Complex patterns of morphological diversity across multiple populations of an urban bird species.

Urbanization presents a natural evolutionary experiment because selection pressures in cities can be strongly mismatched with those found in species' historic habitats. However, some species have managed to adapt and even thrive in these novel conditions. When a species persists across multiple cities, a fundamental question arises: do we see similar traits evolve in similar novel environments? By testing if and how similar phenotypes emerge across multiple urban populations, we can begin to assess the predictability of population response to anthropogenic change. Here, we examine variation within and across multiple populations of a songbird, the dark-eyed junco (Junco hyemalis). We measured morphological variations in juncos across urban and nonurban populations in Southern California. We investigated whether the variations we observed were due to differences in environmental conditions across cities. Bill shape differed across urban populations; Los Angeles and Santa Barbara juncos had shorter, deeper bills than nonurban juncos, but San Diego juncos did not. On the other hand, wing length decreased with the built environment, regardless of the population. Southern Californian urban juncos exhibit both similarities and differences in morphological traits. Studying multiple urban populations can help us determine the predictability of phenotypic evolutionary responses to novel environments.

Evolution Under Thermal Stress Affects Escherichia coli's Resistance to Antibiotics.

Exposure to both antibiotics and temperature changes can induce similar physiological responses in bacteria. Thus, changes in growth temperature may affect antibiotic resistance. Previous studies have found that evolution under antibiotic stress causes shifts in the optimal growth temperature of bacteria. However, little is known about how evolution under thermal stress affects antibiotic resistance. We examined 100+ heat-evolved strains of Escherichia coli that evolved under thermal stress. We asked whether evolution under thermal stress affects optimal growth temperature, if there are any correlations between evolving in high temperatures and antibiotic resistance, and if these strains' antibiotic efficacy changes depending on the local environment's temperature. We found that: (1) surprisingly, most of the heat-evolved strains displayed a decrease in optimal growth temperature and overall growth relative to the ancestor strain, (2) there were complex patterns of changes in antibiotic resistance when comparing the heat-evolved strains to the ancestor strain, and (3) there were few significant correlations among changes in antibiotic resistance, optimal growth temperature, and overall growth.

The evolution of resistance to synergistic multi-drug combinations is more complex than evolving resistance to each individual drug component.

Multidrug antibiotic resistance is an urgent public health concern. Multiple strategies have been suggested to alleviate this problem, including the use of antibiotic combinations and cyclic therapies. We examine how adaptation to (1) combinations of drugs affects resistance to individual drugs, and to (2) individual drugs alters responses to drug combinations. To evaluate this, we evolved multiple strains of drug resistant Staphylococcus epidermidis in the lab. We show that evolving resistance to four highly synergistic combinations does not result in cross-resistance to all of their components. Likewise, prior resistance to one antibiotic in a combination does not guarantee survival when exposed to the combination. We also identify four 3-step and four 2-step treatments that inhibit bacterial growth and confer collateral sensitivity with each step, impeding the development of multidrug resistance. This study highlights the importance of considering higher-order drug combinations in sequential therapies and how antibiotic interactions can influence the evolutionary trajectory of bacterial populations.

Urban birds become less fearful following COVID-19 reopenings.

Following the COVID-19 pandemic, many people around the world stayed home, drastically altering human activity in cities. This exceptional moment provided researchers the opportunity to test how urban animals respond to human disturbance, in some cases testing fundamental questions on the mechanistic impact of urban behaviours on animal behaviour. However, at the end of this 'anthropause', human activity returned to cities. How might each of these strong shifts affect wildlife in the short and long term? We focused on fear response, a trait essential to tolerating urban life. We measured flight initiation distance-at both individual and population levels-for an urban bird before, during and after the anthropause to examine if birds experienced longer-term changes after a year and a half of lowered human presence. Dark-eyed juncos did not change fear levels during the anthropause, but they became drastically less fearful afterwards. These surprising and counterintuitive findings, made possible by following the behaviour of individuals over time, has led to a novel understanding that fear response can be driven by plasticity, yet not habituation-like processes. The pandemic-caused changes in human activity have shown that there is great complexity in how humans modify a behavioural trait fundamental to urban tolerance in animals.

