Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 isolates in rodents.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern1-4. Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral entry, raising concerns that the replication capacity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here we have evaluated the replicative ability and pathogenicity of BA.4 and BA.5 isolates in wild-type Syrian hamsters, human ACE2 (hACE2) transgenic hamsters and hACE2 transgenic mice. We have observed no obvious differences among BA.2, BA.4 and BA.5 isolates in growth ability or pathogenicity in rodent models, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 clinical isolates have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2.

Insecticide resistant Anopheles gambiae have enhanced longevity but reduced reproductive fitness and a longer first gonotrophic cycle.

Widespread insecticide resistance in African malaria vectors raises concerns over the potential to compromise malaria vector control interventions. Understanding the evolution of resistance mechanisms, and whether the selective disadvantages are large enough to be useful in resistance management or designing suitable control strategies is crucial. This study assessed whether insecticide resistance to pyrethroids has an effect on the gonotrophic cycle and reproductive potential of malaria vector Anopheles gambiae. Comparative tests were performed with pyrethroid-resistant and susceptible colonies of Anopheles gambiae colonized from the same geographical area, and the reference Kisumu strain was used as a control. Adult females aged 3 days old were given a blood meal and kept separately for individual egg-laying. The number of days taken to lay eggs post-blood-feeding was recorded to determine the length of the gonotrophic cycle. To measure adult longevity and reproduction potential, newly emerged males and females of equal numbers were aspirated into a cage and females allowed to blood feed daily. The number of eggs laid and the surviving mosquitoes were recorded daily to determine fecundity, net reproduction rate, intrinsic growth rate and adult longevity. Overall, the resistant females had a significantly longer (1.8 days) gonotrophic cycle than susceptible females (F2, 13 = 9. 836, P < 0.01). The proportion of resistant females that laid eggs was lower 31.30% (94/300) compared to 54% (162/300) in the susceptible colony and 65.7% (197/300) in the Kisumu strain. The mean number of eggs laid per female was significantly lower in the resistant colony (88.02 ± 20) compared to the susceptible colony (104.9 ± .28.8) and the Kisumu strain (97.6 ± 34.8). The adult longevity was significantly higher for resistant (39.7 ± 1.6 days) compared to susceptible (29.9 ± 1.7 days) and the Kisumu strain was (29.6 ± 1.1 days) (F2,8 = 45.05, P < 0.0001). Resistant colony exhibited a lower fecundity (4.3 eggs/females/day) and net reproductive rate (2.6 offsprings/female/generation) compared to the susceptible colony (8.6 eggs/female/day; 4.7 offsprings/female/generation respectively) and Kisumu strain (9.7 eggs/female/day; 4.1 offsprings/female/generation respectively). The study suggests high fitness cost on reproductive parameters of pyrethroid-resistant mosquitoes particularly on the duration of gonotrophic cycle, fecundity and net reproductive rate. These fitness costs are likely associated with maintaining both target site and metabolic mechanisms of resistance to pyrethroids. Despite these costs, resistant mosquitoes had longer longevity. These results give insights to understanding the fitness cost of insecticide resistance and thus are critical when predicting the epidemiological impact of insecticide resistance.

The evolutionary maintenance of Lévy flight foraging.

Lévy flight is a type of random walk that characterizes the behaviour of many natural phenomena studied across a multiplicity of academic disciplines; within biology specifically, the behaviour of fish, birds, insects, mollusks, bacteria, plants, slime molds, t-cells, and human populations. The Lévy flight foraging hypothesis states that because Lévy flights can maximize an organism's search efficiency, natural selection should result in Lévy-like behaviour. Empirical and theoretical research has provided ample evidence of Lévy walks in both extinct and extant species, and its efficiency across models with a diversity of resource distributions. However, no model has addressed the maintenance of Lévy flight foraging through evolutionary processes, and existing models lack ecological breadth. We use numerical simulations, including lineage-based models of evolution with a distribution of move lengths as a variable and heritable trait, to test the Lévy flight foraging hypothesis. We include biological and ecological contexts such as population size, searching costs, lifespan, resource distribution, speed, and consider both energy accumulated at the end of a lifespan and averaged over a lifespan. We demonstrate that selection often results in Lévy-like behaviour, although conditional; smaller populations, longer searches, and low searching costs increase the fitness of Lévy-like behaviour relative to Brownian behaviour. Interestingly, our results also evidence a bet-hedging strategy; Lévy-like behaviour reduces fitness variance, thus maximizing geometric mean fitness over multiple generations.

