Phylogenetic Characterization and Prevalence of “Spirobacillus cienkowskii,” a Red-Pigmented, Spiral-Shaped Bacterial Pathogen of Freshwater Daphnia Species

Microscopic examination of the hemolymph from diseased daphniids in 17 lakes in southwestern Michigan and five rock pools in southern Finland revealed the presence of tightly coiled bacteria that bore striking similarities to the drawings of a morphologically unique pathogen, “Spirobacillus cienkowskii,” first described by Elya Metchnikoff more than 100 years ago. The uncultivated microbe was identified as a deeply branching member of the Deltaproteobacteria through phylogenetic analyses of two conserved genes: the 16S rRNA-encoding gene (rrs) and the β-subunit of topoisomerase (gyrB). Fluorescence in situ hybridization confirmed that the rRNA gene sequence originated from bacteria with the tightly coiled morphology. Microscopy and PCR amplification with pathogen-specific primers confirmed infections by this bacterium in four species of Daphnia: Daphnia dentifera, D. magna, D. pulicaria, and D. retrocurva. Extensive field surveys reveal that this bacterium is widespread geographically and able to infect many different cladoceran species. In a survey of populations of D. dentifera in lakes in Michigan, we found the bacterium in 17 of 18 populations studied. In these populations, 0 to 12% of the individuals were infected, with an average of 3% during mid-summer and early autumn. Infections were less common in rock pool populations of D. magna in southern Finland, where the pathogen was found in 5 of 137 populations. The broad geographic distribution, wide host range, and high virulence of S. cienkowskii suggest it plays an important role in the ecology and evolution of daphniids.     

Selective predation and rapid evolution can jointly dampen effects of virulent parasites on Daphnia populations

Parasites are ubiquitous and often highly virulent, yet clear examples of parasite-driven changes in host density in natural populations are surprisingly scarce. Here, we illustrate an example of this phenomenon and offer a theoretically reasonable resolution. We document the effects of two parasites, the bacterium Spirobacillus cienkowskii and the yeast Metschnikowia bicuspidata, on a common freshwater invertebrate, Daphnia dentifera. We show that while both parasites were quite virulent to individual hosts, only bacterial epidemics were associated with significant changes in host population dynamics and density. Our theoretical results may help explain why yeast epidemics did not significantly affect population dynamics. Using a model parameterized with data we collected, we argue that two prominent features of this system, rapid evolution of host resistance to the parasite and selective predation on infected hosts, both decrease peak infection prevalence and can minimize decline in host density during epidemics. Taken together, our results show that understanding the outcomes of host-parasite interactions in this Daphnia-microparasite system may require consideration of ecological context and evolutionary processes and their interaction.     

Epidemiology of a Daphnia-multiparasite system and its implications for the red queen

The Red Queen hypothesis can explain the maintenance of host and parasite diversity. However, the Red Queen requires genetic specificity for infection risk (i.e., that infection depends on the exact combination of host and parasite genotypes) and strongly virulent effects of infection on host fitness. A European crustacean (Daphnia magna)--bacterium (Pasteuria ramosa) system typifies such specificity and high virulence. We studied the North American host Daphnia dentifera and its natural parasite Pasteuria ramosa, and also found strong genetic specificity for infection success and high virulence. These results suggest that Pasteuria could promote Red Queen dynamics with D. dentifera populations as well. However, the Red Queen might be undermined in this system by selection from a more common yeast parasite (Metschnikowia bicuspidata). Resistance to the yeast did not correlate with resistance to Pasteuria among host genotypes, suggesting that selection by Metschnikowia should proceed relatively independently of selection by Pasteuria.     