Meta-analysis of three-stressor combinations on population-level fitness reveal substantial higher-order interactions.

Although natural populations are typically subjected to multiple stressors, most past research has focused on single-stressor and two-stressor interactions, with little attention paid to higher-order interactions among three or more stressors. However, higher-order interactions increasingly appear to be widespread. Consequently, we used a recently introduced and improved framework to re-analyze higher-order ecological interactions. We conducted a literature review of the last 100 years (1920-2020) and reanalyzed 142 ecological three-stressor interactions on species' populations from 38 published papers; the vast majority of these studies were from the past 10 years. We found that 95.8 % (n = 136) of the three-stressor combinations had either not been categorized before or resulted in different interactions than previously reported. We also found substantial levels of emergent properties-interactions that are not due to strong pairwise interactions within the combination but rather uniquely due to all three stressors being combined. Calculating net interactions-the overall accounting for all possible interactions within a combination including the emergent and all pairwise interactions-we found that the most prevalent interaction type is antagonism, corresponding to a smaller than expected effect based on single stressor effects. In contrast, for emergent interactions, the most prevalent interaction type is synergistic, resulting in a larger than expected effect based on single stressor effects. Additionally, we found that hidden suppressive interactions-where a pairwise interaction is suppressed by a third stressor-are found in the majority of combinations (74 %). Collectively, understanding multiple stressor interactions through applying an appropriate framework is crucial for answering fundamental questions in ecology and has implications for conservation biology and population management. Crucially, identifying emergent properties can reveal hidden suppressive interactions that could be particularly important for the ecological management of at-risk populations.

Precocious emergence of cognitive and synaptic dysfunction in 3xTg-AD mice exposed prenatally to ethanol.

Alzheimer's disease (AD) is the most common cause of dementia, affecting approximately 50 million people worldwide. Early life risk factors for AD, including prenatal exposures, remain underexplored. Exposure of the fetus to alcohol (ethanol) is not uncommon during pregnancy, and may result in physical, behavioral, and cognitive changes that are first detected during childhood but result in lifelong challenges. Whether or not prenatal ethanol exposure may contribute to Alzheimer's disease risk is not yet known. Here we exposed a mouse model of Alzheimer's disease (3xTg-AD), bearing three dementia-associated transgenes, presenilin1 (PS1M146V), human amyloid precursor protein (APPSwe), and human tau (TauP301S), to ethanol on gestational days 13.5-16.5 using an established binge-type maternal ethanol exposure paradigm. We sought to investigate whether prenatal ethanol exposure resulted in a precocious onset or increased severity of AD progression, or both. We found that a brief binge-type gestational exposure to ethanol during a period of peak neuronal migration to the developing cortex resulted in an earlier onset of spatial memory deficits and behavioral inflexibility in the progeny, as assessed by performance on the modified Barnes maze task. The observed cognitive changes coincided with alterations to both GABAergic and glutamatergic synaptic transmission in layer V/VI neurons, diminished GABAergic interneurons, and increased ß-amyloid accumulation in the medial prefrontal cortex. These findings provide the first preclinical evidence for prenatal ethanol exposure as a potential factor for modifying the onset of AD-like behavioral dysfunction and set the groundwork for more comprehensive investigations into the underpinnings of AD-like cognitive changes in individuals with fetal alcohol spectrum disorders.

Estimating microbial population data from optical density.

The spectrophotometer has been used for decades to measure the density of bacterial populations as the turbidity expressed as optical density-OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.1. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/mL. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) to show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/mL-rather than OD-is a metric that can be used to directly compare results across experiments, labs, instruments, and species.