Long-term adaptation following influenza A virus host shifts results in increased within-host viral fitness due to higher replication rates, broader dissemination within the respiratory epithelium and reduced tissue damage.

The mechanisms and consequences of genome evolution on viral fitness following host shifts are poorly understood. In addition, viral fitness -the ability of an organism to reproduce and survive- is multifactorial and thus difficult to quantify. Influenza A viruses (IAVs) circulate broadly among wild birds and have jumped into and become endemic in multiple mammalian hosts, including humans, pigs, dogs, seals, and horses. H3N8 equine influenza virus (EIV) is an endemic virus of horses that originated in birds and has been circulating uninterruptedly in equine populations since the early 1960s. Here, we used EIV to quantify changes in infection phenotype associated to viral fitness due to genome-wide changes acquired during long-term adaptation. We performed experimental infections of two mammalian cell lines and equine tracheal explants using the earliest H3N8 EIV isolated (A/equine/Uruguay/63 [EIV/63]), and A/equine/Ohio/2003 (EIV/2003), a monophyletic descendant of EIV/63 isolated 40 years after the emergence of H3N8 EIV. We show that EIV/2003 exhibits increased resistance to interferon, enhanced viral replication, and a more efficient cell-to-cell spread in cells and tissues. Transcriptomics analyses revealed virus-specific responses to each virus, mainly affecting host immunity and inflammation. Image analyses of infected equine respiratory explants showed that despite replicating at higher levels and spreading over larger areas of the respiratory epithelium, EIV/2003 induced milder lesions compared to EIV/63, suggesting that adaptation led to reduced tissue pathogenicity. Our results reveal previously unknown links between virus genotype and the host response to infection, providing new insights on the relationship between virus evolution and fitness.

Carbon Starved Anther modulates sugar and ABA metabolism to protect rice seed germination and seedling fitness.

Seed germination is critical for plant survival and agricultural production, which is affected by both internal seed factors and external environmental conditions. However, the genetic basis and underlying molecular mechanisms of early seed germination in crops remain largely unclear. Here, we report that R2R3 MYB transcription factor Carbon Starved Anther (CSA) is expressed specifically in Oryza sativa embryo and aleurone in response to seed imbibition, peaking at 3-6 h and undetectable by 24-h post-imbibition. CSA seeds germinated more quickly than wild-type rice seeds and had higher levels of amylase activity, glucose, and inactive abscisic acid-glucose ester (ABA-GE), but lower levels of ABA. Through analyzing the CSA-associated transcriptome and CSA binding to downstream target genes, we identified two glycolytic genes as direct CSA targets. CSA inhibits Amylase 3A expression to limit glucose production from starch and activates Os3BGlu6 expression to promote de-conjugation of ABA-GE to ABA; these functions serve to slow germination and improve seedling resilience to abiotic stress in the first 3 weeks of growth. Therefore, this study unveils a protection mechanism conferred by CSA during early seed germination by balancing glucose and ABA metabolism to optimize seed germination and stress response fitness.

Microbiota Influences Fitness and Timing of Reproduction in the Fruit Fly Drosophila melanogaster.

Associated microorganisms ("microbiota") play a central role in determining many animals' survival and reproduction characteristics. The impact of these microbial influences on an animal's fitness, or population growth, in a given environment has not been defined as clearly. We focused on microbiota-dependent host fitness by measuring life span and fecundity in Drosophila melanogaster fruit flies reared individually with 14 different bacterial species. Consistent with previous observations, the different bacteria significantly influenced the timing of fly life span and fecundity. Using Leslie matrices, we show that fly fitness was lowest when the microbes caused the flies to invest in life span over fecundity. Computational permutations showed that the positive fitness effect of investing in reproduction was reversed if fly survival over time was low, indicating that the observed fitness influences of the microbes could be context dependent. Finally, we showed that fly fitness is not influenced by bacterial genes that shape fly life span or fly triglyceride content, a trait that is related to fly survival and reproduction. Also, metagenome-wide association did not identify any microbial genes that were associated with variation in fly fitness. Therefore, the bacterial genetic basis for influencing fly fitness remains unknown. We conclude that bacteria influence a fly's reproductive timing more than total reproductive output and that (e.g., environmental) conditions that influence fly survival likely determine which bacteria benefit fly fitness. IMPORTANCE The ability of associated microorganisms ("microbiota") to influence animal life history traits has been recognized and investigated, especially in the past 2 decades. For many microbial communities, there is not always a clear definition of whether the microbiota or its members are beneficial, pathogenic, or relatively neutral to their hosts' fitness. In this study, we report the influence of individual members of the microbiota on Drosophila melanogaster fitness using Leslie matrices that combine the microbial influences on fly survival and reproduction into a single fitness measure. Our results are consistent with a previous report that, in the laboratory, acetic acid bacteria are more beneficial to the flies than many strains of lactic acid bacteria. We add to the previous finding by showing that this benefit depends on fly survival rate. Together, our work helps to show how the microbiota of a fly influences its laboratory fitness and how these effects may translate to a wild setting.

