Protease inhibitors (PIs) have frequently been found in cyanobacterial blooms and have been shown to affect the major herbivore Daphnia by decreasing growth and inhibiting gut protease activity. However, it has been shown that a clone of Daphnia is able to respond to dietary PIs by increasing its protease gene expression. Such an inducible response might be maternally transferred to the next generation. Therefore, we tested a tolerant clone for maternal transfer of protease gene expression. When exposed to the trypsin inhibitor-producing cyanobacterium Microcystis aeruginosa PCC7806 Mut, Daphnia mothers and their untreated newborns showed an increase in trypsin gene expression compared to naïve mothers grown on control food and their offspring. The maternally transferred increase in gene expression was accompanied by a higher somatic growth rate of the offspring generation from exposed mothers compared to offspring from naïve mothers. This higher growth rate compensated for the lower dry mass of newborns from exposed mothers and led to the same fitness as observed in the offspring of naïve mothers. In nature, clones that can maternally transfer increased protease gene expression should have an advantage over clones that cannot. The selection for such more tolerant clones by naturally occurring PIs might lead to microevolution of natural Daphnia populations, and to local adaptation in the long term. This is the first study to show an adaptive maternal transfer of increased target gene expression in an ecological context.

It is known that cyanobacteria negatively affect herbivores due to their production of toxins such as protease inhibitors. In the present study we investigated potential interspecific differences between two major herbivores, Daphnia magna and Daphnia pulex, in terms of their tolerance to cyanobacteria with protease inhibitors. Seven clones each of D. magna and of D. pulex were isolated from different habitats in Europe and North America. To test for interspecific differences in the daphnids’ tolerance to cyanobacteria, their somatic and population growth rates were determined for each D. magna and D. pulex clone after exposure to varying concentrations of two Microcystis aeruginosa strains. The M. aeruginosa strains NIVA and PCC− contained either chymotrypsin or trypsin inhibitors, but no microcystins. Mean somatic and population growth rates on a diet with 20% NIVA were significantly more reduced in D. pulex than in D. magna. On a diet with 10% PCC−, the population growth of D. pulex was significantly more reduced than that of D. magna. This indicates that D. magna is more tolerant to cyanobacteria with protease inhibitors than D. pulex. The reduction of growth rates was possibly caused by an interference of cyanobacterial inhibitors with proteases in the gut of Daphnia, as many other conceivable factors, which might have been able to explain the reduced growth, could be excluded as causal factors. Protease assays revealed that the sensitivities of chymotrypsins and trypsins to cyanobacterial protease inhibitors did not differ between D. magna and D. pulex. However, D. magna exhibited a 2.3-fold higher specific chymotrypsin activity than D. pulex, which explains the observed higher tolerance to cyanobacterial protease inhibitors of D. magna. The present study suggests that D. magna may control the development of cyanobacterial blooms more efficiently than D. pulex due to differences in their tolerance to cyanobacteria with protease inhibitors.

Lakes are well known for having a pattern of seasonal succession of phytoplankton and zooplankton. The succession of different taxa of phytoplankton results in a succession of zooplankton taxa, and within the genus Daphnia, into a succession of different genotypes (clones). One cause for this succession of Daphnia clones might be the production of digestive protease inhibitors by cyanobacteria, which usually bloom in summer. Here we report seasonal changes in the frequency and the abundance of Daphnia magna haplotypes in a eutrophic lake, which developed a chymotrypsin-inhibitor-producing cyanobacterial bloom in May. These seasonal changes were not related to changes of biotic and abiotic lake parameters. However, a very high content of chymotrypsin inhibitors was observed in May (but not in other months). This was assumed to have exerted a strong punctual selection pressure on the Daphnia population and on the direct targets of the protease inhibitors, i.e. the digestive chymotrypsins of Daphnia. Actually, D. magna from before and during the cyanobacterial bloom showed a different protease pattern on activity stained SDS-PAGE in comparison to clones from the month after the bloom. However, no difference in tolerance, measured as IC50 values, to inhibition by natural lake seston from May was found between the clones from before and after the bloom. Thus, the hypothesis that a seasonal adaptation of D. magna subpopulations from either April/May or June might have occurred could not be proven. This suggests that the Daphnia population investigated here is locally adapted to cyanobacterial protease inhibitors.

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin-inhibitor-containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC(50) values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour-joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.

Daphnia populations often show rapid microevolutionary adaptation to environmental changes. Here, we investigated the possibility that microevolution of Daphnia populations could be driven by natural sestonic Protease Inhibition (PI). We hypothesized that PI changes seasonally, which might lead to concomitant changes in tolerance to PI in a co-occurring Daphnia magna population. In order to test this, seston from a eutrophic pond was sampled regularly over two successive years. Extracts of these freeze-dried samples were used to determine their Inhibitory Potential (IP) on D. magna gut proteases. In the summer seston the IP against chymotrypsins exceeded that of spring seston 200-fold. In order to test for possible impacts on the co-existing D. magna population, we isolated clones before (spring) and after (fall) the peak of the IP. Microsatellite analyses revealed that the two subpopulations were genetically distinct. Individual exposure of three clones from each population to varying concentrations of a cyanobacterium that contains chymotrypsin inhibitors revealed a decrease in population and somatic growth rate for each clone, but no seasonal effects on Daphnia’s tolerance. In order to include maternal effects, we conducted a multi-clonal competition experiment on various cyanobacterial concentrations. However, no evidence for seasonally increased tolerance of D. magna to dietary protease inhibitors could be found.

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour- and time-consuming. Here, we demonstrate high-resolution melting analysis (HRMA) as a cost-effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double-stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.

Daphnia has been shown to acquire tolerance to cyanobacterial toxins within an animals' lifetime and to transfer this tolerance to the next generation. Here we used a strain of the cyanobacterium Microcystis aeruginosa, which contained two chymotrypsin inhibitors (BN920 and CP954), the green alga Scenedesmus obliquus as reference food and a clone of D. magna to investigate the physiological mechanism of acquired tolerance to these cyanobacterial toxins. The intracellular concentrations of CP954 and BN920 were 1550 and 120 μmol l(-1), respectively. When food suspensions of the green alga contained >60% M. aeruginosa, growth rates of D. magna were reduced. When grown on the green alga, three chymotrypsins ranging in mass from 16 to 22 kDa were distinguished in D. magna. Purified BN920 and CP954 specifically inhibited D. magna chymotrypsins. Feeding with encapsulated BN920 resulted in growth depression in D. magna and replacement of the chymotrypsins by three chymotrypsins with smaller molecular mass. With just 20% M. aeruginosa, the same changes in the chymotrypsin pattern as with the pure inhibitor were observed. IC(50) values for inhibition of chymotrypsins of D. magna growing on the green alga were 5.4 nmol l(-1) (BN920) and 7.4 nmol l(-1) (CP954). When D. magna was grown on 20% M. aeruginosa, 2.2-fold higher IC(50) values were observed. This indicated that increased tolerance to these dietary inhibitors was acquired within an animal's lifetime by remodelling the digestive chymotrypsins, which in turn serves as an intra-generational defence against these cyanobacterial inhibitors. This mechanism might be relevant for the transfer of tolerance to the next generation through maternal effects.

BACKGROUND:The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.RESULTS:Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects.CONCLUSION:Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might determine the degree of growth reduction in D. magna in the presence of cyanobacterial protease inhibitors. The rapid response of Daphnia to cyanobacterial protease inhibitors supports the assumption that dietary cyanobacterial protease inhibitors exert a strong selection pressure on Daphnia proteases themselves.