Avoiding dead ends: the experimental evolution of constraint as adaptation to environmental variation in Saccharomyces cerevisiae

Environmental unpredictability results in the evolution of bet-hedging traits, which maximize long-term fitness but are, by definition, suboptimal over short time scales. However, because suboptimal traits are expected to be purged by selection in the shorter term, the persistence of bet hedging remains perplexing. Here, we test the hypothesis that bet hedging persists through the evolution of constraint on short-term adaptation. We experimentally evolve Saccharomyces cerevisiae across two sequential treatments in which the frequency of extreme heat shocks decreases. We predict that experimental evolution under lower frequency heat shocks will result in greater adaptive constraint, or “purge-resistant” bet hedging. Constraint is assayed as evolutionary persistence of heat shock tolerance (HST) under constant benign conditions. As predicted, we find the retention of HST only in lines evolved under reduced frequency detrimental conditions. Results help explain the evolution of bet hedging, and challenge the traditional view that evolutionary constraint is inherently maladaptive.

The Heat Is on: a Simple Method to Increase Genome Editing Efficiency in Plants

Background: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. Results: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions.Conclusions: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

Biological Characterization of Plasmodium falciparum Mitochondrial Heat Shock Protein PfHsp70-3: Possible Involvement in Malaria Pathogenesis

Malaria remains a global health burden accounting for many deaths and illnesses in sub-Saharan Africa notwithstanding many decades of research on the disease. P. falciparum, the causative agent of the most fatal form of malaria, expresses a repertoire of heat shock proteins (Hsp) that cushion the parasite against heat shocks as it shuttles between extreme temperatures in human and mosquito vector hosts. By so doing, such proteins promote parasite’s cytoprotection, survival and pathogenesis. Heat shock proteins are named according to their molecular weights and there are six P. falciparum Hsp70 (PfHsp70) found in various cell compartments with mitochondrial putative PfHsp70-3. Using indirect immunofluorescence, this study established mitochondrial localization of PfHsp70-3 though some more confirmatory studies would be needed in the future. PfHsp70-3 was found to be heat inducible and expressed during all stages of the intra-erythrocytic cycle of parasite development. This could be an indication of PfHsp70-3’s involvement in the infectivity process of P. falciparum by helping the parasite to resist heat shocks during malaria febrile episodes. Generally, the data obtained in this study will enhance the existing knowledge on the biology of P. falciparum mitochondrial heat shock protein functions and open possible avenues for targeting the specificity between PfHsp70-3 and its co-chaperones for drug development. Keywords: Malaria, P. falciparum, Heat shock proteins, PfHsp70-3, pathogenesis

Determination of the Earliest time Point for Heat-shock Inducing Osteoporosis-like Phenotype in the rankl:HSE:CFP Medaka Fish Model for Osteoporosis

The transgenic medaka rankl:HSE:CFP expressing Rankl, a stimulator for osteoclastogenesis - the formation and activation of osteoclasts, bone “eating” cells, under control of a heat inducible promotor has been established as a model for osteoporosis to evaluate antiosteoporosis effects of substances. Transgenic larvae were usually heat-shocked for 90 minutes at 39oC when they were at 9 days post fertilization (dpf) and osteoporosis-like phenotype was analysed when they were at 11 dpf. In this study we investigated whether osteoporosis-like phenotype could be induced in the transgenic larvae when heat-shock was applied at earlier time points, when larvae were at 1, 2, 3, 4, 5, or 6 dpf. Results showed that heat-shocks from the time point of 3 dpf onwards resulted in osteoporosis-like phenotype while heat-shock at 1 or 2 dpf did not affect mineralized bone matrix in 11 dpf larvae. These provide important evidence for study on onset of Rankl induced osteoclasts in fish and help improving experimental procedures using this fish model for osteoporosis.

Mitochondrial responses towards intermittent heat shocks in the Eastern Oyster, Crassostrea virginica

Frequent heat waves caused by climate change can cause physiological stress in many animals, particularly in sessile ectotherms such as bivalves. Most studies characterizing thermal stress in bivalves focus on evaluating the responses to a single stress event. This does not accurately reflect the reality faced by bivalves which are often subject to intermittent heat waves. Here, we investigated the effect of intermittent heat stress on mitochondrial functions of Eastern oyster Crassostrea virginica which play a key role in setting ectotherms’ thermal tolerance. Specifically, we measured changes in mitochondrial oxygen consumption and H2O2 emission rates before, during and after intermittent 7.5°C heat shocks in oysters acclimated to 15°C and 22.5°C. Our results showed that oxygen consumption was impaired following the first heat shock at both acclimation temperatures. After the second heat shock, results for oysters acclimated to 15°C indicated a return to normal. However, oysters acclimated to 22.5°C struggled more with the compounding effects of intermittent heat shocks as denoted with an increase contribution of FAD-linked substrates to mitochondrial respiration as well as high levels of H2O2 emission rates. However, both acclimated populations showed signs of potential recovery ten days after the second heat shock, reflecting a surprising resilience to heat waves by C. virginica. Thus, this study highlights the important role of acclimation in oyster's capacity to weather intermittent heat shock.

Meta-analysis using a Lehmann classifier of obligate seeding species germination in fire-prone Mediterranean Type environments

The generalization that specific seed traits such as dormancy, longevity, or heat-triggered germination of plant species expanding in pyrogenic environments where stochastically but recurrently fire disturbance occurs is a fitness increasing adaptation of obligate seeders dates from the early 20th century. During the last few decades, this hypothesis, qualified as a pyrophytic strategy, is re-evaluated under the lenses of conservation biology and climate change research. The validity of pyrophytism as an equilibrium response to fire vs. the interpretation that the obligate seeding strategy is instead an opportunistic or generalist response to the multitude of abiotic and biotic factors determining the variability and heterogeneity of fire-prone environments such as the Mediterranean Type Ecosystems is indirectly examined and narratively promoted in the renewed fire ecology literature. In this paper, I suggest a need for a typified meta-analysis of the abundant but disparate wealth of research protocols and data to achieve a quantitatively strict understanding of the limits of the contrasting hypotheses. I develop a meta-analytic classifier and test its feasibility and applicability across taxonomic, biologic, and ecological levels of organization, i.e., from the intra-population or inter-individual local level progressively to inter-genus and intra-family levels, across the Mediterranean Basin. Cistaceae species, emblems of the Mediterranean shrublands, are the model for this research. The results of this exercise support the feasibility and flexibility of the Lehmann-type classifier developed. Although Cistus species do respond positively to heat-shocks at the local level, significant variability is uncovered among higher taxa levels and furthermost as the environmental variability increases. The germination variability complicates generalizations when climatic variability and change come into play, questioning long-standing ‘certitudes’ and Mediterranean forest managers and conservation planners' practices.

Temperature and outgroup discrimination

High temperatures have been linked to aggression and different forms of conflict in humans. We consider whether exposure to heat waves increases discriminatory behavior toward outgroups. Using data from two large-scale field experiments in Germany, we find a direct causal effect of exposure to heat shocks on discrimination in helping behavior. As temperature rises, German natives faced with a choice to provide help to strangers in every-day interactions help Muslim immigrants less than they do other German natives, while help rates toward natives are unaffected by temperature. This finding suggests that there may be a physiological basis for discriminatory behavior toward outgroups.

Protein Aggregation as a Bacterial Strategy to Survive Antibiotic Treatment

While protein aggregation is predominantly associated with loss of function and toxicity, it is also known to increase survival of bacteria under stressful conditions. Indeed, protein aggregation not only helps bacteria to cope with proteotoxic stresses like heat shocks or oxidative stress, but a growing number of studies suggest that it also improves survival during antibiotic treatment by inducing dormancy. A well-known example of dormant cells are persisters, which are transiently refractory to the action of antibiotics. These persister cells can switch back to the susceptible state and resume growth in the absence of antibiotics, and are therefore considered an important cause of recurrence of infections. Mounting evidence now suggests that this antibiotic-tolerant persister state is tightly linked to—or perhaps even driven by—protein aggregation. Moreover, another dormant bacterial phenotype, the viable but non-culturable (VBNC) state, was also shown to be associated with aggregation. These results indicate that persisters and VBNC cells may constitute different stages of the same dormancy program induced by progressive protein aggregation. In this mini review, we discuss the relation between aggregation and bacterial dormancy, focusing on both persisters and VBNC cells. Understanding the link between protein aggregation and dormancy will not only provide insight into the fundamentals of bacterial survival, but could prove highly valuable in our future battle to fight them.

Habitat loss, predation pressure and episodic heat-shocks interact to impact arthropods and photosynthetic functioning of microecosystems

Ecosystems face multiple, potentially interacting, anthropogenic pressures that can modify biodiversity and ecosystem functioning. Using a bryophyte–microarthropod microecosystem we tested the combined effects of habitat loss, episodic heat-shocks and an introduced non-native apex predator on ecosystem function (chlorophyll fluorescence as an indicator of photosystem II function) and microarthropod communities (abundance and body size). The photosynthetic function was degraded by the sequence of heat-shock episodes, but unaffected by microecosystem patch size or top-down pressure from the introduced predator. In small microecosystem patches without the non-native predator, Acari abundance decreased with heat-shock frequency, while Collembola abundance increased. These trends disappeared in larger microecosystem patches or when predators were introduced, although Acari abundance was lower in large patches that underwent heat-shocks and were exposed to the predator. Mean assemblage body length (Collembola) was reduced independently in small microecosystem patches and with greater heat-shock frequency. Our experimental simulation of episodic heatwaves, habitat loss and non-native predation pressure in microecosystems produced evidence of individual and potentially synergistic and antagonistic effects on ecosystem function and microarthropod communities. Such complex outcomes of interactions between multiple stressors need to be considered when assessing anthropogenic risks for biota and ecosystem functioning.