the old integument from the head capsule and the carapace. The animals' continued effort to remove the old skin led to the deformation of the newly produced and still soft integument. Additionally, the entire body surface of Daphnia became rapidly covered with particles originating from the food suspension. This was presumably due to the secretion of some sort of body liquid. All described effects strongly inhibited the swimming and feeding abilities of Daphnia. The individuals which had developed the molting disruption died within the next few days. Daphnids which did not receive any food developed clear signs of starvation such as a gradual decrease in swimming activity and a pale coloration but did not experience molting problems similar to those caused by Microcystis. According to the statistical analysis, UWOCC CBS cells with a high microviridin J content, that is, cells adapted to 11 μmol of photons m−2 s−1, had a stronger adverse effect on daphnid molting and survival than did the Microcystis cells with a low cellular microviridin content (UWOCC CBS cultures grown at 25 μmol of photons m−2 s−1). Also, daphnids exposed to microviridin-rich Microcystis died faster than those that were starved (Fig. (Fig.11 and and22).

FIG. 1.
Frequency of molting disruption among individuals fed with either microviridin J-rich or microviridin J-poor Microcystis strain UWOCC CBS (cells grown under 11 or 25 μmol of photons m−2 s−1, respectively). In addition, results ...
FIG. 2.
Mortality among individuals fed with either microviridin J-rich or microviridin J-poor Microcystis strain UWOCC CBS (cells grown under 11 or 25 μmol of photons m−2 s−1, respectively). In addition, results for nonfood controls are ...

Dissolved purified microviridin J caused the same disruptive effect on the molting process of Daphnia as did cells of Microcystis strain UWOCC CBS or UWOCC MRC (3). After 12 h, an increasing number of individuals exposed to 4.5, 6.75, and 12 mg of microviridin J liter−1 showed molting problems identical to those described for experiments with intact Microcystis cells (Fig. (Fig.3).3). Lower microviridin J concentrations failed to induce this effect.

FIG. 3.
Frequency of molting disruption among individuals exposed to 4.5, 6.75, or 12 mg of purified microviridin J per liter of food suspension. All points represent the mean values of three replicates ± the respective standard deviations.

The present findings identified microviridin J as the likely source of the lethal molting disruption newly described by Kaebernick et al. (3) for Daphnia organisms feeding on Microcystis strain UWOCC MRC. Tests with purified microviridin J have proven that solutions of the compound affect daphnid molting in the same manner as that of UWOCC MRC. Further evidence has been provided by the present experiments with Microcystis strain UWOCC CBS. This strain was isolated from an American lake and most likely constitutes a unique genotype compared to its Australian counterpart UWOCC MRC. However, both strains produce microviridin J as their major oligopeptide and disrupt the daphnid molting process. Experiments in the present study have also shown that an increase in the cellular microviridin J content heightens the ability of Microcystis to induce the fatal molting pathology of Daphnia.

There may be several explanations for the molting disruption caused by microviridin J ingested as a component of Microcystis cells. Microviridin J is a very potent inhibitor of daphnid trypsin-like enzymes (8) and will, once released from digested Microcystis cells into the digestive cavity, result in incomplete protein digestion. In turn, Daphnia may become depleted of the amino acids essential for production and sclerotization of a new integument. Molting would also be disrupted if microviridin J inhibited trypsin-like proteases that normally digest the integument to be replaced and also afford its separation from the newly formed skin (2). For this to occur, an uptake of microviridin J from the gut into the blood of Daphnia would be required.

The present findings on the Daphnia toxicity of microviridin J link for the first time an acute toxic effect of cyanobacterial cells to a protease inhibitor contained within those cells. Consequently, hundreds of other cyanobacterial metabolites with activities against proteolytic enzymes (1, 6) must be considered potentially toxic to zooplankton grazers.

Acknowledgments

We express our gratitude to Nils Willumsen for his laboratory assistance.

This study was supported by grant FMRX-CT97-0097 (European Commission) to K.C. M.K. was supported by the Alexander von Humboldt Foundation.

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