Body and Wing Allometries Reveal Flight-Fecundity Tradeoff in Response to Larval Provisioning in Osmia lignaria (Hymenoptera: Megachilidae).

Variation in body size has important implications for physical performance and fitness. For insects, adult size and morphology are determined by larval growth and metamorphosis. Female blue orchard bees, Osmia lignaria, (Say) provision a finite quantity of food to their offspring. In this study, we asked how provision-dependent variation in size changes adult morphology. We performed a diet manipulation in which some larvae were starved in the final instar and some were given unlimited food. We examined the consequences on adult morphology in two ways. First, allometric relationships between major body regions (head, thorax, abdomen) and total body mass were measured to determine relative growth of these structures. Second, morphometrics that are critical for flight (wing area, wing loading, and extra flight power index) were quantified. Head and thorax mass had hyperallometric relationships with body size, indicating these parts become disproportionately large in adults when larvae are given copious provisions. However, abdominal mass and wing area increased hypoallometrically with body size. Thus, large adults had disproportionately lighter abdomens and smaller wing areas than smaller adults. Though both males and females followed these general patterns, allometric patterns were affected by sex. For flight metrics, small adults had reduced wing loading and an increased extra flight power index. These results suggest that diet quantity alters development in ways that affect the morphometric trait relationships in adult O. lignaria and may lead to functional differences in performance.

Evaluation of sex hormone profile and semen parameters in acromegalic male patients.

OBJECTIVE: To investigate the changes in semen quality and bioavailable testosterone concentrations in acromegalic male patients according to their disease activity and compare them with patients with non-functional pituitary adenoma (NFA) and healthy controls (HC). METHODS: Twenty-four acromegalic patients with active disease, 22 acromegalic patients in remission, 10 HCs, and 10 patients with NFA were included. RESULTS: Total and calculated bioavailable testosterone concentrations were lower in patients with pituitary disease. Patients with acromegaly had more severely impaired total testosterone levels and semen parameters in comparison to HCs and patients with NFA. The degree of impairment was more prominent in acromegalic patients with active disease than acromegalic patients in remission. Acromegalic patients in remission had residual impairments in both semen quality and testosterone concentrations. Patients with NFA had the lowest concentrations of calculated bioavailable testosterone, followed by acromegalic patients with active disease and acromegalic patients in remission. Increasing growth hormone (GH) levels were found to be associated with both more severely impaired semen quality and androgen concentrations. CONCLUSION: Growth hormone hypersecretion can disturb reproductive biology and thereof semen quality. The reduction in semen quality and androgen levels may not fully recover upon disease control. Clinicians should be aware of the increased risk of impaired semen parameters and reduced total/bioavailable levels in acromegalic patients, especially in the setting of active disease.

Dual transcriptional analysis reveals adaptation of host and pathogen to intracellular survival of Pseudomonas aeruginosa associated with urinary tract infection.

Long-term survival of bacterial pathogens during persistent bacterial infections can be associated with antibiotic treatment failure and poses a serious public health problem. Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa, which can cause both acute and chronic infections, are particularly challenging due to its high intrinsic resistance to antibiotics. The ineffectiveness of antibiotics is exacerbated when bacteria reside intracellularly within host cells where they can adopt a drug tolerant state. While the early steps of adherence and entry of P. aeruginosa into mammalian cells have been described, the subsequent fate of internalized bacteria, as well as host and bacterial molecular pathways facilitating bacterial long-term survival, are not well defined. In particular, long-term survival within bladder epithelial cells has not been demonstrated and this may have important implications for the understanding and treatment of UTIs caused by P. aeruginosa. Here, we demonstrate and characterize the intracellular survival of wild type (WT) P. aeruginosa inside bladder epithelial cells and a mutant with a disruption in the bacterial two-component regulator AlgR that is unable to survive intracellularly. Using simultaneous dual RNA-seq transcriptional profiling, we define the transcriptional response of intracellular bacteria and their corresponding invaded host cells. The bacterial transcriptional response demonstrates that WT bacteria rapidly adapt to the stress encountered in the intracellular environment in contrast to ΔalgR bacteria. Analysis of the host transcriptional response to invasion suggests that the NF-κB signaling pathway, previously shown to be required for extracellular bacterial clearance, is paradoxically also required for intracellular bacterial survival. Lastly, we demonstrate that intracellular survival is important for pathogenesis of P. aeruginosa in vivo using a model of murine urinary tract infection. We propose that the unappreciated ability of P. aeruginosa to survive intracellularly may play an important role in contributing to the chronicity and recurrence of P. aeruginosa in urinary tract infections.

Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease.

The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Fitness selection in human pluripotent stem cells and interspecies chimeras: Implications for human development and regenerative medicine.

A small number of pluripotent cells within early embryo gives rise to all cells in the adult body, including germ cells. Hence, any mutations occurring in the pluripotent cell population are at risk of being propagated to their daughter cells and could lead to congenital defects or embryonic lethality and pose a risk of being transmitted to future generations. The observation that genetic errors are relatively common in preimplantation embryos, but their levels reduce as development progresses, suggests the existence of mechanisms for clearance of aberrant, unfit or damaged cells. Although early human embryogenesis is largely experimentally inaccessible, pluripotent stem cell (PSC) lines can be derived either from the inner cell mass (ICM) of a blastocyst or by reprogramming somatic cells into an embryonic stem cell-like state. PSCs retain the ability to differentiate into any cell type in vitro and, hence, they represent a unique and powerful tool for studying otherwise intractable stages of human development. The advent of PSCs has also opened up a possibility of developing regenerative medicine therapies, either through PSC differentiation in vitro or by creating interspecies chimeras for organ replacement. Here, we discuss the emerging evidence of cell selection in human PSC populations in vivo and in vitro and we highlight the implications of understanding this phenomenon for human development and regenerative medicine.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness.

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

Reproductive outcomes of intracytoplasmic sperm injection using testicular sperm and ejaculated sperm in patients with AZFc microdeletions: a systematic review and meta-analysis.

Studies have explored the assisted reproductive technology (ART) outcomes of Y-chromosome azoospermia factor c (AZFc) microdeletions, but the effect of sperm source on intracytoplasmic sperm injection (ICSI) remains unknown. To determine the ART results of ICSI using testicular sperm and ejaculated sperm from males with AZFc microdeletions, we searched Embase, Web of Science, and PubMed to conduct a systematic review and meta-analysis. The first meta-analysis results for 106 cycles in five studies showed no significant differences in the live birth rate between the testicular sperm group and the ejaculated sperm group (risk ratio: 0.97, 95% confidence interval [CI]: 0.73-1.28, P = 0.82). The second meta-analysis of 106 cycles in five studies showed no difference in the abortion rate between the testicular sperm group and ejaculated sperm group (risk ratio: 1.06, 95% CI: 0.54-2.06, P = 0.87). The third meta-analysis of 386 cycles in seven studies showed no significant difference in clinical pregnancy rates between the testicular sperm group and the ejaculated sperm group (risk ratio: 1.24, 95% CI: 0.66-2.34, P = 0.50). Inevitable heterogeneity weakened our results. However, our results indicated that testicular sperm and ejaculated sperm yield similar ART outcomes, representing a meaningful result for clinical treatment. More properly designed studies are needed to further confirm our conclusions.

miRNA: local guardians of presynaptic function in plasticity and disease.

Environmental fitness is an essential component of animal survival. Fitness is achieved through responsive physiological plasticity of tissues across the entire body, and particularly in the nervous system. At the molecular level, neural plasticity is mediated via gene-environmental interactions whereby developmental cues and experience dependent input adapt neuronal function to ever changing demands. To this end, neuronal gene regulation must be coupled to changes in neural activity. Seminal discoveries of the 20th century demonstrated neural activity modifies gene expression through calcium-dependent gene transcription. Building on this model, recent work over the last two decades shows that mRNA products of transcriptional programming continue to be regulated in the neuron through the activity-dependent post-transcriptional action of microRNAs (miRNAs). miRNAs are special post-transcriptional regulators that can tune gene expression within the spatial and temporal requirements of synaptic compartments. This mode of gene regulation has proven to be essential for synaptic function and plasticity as miRNA loss of function is highly associated with neural disease. In this review we will discuss current perspective on the link between presynaptic plasticity and miRNA biogenesis in the neuron.

Carryover effects of long-distance avian migration are weaker than effects of breeding environment in a partially migratory bird.

Migration may expose individuals to a wide range of increasing anthropogenic threats. In addition to direct mortality effects, this exposure may influence post-migratory reproductive fitness. Partial migration-where a population comprises migrants and residents-represents a powerful opportunity to explore carryover effects of migration. Studies of partial migration in birds typically examine short-distance systems; here we studied an unusual system where residents breed in mixed colonies alongside long-distance trans-Saharan migrants (lesser kestrels (Falco naumanni) in Spain). Combining geolocator data, stable isotope analysis and resighting data, we examined the effects of this stark difference in migratory strategy on body condition, breeding phenology and breeding success. We monitored four colonies in two regions of southern Spain for five consecutive years (2014-2018), yielding 1962 captures, determining migratory strategy for 141 adult bird-years. Despite a 3000-km difference in distance travelled, we find no effect of strategy on breeding parameters. We find weak evidence for a short-term negative carryover effect of migration on body condition, but this was only apparent in the breeding region with lower primary productivity. Our results indicate that carryover effects of even highly divergent migratory strategies may be minimal relative to effects of conditions experienced on breeding grounds.

Cancer Cell Fitness Is Dynamic.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. SIGNIFICANCE: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Subpopulations of sensorless bacteria drive fitness in fluctuating environments.

Populations of bacteria often undergo a lag in growth when switching conditions. Because growth lags can be large compared to typical doubling times, variations in growth lag are an important but often overlooked component of bacterial fitness in fluctuating environments. We here explore how growth lag variation is determined for the archetypical switch from glucose to lactose as a carbon source in Escherichia coli. First, we show that single-cell lags are bimodally distributed and controlled by a single-molecule trigger. That is, gene expression noise causes the population before the switch to divide into subpopulations with zero and nonzero lac operon expression. While "sensorless" cells with zero preexisting lac expression at the switch have long lags because they are unable to sense the lactose signal, any nonzero lac operon expression suffices to ensure a short lag. Second, we show that the growth lag at the population level depends crucially on the fraction of sensorless cells and that this fraction in turn depends sensitively on the growth condition before the switch. Consequently, even small changes in basal expression can significantly affect the fraction of sensorless cells, thereby population lags and fitness under switching conditions, and may thus be subject to significant natural selection. Indeed, we show that condition-dependent population lags vary across wild E. coli isolates. Since many sensory genes are naturally low expressed in conditions where their inducer is not present, bimodal responses due to subpopulations of sensorless cells may be a general mechanism inducing phenotypic heterogeneity and controlling population lags in switching environments. This mechanism also illustrates how gene expression noise can turn even a simple sensory gene circuit into a bet hedging module and underlines the profound role of gene expression noise in regulatory responses.

Mismatch of thermal optima between performance measures, life stages and species of spiny lobster.

In an ocean warming hotspot off south-east Australia, many species have expanded their ranges polewards, including the eastern rock lobster, Sagmariasus verreauxi. This species is likely extending its range via larval advection into Tasmanian coastal waters, which are occupied by the more commercially important southern rock lobster, Jasus edwardsii. Here, thermal tolerances of these lobster species at two life stages were investigated to assess how they may respond to warming ocean temperatures. We found that the pattern, optimum and magnitude of thermal responses differed between performance measures, life stages and species. Sagmariasus verreauxi had a warmer optimal temperature for aerobic scope and escape speed than J. edwardsii. However, J. edwardsii had a higher magnitude of escape speed, indicating higher capacity for escape performance. There were also differences between life stages within species, with the larval stage having higher variation in optimal temperatures between measures than juveniles. This inconsistency in performance optima and magnitude indicates that single performance measures at single life stages are unlikely to accurately predict whole animal performance in terms of life-time survival and fitness. However, combined results of this study suggest that with continued ocean warming, S. verreauxi is likely to continue to extend its distribution polewards and increase in abundance in Tasmania.