Cyanobacteria facilitate parasite epidemics in Daphnia.

The seasonal dominance of cyanobacteria in the phytoplankton community of lake ecosystems can have severe implications for higher trophic levels. For herbivorous zooplankton such as Daphnia, cyanobacteria have poor nutritional value and some species can produce toxins affecting zooplankton survival and reproduction. Here we present another, hitherto largely unexplored aspect of cyanobacteria, namely that they can increase Daphnia susceptibility to parasites. In a 12-yr monthly time-series analysis of the Daphnia community in Greifensee (Switzerland), we observed that cyanobacteria density correlated significantly with the epidemics of a common gut parasite of Daphnia, Caullerya mesnili, regardless of what cyanobacteria species was present or whether it was colonial or filamentous. The temperature from the previous month also affected the occurrence of Caullerya epidemics, either directly or indirectly by the promotion of cyanobacterial growth. A laboratory experiment confirmed that cyanobacteria increase the susceptibility of Daphnia to Caullerya, and suggested a possible involvement of cyanotoxins or other chemical traits of cyanobacteria in this process. These findings expand our understanding of the consequences of toxic cyanobacterial blooms for lake ecosystems and might be relevant for epidemics experienced by other aquatic species.

Parental and hybrid Daphnia from the D. longispina complex: long-term dynamics in genetic structure and significance of overwintering modes.

In recent decades, hybridization has become a focus of attention because of its role in evolutionary processes. However, little is known about changes in genetic structure within and between parental species and hybrids over time. Here, we studied processes of genetic change in parental species and hybrids from the Daphnia longispina complex (Crustacea, Cladocera) over a period of six years across ten habitats. These cyclical parthenogens respond to fluctuating environments by switching from asexual to sexual reproduction. Importantly, sexually produced diapausing eggs, which resist extreme conditions such as low temperatures and serve as dispersal stages, are produced to a lower extent by hybrids. Long-term microsatellite data revealed clear differences between hybrids and parental species. In hybrids, clonal diversity values were lower, whereas heterozygosity and linkage disequilibrium values were higher compared to parental species. Clonal diversity of hybrids responded to the strength of the winter, with cold winters resulting in few genotypes in the following spring. In time windows when only asexual hybrid females survive, priority effects will favour the establishment of the hybrid offspring before hatchlings from parental diapause eggs can enter the community. The constant high levels of heterozygosity maintained by clonal reproduction in hybrids might lead to their successful establishment over time, when they are able to escape competition from both parental species. Although we found evidence that hybrids diversity depends on fluctuating environments, a direct link between hybrid abundance and the strength of winter was missing. Because of reduced adaptability in clonally reproducing hybrids, multiple factors must contribute to promoting their long-term success in fluctuating environments.

The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

Natural distribution and co-infection patterns of microsporidia parasites in the Daphnia longispina complex.

Microsporidia are intracellular parasites, frequently infecting the planktonic crustacean Daphnia. Questioning the ability to detect and identify microsporidia with conventional microscopic techniques, we applied molecular methods in order to investigate the distribution and co-infection patterns of this parasite among 8 communities of the Daphnia longispina hybrid complex. Eight microsporidian taxa were detected, including 3 that previously had not been characterized genetically. Microsporidian communities from nearby lakes were found to be more similar to each other, apparently due to short distance dispersal via secondary hosts. Moreover, we detected seasonal (but not interannual) changes in microsporidian community structure. With some microsporidia being host-specific, these changes might have resulted from seasonal changes in host taxon and clonal composition. The 2 dominant and closely related parasite species were found mainly in single infections, whereas another pair of related microsporidians was found predominantly in co-infections; suggesting species-level differences in the ability to colonize infected hosts. By applying molecular methods, we were not only able to unambiguously identify parasite taxa but also to reveal multiple infections that otherwise would have remained undetected. Given the increased level of accuracy and sensitivity, we highly recommend molecular approaches in future parasite surveys of Daphnia infections.

Transmission mode affects the population genetic structure of Daphnia parasites.

