Experimental enzyme evolution.
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Administration of histamine phosphate or glycerol trioleate (RE stimulators) to cholesterol-fed rabbits induced identical pattern of changes in cholesterol, phospholipid and triglyceride levels of the blood. However, the incidence of atheroma index in aorta and coronary arteries were significantly higher in histamine-treated and glycerol-trioleate-treated animals compared to cholesterol-fed controls. The rise in these indices appeared to be only partly related to increase in serum beta-lipoproteins but mostly due to increased permeability of the endothelium and internal elastic lamina probably caused by histamine phosphate and glycerol trioleate resulted in greater degree of atheromatous lesions in those groups.
Nonocclusive white mural thrombosis was induced in the abdominal aortae of normolipidemic rabbits by insertion of a polyethylene catheter into the abdominal aortae. The thrombus subsequently organized into intimal thickenings which resembled fibrofatty type atherosclerosis seen in man, showing large numbers of foam cells containing stainable lipid, fatty necrotic centers, cholesterol clefts, calcification, and fibrous caps. The lipid composition of the thrombus and lesions was followed at serial time intervals from 4 hours to 60 weeks. Lipid analysis showed significant concentrations of lipid in the early lesions and with time these lipid concentrations increased and later decreased. These studies demonstrate that the fibrofatty lesions derived from a white thrombus have significant amounts of the same lipids that characterize the atherosclerotic lesions of man, however, there is a lower proportion of cholesterol to the other constituents and a higher proportion of phospholipids. The free cholesterol and cholesteryl ester of the 4-day lesions were much greater than that of platelets alone indicating that significant amounts of plasma are trapped in a thrombus when it forms.
Rat spinal cords have been compressed at the region corresponding to D4-D5 by a 50 mg/cm force. After 5, 30 and 60 min this section has been dissected together with the surrounding cord. The ATPase activity of the homogenates was decreased, suggesting an involvement of this enzyme in the generation of edema.
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Balloon catheter denudation of rat carotid artery that results in significant medial damage is followed by marked intimal smooth muscle cell (SMC) proliferation associated with limited endothelial regrowth. In this report we demonstrate that: (a) SMC of the carotid media, preceding their intimal proliferation, develop a cytoskeletal profile and morphology consistent with a de-differentiated SMC phenotype; and (b) both medial and intimal SMC subsequently revert to a cytoskeletal profile and morphology reflecting incomplete but significant re-differentiation toward normal SMC phenotype. Specifically, early after balloon injury, SMC of the media and those that have migrated into the intima contain decreased amounts of actin, desmin, and tropomyosin and increased amounts of vimentin; moreover, beta-actin becomes the dominant actin isoform, whereas alpha-actin decreases as compared with that found in normal medial SMC. Late after balloon injury, actin is still less abundant, however, desmin, tropomyosin, and vimentin return toward normal values and both medial and intimal SMC again show a predominance of alpha-actin, although the endothelium does not regenerate over the central surface of intimal thickening in this model. The SMC surface to volume ratio significantly decreases early after balloon injury, whereas it is not significantly different late after balloon injury as compared with that of SMC of the normal carotid media. We demonstrate, furthermore that: (c) adjacent luminal SMC are interconnected by gap junctions and develop focal tight junctions, a feature not reported previously to occur in smooth muscle; these cells however do not form any well defined membrane specialization with the leading edge of endothelium, supporting the view that presence of modified SMC on the luminal surface of chronically denuded vessels is not responsible for the cessation of endothelial regrowth.
Swiss Mice infected with Plasmodium berghei berghei and maintained in permanence in a hot environmental temperature undergo a chronic infection whereas controls maintained at the laboratory temperature develop always an acute and lethal infection. The hot environmental temperature does not seem to have any action on the pathogenicity of the parasites. Host defences are stimulated.
Experimental evolution has demonstrated that mesophilic microbes readily adapt to increases in temperature. However, many microbes are psychrotolerant and resistant to cold, which is associated with physiological specializations, suggesting constraints in thermal adaptation. We hypothesized that constraints would limit adaption differently in a mesophilic species (Bacillus thuringiensis) compared with its psychrotolerant relative B. mycoides-with adaptation at cooler temperatures and adaptation at higher temperatures being constrained in each species, respectively. To test this hypothesis, we imposed 140 generations of selection at temperatures at and below the optimum for productivity for both species. The fitness and thermal performance of evolved bacteria showed ancestral thermal niche plays a role in thermal adaptation over this time scale, in support of our hypothesis of adaptive constraints. Temperature-dependent trade-offs appeared common in B. mycoides, with fitness gains associated with decreases in operational niche width; fitness gains at one temperature caused a decrease in the range of temperatures that the bacterium showed appreciable growth. Genome resequencing showed that variation in mutation supply and selection strength could not explain temperature-dependent responses to selection. Importantly, metabolic theory only held true for mesophilic B. thuringiensis, showing abundant but less studied psychrotolerant species could follow different adaptive trajectories.