Long-term dynamics in rock pool Daphnia metapopulations

Metapopulations of Daphnia longispina , D. magna and D. pulex on 16 islands containing 507 rock pools were studied over 17 yr (autumns 1982–1998). Two yearly samples were taken representing early and late summer conditions. A local extinction was assumed when a species was absent from a pool for more than one year. Average yearly extinction rates, calculated from probability of survival in age grouped data, were close to 20%, and number of extinctions was on average balanced by the number of colonizations. Extinction rates during the first year following colonization were 45–50% in all species, but later decreased to low values. In first-year data, single species populations of D. magna had lower extinction rates than in multi-species pools. In later years all species combinations had similar extinction rates. The average proportion of rock pools occupied by D. longispina in any given year was 0.178, by D. magna 0.169 and by D. pulex 0.056. In 22% of cases, coexistence was observed. 71% of the pools were colonized at least once. The distribution of shortest colonization distances was left-skewed, and median distances (7–8 m) were same in all species. The distribution of colonizations mostly corresponded to the expected frequencies calculated as the product of frequencies of source and target pools. Both D. longispina and D. magna colonized pools already occupied with a somewhat higher probability than empty pools. Probably owing to variation of habitat quality, Daphnia populations in some rock pools appear to be less prone to extinction, providing sources of dispersal to patches with higher extinction risk. The system appears to be at an approximate equilibrium, with several characteristics close to classical metapopulation system.     

Rapid evolution and ecological host-parasite dynamics

Traditionally, the termination of parasite epidemics has been attributed to ecological causes: namely, the depletion of susceptible hosts as a result of mortality or acquired immunity. Here, we suggest that epidemics can also end because of rapid host evolution. Focusing on a particular host–parasite system, Daphnia dentifera and its parasite Metschnikowia bicuspidata , we show that Daphnia from lakes with recent epidemics were more resistant to infection and had less variance in susceptibility than Daphnia from lakes without recent epidemics. However, our studies revealed little evidence for genetic variation in infectivity or virulence in Metschnikowia . Incorporating the observed genetic variation in host susceptibility into an epidemiological model parameterized for this system reveals that rapid evolution can explain the termination of epidemics on time scales matching what occurs in lake populations. Thus, not only does our study provide rare evidence for parasite-mediated selection in natural populations, it also suggests that rapid evolution has important effects on short-term host–parasite dynamics.     

Host-parasite coevolution: Insights from the Daphnia-parasite model system

Daphnia and its parasites have become recognized as a model system for studying the epidemiological, evolutionary and genetic interactions between hosts and parasites. The key advantages of the Daphnia-parasite system are the propagation of the host as iso-female lines, that is clonal, but at the same time the possibility to cross lines. Furthermore, Daphnia have diverse parasites, including bacteria, fungi, microsporidia and helminths, which can be kept in culture with the hosts. For two parasites of Daphnia magna, coevolution has been demonstrated phenotypically. Coevolution in D. magna and the bacterium Pasteuria ramosa is consistent with model predictions of coevolution by negative frequency dependent selection, the Red Queen hypothesis. The genetic mechanisms have not yet been elucidated.     

Founder events as determinants of within-island and among-island genetic structure of Daphnia metapopulations

The genetic structure of metapopulations offers insights into the genetic consequences of local extinction and recolonization. We studied allozyme variation in rock pool metapopulations of two species of waterfleas (Daphnia) with the aim to understand how these dynamics influence genetic differentiation. We screened 138 populations of D. magna and 65 populations of D. longispina from an area in the archipelago of southern Finland. The pools from which they were sampled are separated by distances between 1.5 and 4710 m and located on a total of 38 islands. The genetic population structure of the two species was strikingly similar, consistent with their similar metapopulation ecology. The mean F(PT) value (differentiation among pools with respect to the total metapopulation) was 0.55 and a hierarchical analysis showed that genetic differentiation was strong (>0.25) among pools within islands as well as among whole islands. Within islands, pairwise genetic differentiation increased with geographic distance, indicating isolation by distance due to spatially limited dispersal. Previous studies have shown strong founder events occurring during colonization in our metapopulation. We suggest that the genetic population structure in the studied metapopulations is largely explained by three consequences of these founder events: (i) strong drift during colonization, (ii) local inbreeding, which results in hybrid vigour and increased effective migration rates after subsequent immigration, and (iii) effects of selection through hitchhiking of neutral genes with linked loci under selection.     

Physiology of immunity in the water flea Daphnia magna: environmental and genetic aspects of phenoloxidase activity

In an attempt to understand the ecological correlates of immunocompetence in Daphnia magna (Crustacea, Cladocera), we tested for variation in immune function in relation to feeding conditions, host conditions, and host genotype. We investigated both phenotypic (environmental dependent and condition dependent) as well as genotypic aspects of the prophenoloxidase activating system (Pro-POAS), which has been described as a key factor in invertebrate immunity. Daphnia magna is an ideal study system to disentangle phenotypic and genetic variation because females can reproduce clonally. Well-fed Daphnia showed higher phenoloxidase (PO) activity than Daphnia kept at a low food level. Wounding provoked a higher level of PO activity, indicating that the Pro-POAS was condition dependent. Further, we found clonal variation in PO activity among four clones of D. magna isolated from four different populations. The same four clones were tested for their resistance to the bacterial pathogen Pasteuria ramosa. High resistance corresponded to high PO activity. Our results suggest adaptive variation in PO activity and suggest that its expression is costly. These costs may influence the evolution of the PO activity level and the maintenance of its genotypic variation.     

Genetic diversity and genetic differentiation in Daphnia metapopulations with subpopulations of known age

If colonization of empty habitat patches causes genetic bottlenecks, freshly founded, young populations should be genetically less diverse than older ones that may have experienced successive rounds of immigration. This can be studied in metapopulations with subpopulations of known age. We studied allozyme variation in metapopulations of two species of water fleas (Daphnia) in the skerry archipelago of southern Finland. These populations have been monitored since 1982. Screening 49 populations of D. longispina and 77 populations of D. magna, separated by distances of 1.5-2180 m, we found that local genetic diversity increased with population age whereas pairwise differentiation among pools decreased with population age. These patterns persisted even after controlling for several potentially confounding ecological variables, indicating that extinction and recolonization dynamics decrease local genetic diversity and increase genetic differentiation in these metapopulations by causing genetic bottlenecks during colonization. We suggest that the effect of these bottlenecks may be twofold, namely decreasing genetic diversity by random sampling and leading to population-wide inbreeding. Subsequent immigration then may not only introduce new genetic material, but also lead to the production of noninbred hybrids, selection for which may cause immigrant alleles to increase in frequency, thus leading to increased genetic diversity in older populations.     

The relative importance of algae and bacteria as food for Daphnia longispina (Cladocera) in a polyhumic lake

SUMMARY. 1. Grazing on algae and bacteria by the planktonic cladoceran, Daphnia longispina , was studied in a small polyhumic lake with low phytoplankton primary production in southern Finland.
2. D. longispina filtered algae at average rates of 0.09-0.82 ml ind⁻¹ h⁻¹. The filtering rates on bacteria were 26-92% of those on algae in parallel experiments.
3. From June to August algae, including mixotrophic and heterotrophic forms, comprised 56-93% of the food ingested by D. longispina. In mid-September and early October, when the Daphnia population was declining and the algal biomass was low, bacteria comprised 73% and 55% respectively of the food of Daphnia.
4. For D. longispina , an energy pathway via bacteria and bactivorous flagellates is probably a more important link to allochthonous organic matter than direct utilization of epilimnetic bacteria.     

Experimental evolution of field populations of Daphnia magna in response to parasite treatment

Although there is little doubt that hosts evolve to reduce parasite damage, little is known about the evolutionary time scale on which host populations may adapt under natural conditions. Here we study the effects of selection by the microsporidian parasite Octosporea bayeri on populations of Daphnia magna. In a field study, we infected replicated populations of D. magna with the parasite, leaving control populations uninfected. After two summer seasons of experimental evolution (about 15 generations), the genetic composition of infected host populations differed significantly from the control populations. Experiments revealed that hosts from the populations that had evolved with the parasite had lower mortality on exposure to parasite spores and a higher competitive ability than hosts that had evolved without the parasite. In contrast, the susceptibility of the two treatment groups to another parasite, the bacterium Pasteuria ramosa, which was not present during experimental evolution of the populations, did not differ. Fitness assays in the absence of parasites revealed a higher fitness for the control populations, but only under low population density with high resource availability. Overall, our results show that, under natural conditions, Daphnia populations are able to adapt rapidly to the prevailing conditions and that this evolutionary change is specific to the environment.     

Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna

Estimates of phenoloxidase (PO) activity have been suggested as a useful indicator of immunocompetence in arthropods, with the idea that high PO activity would indicate high immunocompetence against parasites and pathogens. Here, we test for variation in PO activity among clones of the planktonic crustacean Daphnia magna and its covariation with susceptibility to infections from four different microparasite species (one bacterium and three microsporidia). Strong clonal variation in PO activity was found within and among populations of D. magna, with 45.6% of the total variation being explained by the clone effect. Quantitative measures of parasite success in infection correlated negatively with PO activity when tested across four host populations. However, these correlations disappeared when the data were corrected for population effects. We conclude that PO activity is not a useful measure of resistance to parasites or of immunocompetence within populations of D. magna. We further tested whether D. magna females that are wounded to induce PO activity are more resistant to infections with the bacterium Pasteuria ramosa than non-wounded controls. We found neither a difference in susceptibility nor a difference in disease progression between the induced group and the control group. These results do not question the function of the PO system in arthropod immune response, but rather suggest that immunocompetence cannot be assessed by considering PO activity alone. Immune response is likely to be a multifactorial trait with various host and parasite characteristics playing important roles in its expression.     

Food quality of heterotrophic bacteria for Daphnia magna: evidence for a limitation by sterols

The quality of heterotrophic bacteria as food for metazoan grazers has been investigated poorly. We conducted growth experiments with juvenile Daphnia magna feeding on different strains of heterotrophic bacteria that represent typical pelagic bacteria of five phylogenetically distinct groups. The bacterial food suspensions were supplemented with cholesterol and/or the polyunsaturated fatty acid eicosapentaenoic acid (EPA), two essential nutrients that are either absent or scarcely represented in bacteria. Our data imply that the selected heterotrophic bacteria are of poor food quality for D. magna, which was indicated either by very low somatic growth rates or by high mortality. However, with four out of six bacterial strains tested, the somatic growth rates increased significantly upon supplementation with cholesterol, which shows that the lack of sterols in bacteria is a major food quality constraint. We did not find clear evidence for a limitation by EPA on bacterial diets within our growth experiments. High mortality was observed when D. magna was fed with Hydrogenophaga sp. or Pseudomonas sp., which indicates that these two bacterial strains are toxic to D. magna. Our findings highlight the limitations of bacteria as a carbon source for Daphnia and point to a so far underestimated diversity of interactions between grazers and its bacterial food.     

Grazer-induced changes in the desmid Staurastrum

In aquatic environments, predator kairomones have been shown to affect morphology of prey species. Past work on the interaction between zooplankton and phytoplankton was based mainly on the Daphnia–Scenedesmus model. Algae of the genus Staurastrum can produce mucilage, causing cell clumping and settling out of the water column. These clumps are too large to be eaten by daphniids. Thus, we hypothesised that this may be a grazer defence. We investigated whether Daphnia magna induces the formation of mucus globules in Staurastrum, how this occurs, and if the formation of clumps inhibits growth in juvenile Daphnia. Eight strains of Staurastrum were used to check whether mucus extrusion is induced by the presence of Daphniaor possibly by a chemical excreted by Daphnia magna. None of the strains reacted to the presence of Daphnia water alone, animals had to be present to induce clumping. Mechanical action (gentle stirring) caused the same strains to clump. The ecological relevance of clumping was then investigated. The different Staurastrum strains were used as food in a growth experiment with ecologically relevant densities of neonates of Daphnia hyalina. These small daphniids did not cause the same clumping observed for Daphnia magna when present in experiments at high densities. We observed that juvenile daphniids grew less well on strains with larger cell size.     

Evidence of phosphorus-limited individual and population growth of Daphnia in a Canadian Shield lake

We performed bag experiments in a Canadian Shield lake with generally high seston (suspended food particles mainly composed of algae) carbon (C):phosphorus (P) ratios, and investigated the responses of individual and population growth of herbivorous Daphnia dentifera on their abundance with (+P) and without (−P) a phosphorus enrichment to lake water. In both treatments, increased abundance of D. dentifera reduced seston C concentration and was accompanied by decreases in population and individual growth rates. However, P-enrichment increased seston P concentration and then reduced seston C:P ratio from 400–700 to ca 100 (by atoms). As a result, both individual and population growth rates were significantly higher in the +P treatment at all animal abundances even though seston C concentrations were similar between the treatments. The magnitude of the growth enhancement by the P-enrichment was independent of animal abundance. Stepwise regression analyses revealed that 71 and 90% of the variance in the population and individual growth rates, respectively, were explained by seston C and P concentrations, and that the contribution of the seston P concentration was roughly the same as that of seston C. Such joint effects of seston C and P indicate that food quality (P content) as well as food quantity (C concentration) can influence Daphnia not only at the level of individual growth but also at the level of population dynamics in P-limited lakes. Our results thus strongly corroborate the hypothesis that the population development of a key herbivore Daphnia in P-limited Canadian Shield lakes is inhibited by the direct effects of P-limited food on individual growth, which weaken the strength of trophic cascading interactions starting from piscivorous fish through planktivorous fish and zooplankton to algae.     