Parasite life cycle variation can shape parasite evolution, by predisposing them towards different population genetic structures. We compared the population genetic structure of two co-occurring parasite species of Daphnia, to collect evidence for their expected transmission modes. The ichthyosporean Caullerya mesnili has a direct life cycle, whereas the microsporidian Berwaldia schaefernai is hypothesized to require passage through a secondary host. The parasites were collected from three geographically isolated Daphnia populations. The nucleotide variation in the internal transcribed spacer (ITS) region was assessed at the within-individual, within-population and among-population levels, using amova. We detected significant partitioning at all levels, except for a lack of among-population variation in Berwaldia. This was confirmed by neighbour-joining and principal component analyses; Caullerya populations were distinct from each other, while there was much overlap among parasite isolates representing different populations of Berwaldia. This all implies a higher amount of gene flow for Berwaldia, consistent with the hypothesized transmission mode.

Higher parasite resistance in Daphnia populations with recent epidemics.

Natural populations often show genetic variation in parasite resistance, forming the basis for evolutionary response to selection imposed by parasitism. We investigated whether previous epidemics selected for higher resistance to novel parasite isolates in a Daphnia galeata-microparasite system by comparing susceptibility of host clones from populations with varying epidemic history. We manipulated resource availability to evaluate whether diet influences Daphnia susceptibility as epidemics are common in nutrient-rich lakes. Exposing clones from 10 lakes under two food treatments to an allopatric protozoan parasite, we found that Daphnia originating from lakes (mainly nutrient rich) with previous epidemics better resist infection. Despite this result, there was a tendency of higher susceptibility in the low food treatment, suggesting that higher resistance of clones from populations with epidemic background is not directly caused by lake nutrient level. Rather, our results imply that host populations respond to parasite-mediated selection by evolving higher parasite resistance.

Virulence, cultivating conditions, and phylogenetic analyses of oomycete parasites in Daphnia.

We describe the infectivity, virulence, cultivating conditions, and phylogenetic positions of naturally occurring oomycete parasites of Daphnia, invertebrates which play a major role in aquatic food webs. Daphnia pulex individuals were found dead and covered by oomycete mycelia when exposed to pond sediments. We were able to extract 4 oomycete isolates from dead Daphnia and successfully cultivate them. Using the ITS and LSU rDNA sequences, we further showed these isolates to be distinct species. The isolates were experimentally demonstrated to be parasitic and not saprobic. After exposure to the parasites, Daphnia mortality was much higher than that reported for Daphnia infected with other known parasite species. Therefore, it is likely that oomycete parasites are important selective pressures in natural Daphnia populations. Moreover, their close phylogenetic relationship to parasites of fish and algae suggests that the stability of aquatic food webs (i.e. fish-Daphnia-algae) might be influenced by the shared parasite communities.

Preparation and characterization of singly labeled ruthenium polypyridine cytochrome c derivatives.

A novel two-step procedure has been developed to prepare cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)]. In the first step, cytochrome c was treated with the mono-N-hydroxysuccinimide ester of 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) to convert positively charged lysine amino groups to negatively charged dcbpy-lysine groups. Singly labeled dcbpy-cytochrome c derivatives were then separated and purified by ion-exchange chromatography. In the second step, the individual dcbpy-cytochrome c derivatives were treated with RuII(bpy)2CO3 to form singly labeled RuII(bpy)2(dcbpy-cytochrome c) derivatives. The specific lysine labeled in each derivative was determined by reverse-phase chromatography of a tryptic digest. All of the derivatives had a strong luminescence emission centered at 662 nm, but the luminescence decay rates were increased relative to that of a non-heme protein control, RuII(bpy)2(dcbpy-lysozyme), which was 1.8 X 10(6) s-1. The luminescence decay rates were found to be 21, 16, 7.2, 5.7, 4.3, 4.3, and 3.5 X 10(6) s-1 for derivatives singly labeled at lysines 13, 72, 25, 7, 39, 86, and 87, respectively. There was an inverse relationship between the luminescence decay rates and the distances between the ruthenium labels and the heme group. The increased luminescence decay rates observed in the cytochrome c derivatives might be due to electron transfer from the excited triplet state of ruthenium to the ferric heme group. However, it is also possible that an energy-transfer mechanism might contribute to the luminescence quenching.(ABSTRACT TRUNCATED AT 250 WORDS)