Evolution of antibiotic resistance impacts optimal temperature and growth rate in Escherichia coli and Staphylococcus epidermidis.

AIMS: Bacterial response to temperature changes can influence their pathogenicity to plants and humans. Changes in temperature can affect cellular and physiological responses in bacteria that can in turn affect the evolution and prevalence of antibiotic-resistance genes. Yet, how antibiotic-resistance genes influence microbial temperature response is poorly understood. METHODS AND RESULTS: We examined growth rates and physiological responses to temperature in two species-E. coli and Staph. epidermidis-after evolved resistance to 13 antibiotics. We found that evolved resistance results in species-, strain- and antibiotic-specific shifts in optimal temperature. When E. coli evolves resistance to nucleic acid and cell wall inhibitors, their optimal growth temperature decreases, and when Staph. epidermidis and E. coli evolve resistance to protein synthesis and their optimal temperature increases. Intriguingly, when Staph. epidermidis evolves resistance to Teicoplanin, fitness also increases in drug-free environments, independent of temperature response. CONCLUSION: Our results highlight how the complexity of antibiotic resistance is amplified when considering physiological responses to temperature. SIGNIFICANCE: Bacteria continuously respond to changing temperatures-whether through increased body temperature during fever, climate change or other factors. It is crucial to understand the interactions between antibiotic resistance and temperature.

No evidence of repeated song divergence across multiple urban and non-urban populations of dark-eyed juncos (Junco hyemalis) in Southern California.

Urbanization can affect species communication by introducing new selection pressures, such as noise pollution and different environmental transmission properties. These selection pressures can trigger divergence between urban and non-urban populations. Songbirds rely on vocalizations to defend territories and attract mates. Urban songbirds have been shown in some species and some populations to increase the frequencies, reduce the length and change other temporal features of their songs. This study compares songs from four urban and three non-urban populations of dark-eyed juncos (Junco hyemalis) throughout Southern California. We examined song length, trill rate, minimum frequency, maximum frequency, peak frequency and frequency bandwidth. We also compared songs recorded from one urban junco population in San Diego nearly two decades ago with recently collected data in 2018-2020. Over all comparisons, we only found significant differences between UCLA and the 2006/2007 UCSD field seasons in minimum and maximum frequencies. These findings partially support and are partially in contrast to previous urban song studies. As urban areas expand, more opportunities will arise to understand urban song divergence in greater detail.

Rapid evolutionary divergence of a songbird population following recent colonization of an urban area.

Colonization of a novel environment by a few individuals can lead to rapid evolutionary change, yet there is scarce evidence of the relative contributions of neutral and selective factors in promoting divergence during the early stages of colonization. Here we explore the role of neutral and selective forces in the divergence of a unique urban population of the dark-eyed junco (Junco hyemalis), which became established on the campus of the University of California at San Diego (UCSD) in the early 1980s. Previous studies based on microsatellite loci documented significant genetic differentiation of the urban population as well as divergence in phenotypic traits relative to nearby montane populations, yet the geographical origin of the colonization and the contributing factors remained uncertain. Our genome-wide single nucleotide polymorphism data set confirmed the marked genetic differentiation of the UCSD population, and we identified the coastal subspecies pinosus from central California as its sister group instead of the neighbouring mountain population. Demographic inference recovered a separation from pinosus as recent as 20-32 generations ago after a strong bottleneck, suggesting a role for drift in genetic differentiation. However, we also found significant associations between habitat variables and genome-wide variants linked to functional genes, some of which have been reported as potentially adaptive in birds inhabiting modified environments. These results suggest that the interplay between founder events and selection may result in rapid shifts in neutral and adaptive loci across the genome, and reveal the UCSD junco population as a case of contemporary evolutionary divergence in an anthropogenic environment.

