Lipofuscin, amyloids, and lipid peroxidation as potential markers of aging in Daphnia.

Accumulation of autofluorescent waste products, amyloids, and products of lipid peroxidation (LPO) are important hallmarks of aging. Until now, these processes have not been documented in Daphnia, a convenient model organism for longevity and senescence studies. We conducted a longitudinal cohort study of autofluorescence and Congo Red (CR) fluorescent staining for amyloids in four clones of D. magna. Additionally, we used a single time point cross-sectional common garden experiment within a single clone in which autofluorescence and BODIPY C11 fluorescence were measured. We observed a robust increase in autofluorescent spots that show diagnostic co-staining by Sudan Black indicating lipofuscin aggregates, particularly in the upper body region. There was also a significant clone-by-age interaction indicating that some genotypes accumulated lipofuscins faster than others. Contrary to predictions, CR fluorescence and lipid peroxidation did not consistently increase with age. CR fluorescence demonstrated a slight non-monotonous relationship with age, achieving the highest values at intermediate ages, possibly due to elimination of physiological heterogeneity in our genetically uniform cohorts. LPO demonstrated a significant ovary status-by-age interaction, decreasing with age when measured in Daphnia with full ovaries (late phase ovarian cycle) and showing no significant trend or slight increase with age when measured during the early phase in the ovarian cycle.

Does geographic variation in thermal tolerance in Daphnia represent trade-offs or conditional neutrality?

Geographic variation in thermal tolerance in Daphnia seems to represent genetic load at the loci specifically responsible for heat tolerance resulting from conditional neutrality. We see no evidence of trade-offs between fitness-related traits at 25 °C vs. 10 °C or between two algal diets across Daphnia magna clones from a variety of locations representing the opposite ends of the distribution of long-term heat tolerance. Likewise, we found no evidence of within-environment trade-offs between heat tolerance and fitness-related traits in any of the environments. Neither short-term and long-term heat tolerance shows any consistent relationship with lipid fluorescence polarization and lipid peroxidation across clones or environments. Pervasive positive correlations between fitness-related traits indicate differences in genetic load rather than trade-off based local adaptation or thermal specialization. For heat tolerance such differences may be caused by either relaxation of stabilizing selection due to lower exposure to high temperature extremes, i.e., conditional neutrality, or by small effective population size followed by the recent range expansion.

Breaking free from thermodynamic constraints: thermal acclimation and metabolic compensation in a freshwater zooplankton species.

Respiration rates of ectothermic organisms are affected by environmental temperatures, and sustainable metabolism at high temperatures sometimes limits heat tolerance. Organisms are hypothesized to exhibit acclimatory metabolic compensation effects, decelerating their metabolic processes below Arrhenius expectations based on temperature alone. We tested the hypothesis that either heritable or plastic heat tolerance differences can be explained by metabolic compensation in the eurythermal freshwater zooplankton crustacean Daphnia magna We measured respiration rates in a ramp-up experiment over a range of assay temperatures (5-37°C) in eight genotypes of D. magna representing a range of previously reported acute heat tolerances and, at a narrower range of temperatures (10-35°C), in D. magna with different acclimation history (either 10 or 25°C). We discovered no difference in temperature-specific respiration rates between heat-tolerant and heat-sensitive genotypes. In contrast, we observed acclimation-specific compensatory differences in respiration rates at both extremes of the temperature range studied. Notably, there was a deceleration of oxygen consumption at higher temperature in 25°C-acclimated D. magna relative to their 10°C-acclimated counterparts, observed in active animals, a pattern corroborated by similar changes in filtering rate and, partly, by changes in mitochondrial membrane potential. A recovery experiment indicated that the reduction of respiration was not caused by irreversible damage during exposure to a sublethal temperature. Response time necessary to acquire the respiratory adjustment to high temperature was lower than for low temperature, indicating that metabolic compensation at lower temperatures requires slower, possibly structural changes.

Rearing Temperature and Fatty Acid Supplementation Jointly Affect Lipid Fluorescence Polarization and Heat Tolerance in Daphnia.

