Lev Yu. Yampolsky

The xenohormesis hypothesis states that consuming stress-exposed food organisms can increase the consumer's tolerance to similar stresses. We tested this hypothesis using the freshwater model herbivore Daphnia magna feeding on the microalga Nannochloropsis limnetica grown at either 25°C (heat stressed), 20°C (presumed optimum) or 12°C (cold stressed). Daphnia magna maintained at either 20°C or 28°C and consuming heat-stressed algae had a significantly higher short-term heat tolerance. Furthermore, D. magna kept at either 12°C or 28°C (but not at 20°C) achieved a significantly higher lifespan when feeding on heat-stressed algae compared with D. magna feeding on non-stressed or cold-stressed algae, with fecundity being higher on cold-stressed food. This was accompanied by higher antioxidant capacity in D. magna fed heat-stressed algae, but no differences in lipid peroxidation were observed. Fatty acid analysis revealed that D. magna fed heat-stressed algae contained significantly lower levels of the essential polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), which is thought to be particularly susceptible to lipid peroxidation, than their counterparts feeding on algae grown at 20°C. We conclude that the lower EPA level in D. magna fed heat-stressed algae has caused the higher heat tolerance in D. magna by altering PUFA-mediated membrane properties, i.e. increasing membrane rigidity. Our findings suggest that the consumption of heat-stressed algae can provide heat tolerance and lifespan benefits in zooplankton, but at the cost of reduced fecundity. Further studies are needed to better understand the influence of heat waves on trophic interaction at the phytoplankton–zooplankton interface.

Aging is a multifaceted process that occurs on the background of and is significantly driven by transcriptional and cell type abundance changes. Aging-related transcriptional changes may include reduced transcription of maintenance, repair and DNA methylation genes, de-differentiation, or increased transcription of transposons. Unbiased detection of these changes and full understanding of their physiological effects requires single-cell resolution. We studied single-nuclei transcriptomes of a model microcrustacean Daphnia magna sampled from 3 age- and reproductive status groups: young, old reproductively senescent, and old, regaining reproductive function late in life. We detected 17 cell clusters, some identifiable as ovary or fat body-, midgut-, epithelium-, and neural tissue-related, some escaping unambiguous identification. At the same time, some well-characterized cell types were not detected, such as hemocytes or myocytes. We detect significant changes of cell type abundance with age and with reproductive “rejuvenation” in ovary- and gut-related clusters. We also detect several patterns of functional transcriptional differences between age classes with nearly all cell types, with changes between old reproductive and old non-reproductive Daphnia often reversing age-related changes, and often in good concordance with previous bulk RNAseq data.

Survival of aquatic invertebrates relies on the resistance of dormant stages to environmental stress and the synchronization of dormancy with seasonal changes. We investigated hatching patterns in Daphnia magna from four Mediterranean populations that experience seasonal drying. Ephippial eggs were either transferred directly to hatching-stimulation conditions or first exposed to one of the following storage treatments: wet, dark storage at 20 °C or 1 °C; or dry, dark storage at 20, 40, or 60 °C. Surprisingly, the most successful and synchronous hatching occurred after 2-month dark, cold (1 °C) storage at the temperature that rarely, if ever, occurs in their natural habitats. Dry storage led to more gradual hatching. Early diapause termination was observed in two of the studied populations, with part of the eggs hatching prior to storage and within a few days of ephippium production. Across all populations, ephippial eggs demonstrated high heat resistance and dry storage temperature had no significant effect on hatching rates or egg mortality. Despite similar natural environmental pressures, populations differed significantly in their hatching timing, suggesting fine-scale local or temporal adaptation in dormancy regulation.

Detailed knowledge of transcriptional responses to environmental cues or developmental stimuli requires single-cell resolution. We performed 2 single-cell RNAseq experiments of adult females and males of Daphnia magna, a freshwater plankton crustacean which is both a classic and emerging new model for eco-physiology, toxicology, and evolutionary genomics. We were able to identify >25 distinct cell types about half of which could be functionally annotated. First, we identified ovaries- and testis-related cell types by focusing on female- and male-specific clusters. Second, we compared markers between cell clusters and bulk RNAseq data on transcriptional profiles of early embryos, circulating hemocytes, midgut, heads (containing brain, eyes, muscles, and hepatic caeca), antennae II, and carapace. Finally, we compared transcriptional profiles of Daphnia cell clusters with orthologous markers of 200+ cell types annotated in Drosophila cell atlas. This allowed us to recognize striated myocytes, enterocytes, cuticular cells, as well as 9 different neuron types, including photoreceptors. Several well-defined clusters showed a significant enrichment in markers of both hemocytes and either fat body, or ovaries, or certain neuron types of Drosophila, but not with bulk RNAseq data from circulating hemocytes. This allowed us to hypothesize the existence of noncirculating, fat body-, ovary-, or neuron-associated populations of hemocytes in Daphnia. The circulating hemocytes express numerous cuticular proteins suggesting their role, in addition to macrophagy, in wound repair. Our data will be useful as a baseline resource for researchers using Daphnia to answer questions in ecophysiology, toxicology and biology of adaptation to changing environment.