L-Type Calcium Channels Contribute to Ethanol-Induced Aberrant Tangential Migration of Primordial Cortical GABAergic Interneurons in the Embryonic Medial Prefrontal Cortex.

Exposure of the fetus to alcohol (ethanol) via maternal consumption during pregnancy can result in fetal alcohol spectrum disorders (FASD), hallmarked by long-term physical, behavioral, and intellectual abnormalities. In our preclinical mouse model of FASD, prenatal ethanol exposure disrupts tangential migration of corticopetal GABAergic interneurons (GINs) in the embryonic medial prefrontal cortex (mPFC). We postulated that ethanol perturbed the normal pattern of tangential migration via enhancing GABAA receptor-mediated membrane depolarization that prevails during embryonic development in GABAergic cortical interneurons. However, beyond this, our understanding of the underlying mechanisms is incomplete. Here, we tested the hypothesis that the ethanol-enhanced depolarization triggers downstream an increase in high-voltage-activated nifedipine-sensitive L-type calcium channel (LTCC) activity and provide evidence implicating calcium dynamics in the signaling scheme underlying the migration of embryonic GINs and its aberrance. Tangentially migrating Nkx2.1+ GINs expressed immunoreactivity to Cav1.2, the canonical neuronal isoform of the L-type calcium channel. Prenatal ethanol exposure did not alter its protein expression profile in the embryonic mPFC. However, exposing ethanol concomitantly with the LTCC blocker nifedipine prevented the ethanol-induced aberrant migration both in vitro and in vivo In addition, whole-cell patch clamp recording of LTCCs in GINs migrating in embryonic mPFC slices revealed that acutely applied ethanol potentiated LTCC activity in migrating GINs. Based on evidence reported in the present study, we conclude that calcium is an important intracellular intermediary downstream of GABAA receptor-mediated depolarization in the mechanistic scheme of an ethanol-induced aberrant tangential migration of embryonic GABAergic cortical interneurons.

Antibiotics Shift the Temperature Response Curve of Escherichia coli Growth.

Temperature variation-through time and across climatic gradients-affects individuals, populations, and communities. Yet how the thermal response of biological systems is altered by environmental stressors is poorly understood. Here, we quantify two key features-optimal temperature and temperature breadth-to investigate how temperature responses vary in the presence of antibiotics. We use high-throughput screening to measure growth of Escherichia coli under single and pairwise combinations of 12 antibiotics across seven temperatures that range from 22°C to 46°C. We find that antibiotic stress often results in considerable changes in the optimal temperature for growth and a narrower temperature breadth. The direction of the optimal temperature shifts can be explained by the similarities between antibiotic-induced and temperature-induced damage to the physiology of the bacterium. We also find that the effects of pairs of stressors in the temperature response can often be explained by just one antibiotic out of the pair. Our study has implications for a general understanding of how ecological systems adapt and evolve to environmental changes. IMPORTANCE The growth of living organisms varies with temperature. This dependence is described by a temperature response curve that is described by an optimal temperature where growth is maximized and a temperature range (termed breadth) across which the organism can grow. Because an organism's temperature response evolves or acclimates to its environment, it is often assumed to change over only evolutionary or developmental timescales. Counter to this, we show here that antibiotics can quickly (over hours) change the optimal growth temperature and temperature breadth for the bacterium Escherichia coli. Moreover, our results suggest a shared-damage hypothesis: when an antibiotic damages similar cellular components as hot (or cold) temperatures do, this shared damage will combine and compound to more greatly reduce growth when that antibiotic is administered at hot (or cold) temperatures. This hypothesis could potentially also explain how temperature responses are modified by stressors other than antibiotics.

Hidden suppressive interactions are common in higher-order drug combinations.

