Molecular Identification and Hidden Diversity of Novel Daphnia Parasites from European Lakes

Parasites play important roles in local population dynamics and genetic structure. However, due to insufficient diagnostic tools, detailed host-parasite interactions may remain concealed by hidden parasite diversity in natural systems. Microscopic examination of 19 European lake Daphnia populations revealed the presence of three groups of parasites: fungi, microsporidia, and oomycetes. For most of these parasites no genetic markers have been described so far. Based on sequence similarities of the nuclear small-subunit and internal transcribed spacer (ITS) rRNA gene regions, one fungus, four microsporidian, and nine oomycete taxa were discovered in 147 infected Daphnia (and/or three other zooplankton crustaceans). Additionally, cloning of rRNA gene regions revealed parasite sequence variation within host individuals. This was most pronounced in the ITS region of one microsporidian taxon, where the within-host sequence variation ranged from 1.7% to 5.3% polymorphic sites for parasite isolates from 14 different geographical locations. Interestingly, the parasite isolates from close locations grouped together based on sequence similarities, suggesting that there was parasite dispersal. Taken together, the data obtained in this study revealed hidden diversity of parasite communities in Daphnia lake populations. Moreover, a higher level of resolution for identifying parasite strains makes it possible to test new hypotheses with respect to parasite dispersal, transmission routes, and coinfection.During the last decade, microparasites of Daphnia species, which are small zooplankton crustaceans, have become a popular study system in ecological and evolutionary research (for a review, see reference 15). It has been shown both in the field and under controlled laboratory conditions that parasites have a substantial impact on Daphnia fitness (7, 21, 52). Parasite-induced reductions in Daphnia population density (11, 12) or even population crashes (17) might result in disruptions of aquatic food webs, as daphnids play important roles as main phytoplankton grazers and as a major food of planktivorous fish (27). Moreover, as infections are often genotype specific (6, 8), they can lead to changes in the gene pool of a Daphnia population (7, 14), sometimes significantly increasing the genetic diversity of the host population (12, 54). Thus, Daphnia parasites cause not only ecological but also evolutionary changes in aquatic systems.Conclusions regarding the importance of parasites in natural systems require powerful tools to detect and properly identify parasite taxa. Thus far, few species-specific molecular markers have been developed for Daphnia parasites (33, 38, 39, 41) and then used in experimental studies (3). In surveys of natural Daphnia populations, parasite identification has been based primarily on microscopic examination (4, 5, 29, 52), with only one exception (32). The parasites recorded in natural populations of Daphnia are thus considered members of certain taxa, or even species, without genetic confirmation. The fact that molecular markers are not used to characterize Daphnia infections makes it difficult to compare epidemic patterns across different habitats and/or various field surveys, as parasites cannot be unambiguously identified by microscopic examination alone. Even if microscopic identification is theoretically possible (for example, by examining ultrastructural morphology by electron microscopy [37]), this approach is not feasible for routine analysis. Consequently, classification of parasites that actually belong to different taxa in the same group might introduce noise into field surveys, as parasite taxa differ widely in virulence and host range (for a review, see reference 15).Most of the known Daphnia parasites that have been described were obtained from small temporary ponds and rock pools (4, 16, 43). In permanent lakes, lower parasite diversity was assumed, mainly because increased fish predation reduces the population density of potential hosts (18), whereas high host density is a crucial determinant of epidemic spread (1, 2, 45). In addition, infected Daphnia spp. are more vulnerable to fish that hunt visually due to loss of their transparent appearance (11, 13). On the other hand, it was recently shown that even if Daphnia host density was reduced by selective fish predation, the prevalence of infection did not decline, probably due to the very high rates of transmission of the parasite that was observed (11). In contrast, we expected that the highly heterogeneous biotic and abiotic conditions in permanent lakes (27) would provide a variety of niches (for a review, see reference 47), which also favor a high level of parasite diversity. Therefore, Czech canyon-shaped reservoirs were chosen as our main study systems, because in these lakes environmental gradients are particularly pronounced in both the horizontal and vertical dimensions (42). Moreover, the Daphnia communities of these reservoirs are dominated by members of the Daphnia longispina complex (35), taxa which have previously been shown to be infected by a variety of parasites (52).The results of our study revealed a high level of diversity of Daphnia parasites in permanent lakes. Fourteen different parasite taxa were detected using nuclear small-subunit (SSU) and internal transcribed spacer (ITS) rRNA gene sequence information. In addition, a high level of sequence variation was observed in the ITS region of one microsporidian taxon. Thus, molecular markers are now available which allow discrimination with high resolution among and within parasite taxa and provide tools to address more detailed questions concerning lake Daphnia-microparasite systems.     

Der Asiatische Marienkäfer als Modell

The harlequin ladybird as a model in invasion biology The harlequin ladybird Harmonia axyridis has been established as a promined model in invasion biology because it particularly suited to test hypotheses explaining the successful spread of some introduced species. Recent research elucidated that H. axyridis carries parasites which can infect native ladybird species if the feed on its eggs or larvae within intraguild predation. Besides these “bioweapons” H. axyridis accumulates small molecules in its hemolymph which provide as chemical weapons protection against pathogens and predators. The application of state of the art genetic tools will help to elucidate adaptations mediating invasive success at the molecular level.     

Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly‐ubiquitinated by the proteasome‐associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat‐shock or arsenite treatment, when poly‐ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin‐conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.     

Estimation of Density-Dependent Mortality of Juvenile Bivalves in the Wadden Sea

We investigated density-dependent mortality within the early months of life of the bivalves Macoma balthica (Baltic tellin) and Cerastoderma edule (common cockle) in the Wadden Sea. Mortality is thought to be density-dependent in juvenile bivalves, because there is no proportional relationship between the size of the reproductive adult stocks and the numbers of recruits for both species. It is not known however, when exactly density dependence in the pre-recruitment phase occurs and how prevalent it is. The magnitude of recruitment determines year class strength in bivalves. Thus, understanding pre-recruit mortality will improve the understanding of population dynamics. We analyzed count data from three years of temporal sampling during the first months after bivalve settlement at ten transects in the Sylt-Rømø-Bay in the northern German Wadden Sea. Analyses of density dependence are sensitive to bias through measurement error. Measurement error was estimated by bootstrapping, and residual deviances were adjusted by adding process error. With simulations the effect of these two types of error on the estimate of the density-dependent mortality coefficient was investigated. In three out of eight time intervals density dependence was detected for M. balthica, and in zero out of six time intervals for C. edule. Biological or environmental stochastic processes dominated over density dependence at the investigated scale.     

Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects

Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.     

Traits and phylogenetic history contribute to network structure across Canadian plant–pollinator communities

Interaction webs, or networks, define how the members of two or more trophic levels interact. However, the traits that mediate network structure have not been widely investigated. Generally, the mechanism that determines plant-pollinator partnerships is thought to involve the matching of a suite of species traits (such as abundance, phenology, morphology) between trophic levels. These traits are often unknown or hard to measure, but may reflect phylogenetic history. We asked whether morphological traits or phylogenetic history were more important in mediating network structure in mutualistic plant-pollinator interaction networks from Western Canada. At the plant species level, sexual system, growth form, and flower symmetry were the most important traits. For example species with radially symmetrical flowers had more connections within their modules (a subset of species that interact more among one another than outside of the module) than species with bilaterally symmetrical flowers. At the pollinator species level, social species had more connections within and among modules. In addition, larger pollinators tended to be more specialized. As traits mediate interactions and have a phylogenetic signal, we found that phylogenetically close species tend to interact with a similar set of species. At the network level, patterns were weak, but we found increasing functional trait and phylogenetic diversity of plants associated with increased weighted nestedness. These results provide evidence that both specific traits and phylogenetic history can contribute to the nature of mutualistic interactions within networks, but they explain less variation between networks.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.