Daphnia is a classic model organism for the study of circulation and hemocyte biology. Despite the ease of observation and separation of hemocytes, it is not known whether the circulating hemocytes continue to be produced throughout Daphnia lifespan, and if yes, whether they are generated in any specific hemopoietic tissue or represent a reproducing population as known in some arthropods. We detected de novo DNA synthesis by means of EdU staining in a significant portion of circulating hemocytes in adult D. magna . This portion did not significantly vary among phases of molting/ovary cycle and showed only a slight decrease with age. We did not detect any matching newly synthesized DNA anywhere in any other tissues of adult daphnids, eliminating the hypothesis of the existence of a population of hemopoietic stem cells. Rather, it appears that circulating hemocytes are a reproducing population with mitoses continuing throughout life. Hemocytes showed average ploidy of 4, with a considerable variation around that value indicating that either some endomitoses were also present or a significant portion of hemocytes were sampled in G2. However, while there was no difference in total DNA content measured by Hoechst fluorescence between hemocytes that showed and did not show recent DNA synthesis, among EdU-positive hemocytes there was a positive correlation between Hoechst and EdU fluorescence, indicating that many cells were sampled during S-phase. It is not known whether the total number of hemocytes increases with age or the observed proliferation compensates for losses of hemocytes, as well as whether the EdU-positive hemocytes represent a separate mitotically active stem subpopulation of circulating hemocytes.

NAD + homeostasis is an important determinant of lifespan and may be a key mechanism of caloric restriction (CR) expansion of lifespan. Ketone bodies such as beta-hydroxybutyrate (BHB) that regulate NAD + abundance and NAD + precursors such nicotinamide mononucleotide (NMN), are known to extend life in experimental animals and ameliorate age-related conditions in humans. We tested the hypothesis that chronic BHB and NMN exposure can extend lifespan similarly to the effect of CR treatment in a model organism Daphnia, a freshwater zooplankton crustacean. We also measured fecundity, lipofuscin accumulation, and lipid investments into offspring in Daphnia fed the full diet, full diet with BHB, NMN, and combined treatments, and fed the CR diet (25% of the full diet). We show that BHB exposure, but not NMN exposure, reduces early life mortality in fully fed Daphnia to levels similar to those observed under CR without compromising fecundity. We also observed that in a combined exposure cohort, NMN nearly eliminates the beneficial effect of BHB. None of the treatments affected lipofuscin accumulation, but the NMN and the combined treatment mimicked the effect of CR on neonate size in older females. An RNAseq experiment comparing the two diets and the two exposure treatments showed showed that BHB-treated Daphnia change expression of a variety of genes, including genes with known longevity extending effects, but differential expression of few genes is consistent with the effects of CR and their functionality is not clear.

NAD+ homeostasis is an important determinant of lifespan and may be a key mechanism of caloric restriction (CR) expansion of lifespan. Ketone bodies such as beta-hydroxybutyrate (BHB) that regulate NAD+ abundance and NAD+ precursors such nicotinamide mononucleotide (NMN), as are known to extend life in experimental animals and ameliorate age-related conditions in humans. We tested the hypothesis that chronic BHB and NMN exposure separately or in combination can extend lifespan in a model organism Daphnia, a freshwater zooplankton crustacean with the magnitude similar to that of the CR treatment. We also measured fecundity, lipofuscin accumulation, and lipid investments into offspring in Daphnia fed the full diet, full diet with BHB, NMN, and combined treatments, and fed the CR diet (25% of the full diet). We also conducted an RNAseq experiment comparing the two diets and the two exposure treatments. We show that BHB exposure, but not NMN exposure reduces early life mortality in Daphnia fed the full diet to levels similar to those observed under CR without compromising fecundity. We also observed that in a combined exposure cohort, NMN nearly eliminates the beneficial effect of BHB. None of the treatments affected lipofuscin accumulation, but the NMN and the combined treatment mimicked the effect of CR on neonate size in older females. We show that BHB-treated Daphnia change expression of a variety of genes, including genes with known longevity extending effects, but differential expression of few genes is consistent with the effects of CR and their functionality is not clear.