The rapid increase of multi-drug resistant bacteria has led to a greater emphasis on multi-drug combination treatments. However, some combinations can be suppressive-that is, bacteria grow faster in some drug combinations than when treated with a single drug. Typically, when studying interactions, the overall effect of the combination is only compared with the single-drug effects. However, doing so could miss "hidden" cases of suppression, which occur when the highest order is suppressive compared with a lower-order combination but not to a single drug. We examined an extensive dataset of 5-drug combinations and all lower-order-single, 2-, 3-, and 4-drug-combinations. We found that a majority of all combinations-54%-contain hidden suppression. Examining hidden interactions is critical to understanding the architecture of higher-order interactions and can substantially affect our understanding and predictions of the evolution of antibiotic resistance under multi-drug treatments.

Nests in the cities: adaptive and non-adaptive phenotypic plasticity and convergence in an urban bird.

Phenotypic plasticity plays a critical role in adaptation to novel environments. Behavioural plasticity enables more rapid responses to unfamiliar conditions than evolution by natural selection. Urban ecosystems are one such novel environment in which behavioural plasticity has been documented. However, whether such plasticity is adaptive, and if plasticity is convergent among urban populations, is poorly understood. We studied the nesting biology of an 'urban-adapter' species, the dark-eyed junco (Junco hyemalis), to understand the role of plasticity in adapting to city life. We examined (i) whether novel nesting behaviours are adaptive, (ii) whether pairs modify nest characteristics in response to prior outcomes, and (iii) whether two urban populations exhibit similar nesting behaviour. We monitored 170 junco nests in urban Los Angeles and compared our results with prior research on 579 nests from urban San Diego. We found that nests placed in ecologically novel locations (off-ground and on artificial surfaces) increased fitness, and that pairs practiced informed re-nesting in site selection. The Los Angeles population more frequently nested off-ground than the San Diego population and exhibited a higher success rate. Our findings suggest that plasticity facilitates adaptation to urban environments, and that the drivers behind novel nesting behaviours are complex and multifaceted.

Using a newly introduced framework to measure ecological stressor interactions.

Understanding how stressors combine to affect population abundances and trajectories is a fundamental ecological problem with increasingly important implications worldwide. Generalisations about interactions among stressors are challenging due to different categorisation methods and how stressors vary across species and systems. Here, we propose using a newly introduced framework to analyse data from the last 25 years on ecological stressor interactions, for example combined effects of temperature, salinity and nutrients on population survival and growth. We contrast our results with the most commonly used existing method - analysis of variance (ANOVA) - and show that ANOVA assumptions are often violated and have inherent limitations for detecting interactions. Moreover, we argue that rescaling - examining relative rather than absolute responses - is critical for ensuring that any interaction measure is independent of the strength of single-stressor effects. In contrast, non-rescaled measures - like ANOVA - find fewer interactions when single-stressor effects are weak. After re-examining 840 two-stressor combinations, we conclude that antagonism and additivity are the most frequent interaction types, in strong contrast to previous reports that synergy dominates yet supportive of more recent studies that find more antagonism. Consequently, measuring and re-assessing the frequency of stressor interaction types is imperative for a better understanding of how stressors affect populations.

Compounding Effects of Climate Warming and Antibiotic Resistance.

Bacteria have evolved diverse mechanisms to survive environments with antibiotics. Temperature is both a key factor that affects the survival of bacteria in the presence of antibiotics and an environmental trait that is drastically increasing due to climate change. Therefore, it is timely and important to understand links between temperature changes and selection of antibiotic resistance. This review examines these links by synthesizing results from laboratories, hospitals, and environmental studies. First, we describe the transient physiological responses to temperature that alter cellular behavior and lead to antibiotic tolerance and persistence. Second, we focus on the link between thermal stress and the evolution and maintenance of antibiotic resistance mutations. Finally, we explore how local and global changes in temperature are associated with increases in antibiotic resistance and its spread. We suggest that a multidisciplinary, multiscale approach is critical to fully understand how temperature changes are contributing to the antibiotic crisis.

Evolution of antibiotic cross-resistance and collateral sensitivity in Staphylococcus epidermidis using the mutant prevention concentration and the mutant selection window.