Supplementation with sterols improves food quality of a ciliate for Daphnia magna

Experimental results provide evidence that trophic interactions between ciliates and Daphnia are constrained by the comparatively low food quality of ciliates. The dietary sterol content is a crucial factor in determining food quality for Daphnia. Ciliates, however, presumably do not synthesize sterols de novo. We hypothesized that ciliates are nutritionally inadequate because of their lack of sterols and tested this hypothesis in growth experiments with Daphnia magna and the ciliate Colpidium campylum. The lipid content of the ciliate was altered by allowing them to feed on fluorescently labeled albumin beads supplemented with different sterols. Ciliates that preyed upon a sterol-free diet (bacteria) did not contain any sterols, and growth of D. magna on these ciliates was poor. Supplementation of the ciliates' food source with different sterols led to the incorporation of the supplemented sterols into the ciliates' cells and to enhanced somatic growth of D. magna. Sterol limitation was thereby identified as the major constraint of ciliate food quality for Daphnia. Furthermore, by supplementation of sterols unsuitable for supporting Daphnia growth, we provide evidence that ciliates as intermediary grazers biochemically upgrade unsuitable dietary sterols to sterols appropriate to meet the physiological demands of Daphnia.     

Parasite-mediated selection in experimental Daphnia magna populations

Abstract It has been suggested that parasites are a strong selecting force for their hosts and therefore may alter the outcome of competition among host genotypes. We tested the extent to which parasite-mediated selection by different parasite species influenced competition among clones of the cyclic parthenogen Daphnia magna . We monitored clone frequency changes in laboratory microcosm populations consisting of 21 D. magna clones. Parasite treatments (two microsporidians, Glugoides intestinalis and Ordospora colligata ) and a parasite-free control treatment were followed over a nine-month period. A further treatment with the bacterium Pasteuria ramosa failed. We found significant differences in clonal success among the treatments: the two parasite treatments differed from the control treatment and from each other. Additionally, we measured the clone-specific population carrying capacity, competitive ability against tester clones, and reproductive success of infected and uninfected females to test whether they correlate with clonal success in the microcosms. The clone-specific competitive ability was a good predictor of clonal success in the microcosms, but clonal carrying capacity and host reproductive success were not. Our study shows that parasite-mediated selection can strongly alter the outcome of clonal competition. The results suggest that parasites may influence microevolution in Daphnia populations during periods of asexual reproduction.     

Enzyme Variability in Natural Populations of DAPHNIA MAGNA II. Genotypic Frequencies in Permanent Populations

In permanent habitats populations of the cyclical parthenogen, Daphnia magna, reproduce by continued parthenogenesis and are subject to only sporadic sexual recruitment. The genetic effects of this breeding system have been investigated by analyzing allozyme frequencies in thirteen populations of D. magna.-Genotypic frequencies at polymorphic loci characteristically deviated markedly from Hardy-Weinberg proportions and gametic phase imbalance between loci was frequent. Genotypic frequencies were subject to violent, selectively determined oscillations over short periods of time. These observations suggest that permanent populations of D. magna ordinarily consist of a limited number of highly structured genotypes. The adaptational advantages offered by such structuring may have been a major selective factor favoring the evolution of cyclical parthenogenesis.     

Density and size distribution of Daphnia populations at different fish predation levels

Density and size structure changes of natural daphniids populations were studied in enclosures with a different level of fish predation. Daphnia pulicaria was totaly removed in high predation variants, and the differences of the mean body length both in adults and juveniles are apparent between low predation enclosure and enclosure without fish. Daphnia galeata was replaced by D. magna and D. pulicaria in the enclosure without fish. The decline of densities and the substantial (30–50%) and fast (during 12 days) shift of mean body length both in adults and juveniles of D. galeata was induced by the fish (carp fry) introduction to the high predation enclosures.