The homeoviscous adaptation hypothesis states that the relative abundance of polyunsaturated fatty acids (PUFAs) in membrane phospholipids of ectothermic organisms decreases with increasing temperatures to maintain vital membrane properties. We reared Daphnia magna at 15°, 20°, and 25°C and increasing dietary concentrations of the long-chain PUFA eicosapentaenoic acid (EPA) to test the hypothesis that the well-documented increase in heat tolerance of high-temperature-reared Daphnia is due to a reduction in body PUFA concentrations. Heat tolerance was assessed by measuring the time to immobility at a lethally high temperature (Timm at 37°C), and whole body lipid fluorescence polarization (FP) was used as an estimate of membrane fluidity. At all rearing temperatures, EPA supplementation resulted in an increase in the relative abundance of EPA in body tissues, but only at 15° and 25°C did this result in a decrease in heat tolerance, and only at 20°C was this associated with an increase in membrane fluidity (i.e., decrease in FP). Overall, however, the degree of tissue fatty acid unsaturation correlated well with heat tolerance and FP. Our results support the homeoviscous adaptation hypothesis by showing that cold-reared Daphnia accumulate PUFAs within their body tissues and thus are more susceptible to heat than hot-reared Daphnia accumulating fewer PUFAs. However, our data also point out that further studies are required that elucidate the complex relationships between PUFA supply, membrane fluidity, and heat tolerance in ectotherms.

Untangling the effects of codon mutation and amino acid exchangeability.

Determining the relative contributions of mutation and selection to evolutionary change is a matter of great practical and theoretical significance. In this paper, we examine relative contributions of codon mutation rates and amino acid exchangeability on the frequencies of each type of amino acid difference in alignments of distantly related proteins, alignments of closely related proteins, and among human SNPs, using a model that incorporates prior estimates of mutation and exchangeability parameters. For the operational exchangeability of amino acids in proteins, we use EX, a measure of protein-level effects from a recent statistical meta-analysis of nearly 10,000 experimental amino acid exchanges. EX is both free of mutational effects and more powerful than commonly used "biochemical distance" measures (1). For distant protein relationships, mutational effects (genetic code, transition/transversion bias) and operational exchangeability (EX) account for roughly equal portions of variance in off-diagonal values, the complete model accounting for R2 = 0.35 of the variance. For human/chimpanzee alignments representing closely related proteins relationships, mutational effects (including CpG bias) account for 0.52 of the variance; adding EX to the model increases this to 0.67. For natural variation in human proteins, the variance explained by mutational effects alone, and by mutational effects and operational exchangeability are, respectively, 0.66 and 0.70 for SNPs in HGVBase, and 0.56 and 0.60 for disease-causing missense variants in HGMD. Thus, exchangeability has a stronger relative effect for distant protein evolution than for the cases of closely related proteins or of population variation. A more detailed model for the hominid data suggests that 1) there is a threshold in EX below which substitutions are highly unlikely to be accepted, corresponding to roughly 30 % relative protein activity; 2) selection against missense mutants is a slightly convex function of protein activity, not changing much as long as protein activity is low; and 3) the probability of disease-causing effects decreases nearly linearly with EX.

Bias in the introduction of variation as an orienting factor in evolution.

According to New Synthesis doctrine, the direction of evolution is determined by selection and not by "internal causes" that act by way of propensities of variation. This doctrine rests on the theoretical claim that because mutation rates are small in comparison to selection coefficients, mutation is powerless to overcome opposing selection. Using a simple population-genetic model, this claim is shown to depend on assuming the prior availability of variation, so that mutation may act only as a "pressure" on the frequencies of existing alleles, and not as the evolutionary process that introduces novelty. As shown here, mutational bias in the introduction of novelty can strongly influence the course of evolution, even when mutation rates are small in comparison to selection coefficients. Recognizing this mode of causation provides a distinct mechanistic basis for an "internalist" approach to determining the contribution of mutational and developmental factors to evolutionary phenomena such as homoplasy, parallelism, and directionality.

The effect of cryopreservation on the lethal mutation rate in Drosophila melanogaster.

Although cryopreservation is routinely used for the storage of a range of biological organisms, few studies have been conducted to determine whether cryopreservation increases the frequency of mutation. A procedure for the cryopreservation of Drosophila melanogaster embryos has recently been developed. Cryopreservation of D. melanogaster is of special interest to geneticists and evolutionary biologists because it would make it possible to assay control and experimental populations simultaneously during long-term studies. Before cryopreserved embryos can be used for such studies, it is first necessary to show that cryopreservation is not mutagenic. We tested for mutagenic effects or cryopreservation in D. melanogaster embryos with an X-linked, recessive lethal assay. The mutation rates of cryopreserved and control flies were not significantly different. We can be 95% certain that cryopreservation does not increase mutation by a factor greater than 2.39. This is the first quantitative estimate of the mutagenic effect of cryopreservation on the germ line of a metazoan. The results are reassuring when considering the genetic impact of cryopreservation on mammalian gametes and embryos.