Emerging evidence suggests mtDNA haplotypes contribute to fitness variation and local adaptation, with directional thermal selection and negative frequency-dependent selection shaping haplotype diversity. However, their interplay remains unexplored. We conducted experimental evolution using Drosophila melanogaster populations from opposite ends of an Australian latitudinal cline (Melbourne and Townsville), exposing them to contrasting temperatures (17°C versus 27°C) and varying starting frequencies of two mtDNA haplotypes (A1 and B1) that occur at appreciable frequencies in these populations. We paired this with population genetic simulations to estimate selection and its influence on haplotype trajectories. Haplotype frequencies were influenced by interactions involving temperature, starting frequency, and nuclear genomic background (Melbourne, Townsville, or admixed). Although prior work predicted A1 should be favoured at the warmer temperature and B1 at the cooler temperature, A1 was generally favoured across both temperatures. Simulations supported directional selection in populations evolving at 17°C in the Melbourne background; otherwise dynamics were best explained by balancing selection shaped by negative frequency-dependent fitness effects. Patterns also varied across nuclear backgrounds, suggestive of mito-nuclear epistasis. These findings challenge a simple thermal adaptation model of mtDNA dynamics, suggesting that mtDNA evolution is shaped by interacting effects of temperature, frequency-dependence, nuclear background and experimental environment.
UNLABELLED: Predator-mediated selection is an important ecological force shaping bacterial evolution, but its effects on genomic adaptation and virulence in opportunistic pathogens are not fully understood. Here, we used experimental evolution to study how exposure to the ciliate predator Tetrahymena thermophila affects Pseudomonas aeruginosa. Replicate populations were evolved for 60 days with or without the predator, followed by whole-genome shotgun metagenomic sequencing and phenotypic analyses. Both treatments showed strong selection and evidence of parallel evolution at gene and nucleotide levels, indicating constrained adaptation. However, predator exposure altered evolutionary dynamics. Predator-evolved populations showed a wider distribution of mutation frequencies, with many mutations persisting at intermediate frequencies, consistent with increased clonal interference and ongoing competition among lineages. In contrast, populations evolved without predators showed more high-frequency mutations, consistent with selective sweeps, although some low-frequency variants remained. Despite substantial genomic change, phenotypic outcomes were variable. Virulence in an invertebrate host model did not consistently increase. Instead, evolved isolates showed context-dependent changes, including modest decreases or occasional increases. Competition assays also showed no consistent fitness advantage for predator-evolved isolates, suggesting trade-offs between predator resistance and growth in other environments. Overall, predator-mediated selection reshaped evolutionary dynamics by maintaining diversity and altering the balance of lineages rather than producing uniform increases in virulence. These results highlight how ecological complexity influences adaptive evolution and the context-dependent nature of pathogen traits. IMPORTANCE: Opportunistic pathogens such as Pseudomonas aeruginosa often evolve in environmental settings before infecting hosts, raising questions about how ecological interactions influence virulence. Predator-mediated selection has been suggested to increase virulence via coincidental evolution, but evidence is inconsistent. Here, we show that exposure to a eukaryotic predator does not consistently elevate virulence but does reshape evolutionary dynamics by altering how mutations spread in populations. Predator-exposed populations retained more intermediate-frequency mutations, consistent with increased clonal interference and ongoing competition among lineages, whereas non-predator populations were dominated by selective sweeps. These differences were also reflected in functional targets of adaptation, with predator exposure favoring mutations in genes involved in environmental sensing and interaction. Together, these findings suggest that ecological complexity shapes the dynamics of adaptation rather than driving a single evolutionary outcome, highlighting that virulence is an emergent property influenced by underlying evolutionary processes.
Climate change is increasing the risk of emerging parasites. However, whether more virulent variants will spread during climate-driven outbreaks remains unclear. Here, we aimed to explore the short-term trajectory of parasite evolution-at the phenotypic and genomic scales-across environmentally relevant temperatures in a thermally mismatched host-parasite interaction. We experimentally evolved a wild parasitic bacterium (Leucobacter musarum), across the thermal range (20°C-30°C) and extremes (35°C) of Cabo Verde-the site of field collection-in a Caenorhabditis elegans host strain. Starting from a single bacterial isolate, we then tracked phenotypic and de novo genomic changes that arose across replicate populations following ten passages of experimental evolution. We found that at 25°C, warm for the host but an average temperature for the parasite, host-mediated selection favoured higher virulence and genomic diversification by the end of the experiment. At hot temperatures, towards the limit of host survival, virulence was maintained across all parasite populations. Parasites evolved at hot temperatures also displayed a latent virulence boost, deadlier once hosts experienced a heatwave. Patterns of molecular evolution were constrained to parallel changes in fewer loci at extreme temperatures. Our findings suggest that shifting environmental temperatures will leave phenotypic and genomic signatures on evolving parasites.
Endosymbiosis is a major driver of evolutionary innovation and underpins the function of diverse ecosystems. The origins and evolution of endosymbiosis are challenging to study experimentally due to the short-lived culturability of many microbial strains derived from endosymbiotic interactions. The facultative endosymbiosis between the ciliate, Paramecium bursaria, and the green alga, Micractinium conductrix (Chlorellaceae, Trebouxiophyceae), is ecologically widespread and has emerged as a powerful lab-tractable model system. This endosymbiosis is founded upon a reciprocal nutrient exchange, but each of the species can be cultured independently enabling quantification of symbiotic fitness effects, new partnerships to be generated in the lab, and co-associations to be subject to experimental evolution. To date, evolve-and-resequence approaches have been limited due to a lack of high-quality genome assemblies enabling gene variants to be identified. Here, we report a near telomere-to-telomere genome assembly for M. conductrix 186b, using a range of sequencing technologies. Comparative analysis shows that this is one of the most complete Chlorellaceae algal genome assemblies available to date. To aid accurate gene calling and annotation, we conducted both RNAseq and Iso-Seq transcriptome sequencing experiments. Collectively, these 'omics datasets will facilitate: (i) comparative genomics studies of endosymbiont evolution, (ii) evolve-and-resequence experiments, (iii) genome-scale metabolic modeling studies, and (iv) identification of targets for genetic modification experiments and biotechnological applications.
Antimicrobial resistance (AMR) is a threat to modern medicine. To combat AMR pathogens, natural predators like bacteriophages and predatory bacteria have gained interest recently. Predatory bacterium Bdellovibrio bacteriovorus is ubiquitous and has a broad prey range. It is particularly potent at killing many AMR Gram-negative bacterial pathogens featured on the WHO priority list. However, it is currently unclear whether prey bacteria can evolve genetically-determined resistance against predation by B. bacteriovorus. Here, we show that the model bacterium Escherichia coli K-12 consistently evolves resistance against B. bacteriovorus during experimental evolution. Selection for resistance scaled positively with predation pressure and was widespread after two cycles of predator exposure. Similar to antibiotics, predation resistance was costly, manifesting in a trade-off between predation resistance and fitness in the absence of predators. Genetic analysis combined with proteomics identified mutations that lead to the down-regulation of the outer membrane porin OmpF as a common resistance mechanism. In addition, a rarer mutation in cell envelope lipopolysaccharide-modifying enzyme WaaF also conferred predation resistance, likely by a pleiotropic effect, which included OmpF down regulation. While our study uncovers evolutionary and mechanistic aspects of prey escape from predation, it also highlights that the high cost of resistance reflects a handicap for the pathogen and can thus be exploited to increase treatment sustainability. Altogether, our work generates essential knowledge in ecologically important predator-prey interactions and can advance predatory bacteria as "living antibiotics" to combat AMR.
Theory suggests sexual selection will enhance population viability by purging deleterious alleles. However, direct genomic evidence for this fundamental idea is scarce and contradictory. We combined long-term experimental evolution with whole-genome resequencing to directly test how sexual selection affects mutation load, genomic divergence, and extinction risk in small populations (maximum Ne = 40) of Tribolium castaneum. After 156 generations, populations evolving under strong sexual selection carried substantially fewer deleterious alleles than populations under weak sexual selection, based on both individual-level estimates of missense and nonsense variants and population-level Rxy analyses, indicating more efficient purging of deleterious alleles. In contrast, nucleotide diversity and runs of homozygosity were similar across treatments, indicating that purging acted most strongly on deleterious variation, and that reduced mutation load in these small populations under strong sexual selection was not explained by demographic effects. Importantly, population-level mutation load estimates best explained extinction risk under inbreeding, directly linking sexual selection to purging and population viability. Genome scans of high and low sexual selection populations revealed peaks of divergence, which included genes involved in courtship, sex discrimination, and seminal fluid proteins. Our results provide direct genomic evidence that sexual selection can reduce mutation load without eroding standing genetic diversity and thus adaptive potential, while driving adaptive divergence in reproductive traits. This beneficial purging may help explain the widespread prevalence of sexual reproduction in nature despite inherent costs and have important ramifications as to how we manage populations of conservation concern.