In bacteria, evolution of resistance to one antibiotic is frequently associated with increased resistance (cross-resistance) or increased susceptibility (collateral sensitivity) to other antibiotics. Cross-resistance and collateral sensitivity are typically evaluated at the minimum inhibitory concentration (MIC). However, these susceptibility changes are not well characterized with respect to the mutant prevention concentration (MPC), the antibiotic concentration that prevents a single-step mutation from occurring. We measured the MIC and the MPC for Staphylococcus epidermidis and 14 single-drug resistant strains against seven antibiotics. We found that the MIC and the MPC were positively correlated but that this correlation weakened if cross-resistance did not evolve. If any type of resistance did evolve, the range of concentrations between the MIC and the MPC tended to shift right and widen. Similar patterns of cross-resistance and collateral sensitivity were observed at the MIC and MPC levels, though more symmetry was observed at the MIC level. Whole-genome sequencing revealed mutations in both known-target and nontarget genes. Moving forward, examining both the MIC and the MPC may lead to better predictions of evolutionary trajectories in antibiotic-resistant bacteria.

Evolutionary consequences of feedbacks between within-host competition and disease control.

Lay Summary: Competition often occurs among diverse parasites within a single host, but control efforts could change its strength. We examined how the interplay between competition and control could shape the evolution of parasite traits like drug resistance and disease severity.

Antibiotic combination therapy against resistant bacterial infections: synergy, rejuvenation and resistance reduction.

Introduction: Anti-Microbial Resistance (AMR) is a pandemic which threatens modern medicine. There is a lack of effective drug treatment due to the slow pace, high cost and low achievable sales prices of new antibiotic monotherapies. New hope comes in the shape of antibiotic combination therapy, which although used by mother nature, is under-explored and could provide the solution to AMR.Areas covered: We performed a search of Pubmed and Medline using the keywords 'combination therapy', 'antimicrobial resistance' for articles between 1930 and 2019, as supplemented with other relevant references to our knowledge. We have reviewed the theoretical considerations for combination development and examine the existing and future clinical indications of combination therapies. We have discussed the potential of antibiotic combinations to provide therapeutic synergy, rejuvenating the effectiveness of old antibiotics to which the bacteria had developed resistance previously. We have examined the current thinking and evidence on resistance reduction using combination therapies, with a review on toxicity and drug-drug antagonism.Expert opinion: Antibiotic combination therapy, exploiting synergies, old-drug rejuvenation and resistance reduction could provide the solution to AMR. The number of pharmaceutical companies in this area is likely to expand, bringing promising combinations to the bedside, to save millions of lives worldwide.

Prenatal Exposure to Ethanol Alters Synaptic Activity in Layer V/VI Pyramidal Neurons of the Somatosensory Cortex.

Fetal alcohol spectrum disorder (FASD) encompasses a range of cognitive and behavioral deficits, with aberrances in the function of cerebral cortical pyramidal neurons implicated in its pathology. However, the mechanisms underlying these aberrances, including whether they persist well beyond ethanol exposure in utero, remain to be explored. We addressed these issues by employing a mouse model of FASD in which pregnant mice were exposed to binge-type ethanol from embryonic day 13.5 through 16.5. In both male and female offspring (postnatal day 28-32), whole-cell patch clamp recording of layer V/VI somatosensory cortex pyramidal neurons revealed increases in the frequency of excitatory and inhibitory postsynaptic currents. Furthermore, expressing channelrhodopsin in either GABAergic interneurons (Nkx2.1Cre-Ai32) or glutamatergic pyramidal neurons (Emx1IRES Cre-Ai32) revealed a shift in optically evoked paired-pulse ratio. These findings are consistent with an excitatory-inhibitory imbalance with prenatal ethanol exposure due to diminished inhibitory but enhanced excitatory synaptic strength. