Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna

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 IC₅₀ 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.     

Context dependency of infectious disease: the cyanobacterium Microcystis aeruginosa decreases white bacterial disease in Daphnia magna

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Interclonal proteomic responses to predator exposure in Daphnia magna may depend on predator composition of habitats

Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterizing the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the water flea, is a textbook example for predator‐induced phenotypic plastic defences; however, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages. We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. To get a more comprehensive idea of this phenomenon, we studied four genotypes with five biological replicates each, originating from habitats characterized by different predator composition, ranging from predator‐free habitats to habitats containing T. cancriformis. We analysed the morphologies as well as proteomes of predator‐exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high‐throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype‐dependent manner. Proteins connected to genotype‐dependent responses were related to the cuticle, protein synthesis and calcium binding, whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype‐dependent responses at the proteome level were most distinct for the only genotype that shares its habitat with Triops. Altogether, our study provides new insights concerning genotype‐dependent and general molecular processes involved in predator‐induced phenotypic plasticity in D. magna.     

Parasite‐mediated selection in a natural metapopulation of Daphnia magna

Parasite‐mediated selection varying across time and space in metapopulations is expected to result in host local adaptation and the maintenance of genetic diversity in disease‐related traits. However, nonadaptive processes like migration and extinction‐(re)colonization dynamics might interfere with adaptive evolution. Understanding how adaptive and nonadaptive processes interact to shape genetic variability in life‐history and disease‐related traits can provide important insights into their evolution in subdivided populations. Here we investigate signatures of spatially fluctuating, parasite‐mediated selection in a natural metapopulation of Daphnia magna. Host genotypes from infected and uninfected populations were genotyped at microsatellite markers, and phenotyped for life‐history and disease traits in common garden experiments. Combining phenotypic and genotypic data a Q_ST–F_ST‐like analysis was conducted to test for signatures of parasite mediated selection. We observed high variation within and among populations for phenotypic traits, but neither an indication of host local adaptation nor a cost of resistance. Infected populations have a higher gene diversity (Hs) than uninfected populations and Hs is strongly positively correlated with fitness. These results suggest a strong parasite effect on reducing population level inbreeding. We discuss how stochastic processes related to frequent extinction‐(re)colonization dynamics as well as host and parasite migration impede the evolution of resistance in the infected populations. We suggest that the genetic and phenotypic patterns of variation are a product of dynamic changes in the host gene pool caused by the interaction of colonization bottlenecks, inbreeding, immigration, hybrid vigor, rare host genotype advantage and parasitism. Our study highlights the effect of the parasite in ameliorating the negative fitness consequences caused by the high drift load in this metapopulation.     

Temporal genetic stability in natural populations of the waterflea Daphnia magna in response to strong selection pressure

Studies monitoring changes in genetic diversity and composition through time allow a unique understanding of evolutionary dynamics and persistence of natural populations. However, such studies are often limited to species with short generation times that can be propagated in the laboratory or few exceptional cases in the wild. Species that produce dormant stages provide powerful models for the reconstruction of evolutionary dynamics in the natural environment. A remaining open question is to what extent dormant egg banks are an unbiased representation of populations and hence of the species’ evolutionary potential, especially in the presence of strong environmental selection. We address this key question using the water flea Daphnia magna, which produces dormant stages that accumulate in biological archives over time. We assess temporal genetic stability in three biological archives, previously used in resurrection ecology studies showing adaptive evolutionary responses to rapid environmental change. We show that neutral genetic diversity does not decline with the age of the population and it is maintained in the presence of strong selection. In addition, by comparing temporal genetic stability in hatched and unhatched populations from the same biological archive, we show that dormant egg banks can be consulted to obtain a reliable measure of genetic diversity over time, at least in the multidecadal time frame studied here. The stability of neutral genetic diversity through time is likely mediated by the buffering effect of the resting egg bank.     

Carapace epithelia are rich in large filamentous actin bundles in Daphnia magna,Daphnia pulex,and Sida crystallina (Crustacea: Cladocera)

Cladocerans (water fleas) are planktonic crustaceans that typically have a bivalved carapace. Each valve of the carapace consists of two cuticle‐secreting epithelial layers that are separated by a hemolymphatic chamber and joined by pillar structures. Ultrastructural analyses in several species of Cladocera have shown that the carapace epithelia and pillars contain filamentous structures of unknown composition. In the present study we used a fluorescent phalloidin conjugate to show that the carapaces of three cladocerans, Daphnia magna, D. pulex, and Sida crystallina, are rich in large bundles of filamentous actin (F‐actin). In D. magna we employed confocal microscopy and orthogonal views of three‐dimensional reconstructions to show that these bundles extend radially from foci in the pillars towards the integument surfaces, and their structure is consistent with that of contractile stress fibers. Using a fluorescent lipophilic stain, DiOC₆(3), we show that the F‐actin bundles are distributed in membrane‐rich regions within the carapace epithelia, and that, in the superficial epithelium, these may be large membrane‐bound organelles. In D. magna, the F‐actin bundles are present in embryonic, juvenile instar, and adult, developmental stages, and through development the bundles become larger, contain more F‐actin, and become more widely spaced. We present an alignment of the deduced amino acid sequences of six putative D. pulex actin genes, and discuss the implications that their respective sequences have on the likelihood of their inclusion into the F‐actin bundles of the carapace. Our identification of these large F‐actin bundles within the pillars of three cladocerans provides new insight into the role these structures play in influencing carapace dynamics within this order.     

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub‐cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter‐ and intra‐molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle‐specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.     

Inbreeding and adaptive plasticity: an experimental analysis on predator‐induced responses in the water flea Daphnia

Several studies have emphasized that inbreeding depression (ID) is enhanced under stressful conditions. Additionally, one might imagine a loss of adaptively plastic responses which may further contribute to a reduction in fitness under environmental stress. Here, we quantified ID in inbred families of the cyclical parthenogen Daphnia magna in the absence and presence of fish predation risk. We test whether predator stress affects the degree of ID and if inbred families have a reduced capacity to respond to predator stress by adaptive phenotypic plasticity. We obtained two inbred families through clonal selfing within clones isolated from a fish pond. After mild purging under standardized conditions, we compared life history traits and adaptive plasticity between inbred and outbred lineages (directly hatched from the natural dormant egg bank of the same pond). Initial purging of lineages under standardized conditions differed among inbred families and exceeded that in outbreds. The least purged inbred family exhibited strong ID for most life history traits. Predator‐induced stress hardly affected the severity of ID, but the degree to which the capacity for adaptive phenotypic plasticity was retained varied strongly among the inbred families. The least purged family overall lacked the capacity for adaptive phenotypic plasticity, whereas the family that suffered only mild purging exhibited a potential for adaptive plasticity that was comparable to the outbred population. We thus found that inbred offspring may retain the capacity to respond to the presence of fish by adaptive phenotypic plasticity, but this strongly depends on the parental clone engaging in selfing.     

Direct and transgenerational effects of an experimental heatwave on early life stages in a freshwater snail

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A novel approach to parasite population genetics: Experimental infection reveals geographic differentiation,recombination and host‐mediated population structure in Pasteuria ramosa,a bacterial parasite of Daphnia

The population structure of parasites is central to the ecology and evolution of host‐parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well‐separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host—a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host‐parasite system.     

Grandparental effects in marine sticklebacks: transgenerational plasticity across multiple generations

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short‐term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry‐over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.     

Ecological and evolutionary factors of intraspecific variation in inducible defenses: Insights gained from Daphnia experiments

Phenotypic variation among individuals and species is a fundamental principle of natural selection. In this review, we focus on numerous experiments involving the model species Daphnia (Crustacea) and categorize the factors, especially secondary ones, affecting intraspecific variations in inducible defense. Primary factors, such as predator type and density, determine the degree to which inducible defense expresses and increases or decreases. Secondary factors, on the other hand, act together with primary factors to inducible defense or without primary factors on inducible defense. The secondary factors increase intraspecies variation in inducible defense, and thus, the level of adaptation of organisms varies within species. Future research will explore the potential for new secondary factors, as well as the relative importance between factors needs to be clarified.     