Microbial populations strongly shape their environment, which can re-route adaptation toward organism-generated fitness optima. However, the conditions that promote these eco-evolutionary feedbacks are unclear. Here, we used experimental evolution to test whether high population density, by strengthening niche construction, drives eco-evolutionary feedbacks in the bacterial pathogen Pseudomonas aeruginosa (Pa) MPAO1. We tested for adaptation to organism-modified environments by measuring the relative performance of ancestral and endpoint populations in filtrate generated by each evolutionary line sampled across generations. Contrary to expectations, we found that endpoint populations had higher performance than the ancestral strain in filtrate across nearly all evolutionary lines regardless of population density. This was caused by the emergence of hyperactive filamentous bacterio(phage) mutants during experimental passaging that inhibited the ancestral strain but not endpoint populations in modified media. Hyperactive phages emerged from one of two avirulent prophages in MPAO1's genome (Pf4 or Pf6). Hyperactive phages drove the evolution of phage resistance in bacterial populations via mutations in the type IV pilus (TIVP), the phage's binding receptor. In a follow-up experiment, we showed that these TIVP mutations pleiotropically reduced motility and conferred resistance to a TIVP-targeting virulent phage, both of which are important traits for Pa infection and treatment. Overall, this work suggests that filamentous phage evolution can drive eco-evolutionary feedbacks in bacterial populations, causing phenotypic and genetic changes that would not be anticipated from adaptation to the extrinsic environment alone.
Evolutionary repair refers to the compensatory evolution that follows perturbations in cellular processes. While evolutionary trajectories are often reproducible, other studies suggest they are shaped by genotype-by-environment (GxE) interactions. Here, we test the predictability of evolutionary repair in response to DNA replication stress-a severe perturbation impairing the conserved mechanisms of DNA synthesis, resulting in genetic instability. We conducted high-throughput experimental evolution on Saccharomyces cerevisiae experiencing constitutive replication stress, grown under different glucose availability. We found that glucose levels impact the physiology and adaptation rate of replication stress mutants. However, the genetics of adaptation show remarkable robustness across environments. Recurrent mutations collectively recapitulated the fitness of evolved lines and are advantageous across macronutrient availability. We also identified a novel role of the mediator complex of RNA polymerase II in adaptation to replicative stress. Our results highlight the robustness and predictability of evolutionary repair mechanisms to DNA replication stress and provide new insights into the evolutionary aspects of genome stability, with potential implications for understanding cancer development.
In the present article we examine clonality in virus evolution. Most viruses retain an active recombination machinery as a potential means to initiate new levels of genetic exploration that go beyond those attainable solely by point mutations. However, despite abundant recombination that may be linked to molecular events essential for genome replication, herein we provide evidence that generation of recombinants with altered biological properties is not essential for the completion of the replication cycles of viruses, and that viral lineages (near-clades) can be defined. We distinguish mechanistically active but inconsequential recombination from evolutionarily relevant recombination, illustrated by episodes in the field and during experimental evolution. In the field, recombination has been at the origin of new viral pathogens, and has conferred fitness advantages to some viruses once the parental viruses have attained a sufficient degree of diversification by point mutations. In the laboratory, recombination mediated a salient genome segmentation of foot-and-mouth disease virus, an important animal pathogen whose genome in nature has always been characterized as unsegmented. We propose a model of continuous mutation and recombination, with punctuated, biologically relevant recombination events for the survival of viruses, both as disease agents and as promoters of cellular evolution. Thus, clonality is the standard evolutionary mode for viruses because recombination is largely inconsequential, since the decisive events for virus replication and survival are not dependent on the exchange of genetic material and formation of recombinant (mosaic) genomes.
1. Structural changes in the thymus during the evolution experimental renal hypertension were investigated to determine their possible role in the genesis of hypertensive vascular disease. 2. The thymus, adrenal glands and the progression of hypertensive vascular lesions were investigated in rats during the first 30 days after occlusion of the aorta between the two renal arteries. 3. Hypertension was initially accompanied by marked atrophy of the thymus, most pronounced 9 days after operation. During this time, the adrenal glands doubled in size and the heart became enlarged. 4. After 21 days the thymus regenerated and became hypertrophic. Histological features of hyperactivity accompanied by infiltration of plasma cells were evident, while the adrenal glands remained enlarged. 5. The observed structural changes of the regenerated thymus in the presence of sustained adrenal hypertrophy indicate that the thymus may contribute to the production of hypertensive vascular disease.