Maternal predator‐exposure affects offspring size at birth but not telomere length in a live‐bearing fish

The perception of predation risk could affect prey phenotype both within and between generations (via parental effects). The response to predation risk could involve modifications in physiology, morphology, and behavior and can ultimately affect long‐term fitness. Among the possible modifications mediated by the exposure to predation risk, telomere length could be a proxy for investigating the response to predation risk both within and between generations, as telomeres can be significantly affected by environmental stress. Maternal exposure to the perception of predation risk can affect a variety of offspring traits but the effect on offspring telomere length has never been experimentally tested. Using a live‐bearing fish, the guppy (Poecilia reticulata), we tested if the perceived risk of predation could affect the telomere length of adult females directly and that of their offspring with a balanced experimental setup that allowed us to control for both maternal and paternal contribution. We exposed female guppies to the perception of predation risk during gestation using a combination of both visual and chemical cues and we then measured female telomere length after the exposure period. Maternal effects mediated by the exposure to predation risk were measured on offspring telomere length and body size at birth. Contrary to our predictions, we did not find a significant effect of predation‐exposure neither on female nor on offspring telomere length, but females exposed to predation risk produced smaller offspring at birth. We discuss the possible explanations for our findings and advocate for further research on telomere dynamics in ectotherms.     

Phenotypic integration plasticity in Daphnia magna: an integral facet of G × E interactions

Phenotypic integration can be defined as the network of multivariate relationships among behavioural, physiological and morphological traits that describe the organism. Phenotypic integration plasticity refers to the change in patterns of phenotypic integration across environments or ontogeny. Because studies of phenotypic plasticity have predominantly focussed on single traits, a G × E interaction is typically perceived as differences in the magnitude of trait expression across two or more environments. However, many plastic responses involve coordinated responses in multiple traits, raising the possibility that relative differences in trait expression in different environments are an important, but often overlooked, source of G × E interaction. Here, we use phenotypic change vectors to statistically compare the multivariate life‐history plasticity of six Daphnia magna clones collected from four disparate European populations. Differences in the magnitude of plastic responses were statistically distinguishable for two of the six clones studied. However, differences in phenotypic integration plasticity were statistically distinguishable for all six of the clones studied, suggesting that phenotypic integration plasticity is an important component of G × E interactions that may be missed unless appropriate multivariate analyses are used.     

Intrapopulation variability in a functional trait: Susceptibility of Daphnia to limitation by dietary fatty acids

1. Functional traits are measurable characteristics of an organism that have an impact on its fitness. Variation in functional traits between and among species has been suggested to represent the basis for competition and selection, thus allowing for evolution in natural populations.
2. In freshwater ecosystems, the availability of essential polyunsaturated fatty acids (PUFAs), in particular ω3‐ and ω6‐PUFAs, determines the food quality of phytoplankton for the herbivorous zooplankton Daphnia, an unselective filter feeder. The content of such essential PUFAs in the phytoplankton is thus a functional phytoplankton trait affecting the trophic transfer efficiency and dynamics at the pelagic plant–herbivore interface.
3. In turn, the susceptibility of consumers to become limited by the availability of essential PUFAs is a fitness‐determining trait of Daphnia genotypes, and variability of this herbivore trait may thus affect the daphnids’ intrapopulation competition. To estimate the intrapopulation variation in susceptibility, we isolated clonal lines of Daphnia longispina from a natural population and compared the strength of their limitation by dietary PUFA availability via standardised laboratory growth assays. We used a liposome supplementation technique to enrich a PUFA‐poor green alga with essential ω3‐ and ω6‐PUFAs and determined juvenile somatic growth rate of different D. longispina genotypes as a fitness proxy.
4. As expected, D. longispina genotypes that coexisted in a natural population differed markedly in their specific patterns of susceptibility to dietary PUFA availability. On average, the D. longispina population was more strongly susceptible to limitations in the availability of the ω6‐PUFA arachidonic acid (20:4ω6) than to limitations in the availability of ω3‐PUFAs α‐linolenic acid (18:3ω3) and eicosapentaenoic acid (20:5ω3).
5. The ability to cope with PUFA limitation is thus a crucial trait that can probably affect intraspecific competition and Daphnia population structure. Therefore, we suggest that such intrapopulation variation in susceptibility to absence of dietary PUFAs might be one of the driving forces of natural selection and local adaptation among freshwater zooplankton.

     

Extinction hazards in experimental Daphnia magna populations: effects of genotype diversity and environmental variation

Extinction is ubiquitous in natural systems and the ultimate fate of all biological populations. However, the factors that contribute to population extinction are still poorly understood, particularly genetic diversity and composition. A laboratory experiment was conducted to examine the influences of environmental variation and genotype diversity on persistence in experimental Daphnia magna populations. Populations were initiated in two blocks with one, two, three, or six randomly selected and equally represented genotypes, fed and checked for extinction daily, and censused twice weekly over a period of 170 days. Our results show no evidence for an effect of the number of genotypes in a population on extinction hazard. Environmental variation had a strong effect on hazards in both experimental blocks, but the direction of the effect differed between blocks. In the first block, variable environments hastened extinction, while in the second block, hazards were reduced under variable food input. This occurred despite greater fluctuations in population size in variable environments in the second block of our experiment. Our results conflict with previous studies, where environmental variation consistently increased extinction risk. They are also at odds with previous studies in other systems that documented significant effects of genetic diversity on population persistence. We speculate that the lack of sexual reproduction, or the phenotypic similarity among our experimental lines, might underlie the lack of a significant effect of genotype diversity in our study.     

Chemical egg defence in the large milkweed bug,Oncopeltus fasciatus,derives from maternal but not paternal diet

Many herbivorous insects sequester defensive compounds from their host‐plants and incorporate them into their eggs to protect them against predation. Here, we investigate whether transmission of cardenolides from the host‐diet to the eggs is maternal, paternal, or biparental in the large milkweed bug, Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae). We reared individual bugs on either milkweed seeds [MW; Asclepias syriaca L. (Apocynaceae)] that contain cardenolides, or on sunflower seeds [SF; Helianthus annuus L. (Asteraceae)] that do not contain cardenolides. We mated females and males so that all four maternal/paternal diet combinations were represented: MW/MW, MW/SF, SF/MW, and SF/SF. Using larvae of the common green lacewing, Chrysoperla (Chrysopa) carnea (Stevens) (Neuroptera: Chrysopidae), we conducted two‐choice predation trials to assess whether maternal, paternal, or biparental transmission of cardenolides into the eggs of O. fasciatus increased protection against predation. Furthermore, we used high performance liquid chromatography (HPLC) to assess putative cardenolide content of eggs from the various parental diet treatment groups. The predation trials suggested that regardless of male diet, eggs were afforded better protection when females had been raised on milkweed. However, many eggs were at least partially consumed. This suggests that although chemical defence of eggs does not guarantee protection to eggs on an individual basis, they may increase the probability that some eggs in a clutch are left intact thereby potentially conferring a fitness advantage to more offspring than if eggs are left unprotected. Based on HPLC analysis we found that maternal contribution of cardenolides was significantly greater than paternal contribution of cardenolides to the eggs, supporting the results of our predation trials that a maternal diet of milkweed makes eggs more distasteful than a paternal diet of milkweed.     

Exposure to natural pathogens reveals costly aphid response to fungi but not bacteria

Immune responses are costly, causing trade‐offs between defense and other host life history traits. Aphids present a special system to explore the costs associated with immune activation since they are missing several humoral and cellular mechanisms thought important for microbial resistance, and it is unknown whether they have alternative, novel immune responses to deal with microbial threat. Here we expose pea aphids to an array of heat‐killed natural pathogens, which should stimulate immune responses without pathogen virulence, and measure changes in life‐history traits. We find significant reduction in lifetime fecundity upon exposure to two fungal pathogens, but not to two bacterial pathogens. This finding complements recent genomic and immunological studies indicating that pea aphids are missing mechanisms important for bacterial resistance, which may have important implications for how aphids interact with their beneficial bacterial symbionts. In general, recent exploration of the immune systems of non‐model invertebrates has called into question the generality of our current picture of insect immunity. Our data highlight that taking an ecological approach and measuring life‐history traits to a broad array of pathogens provides valuable information that can complement traditional approaches.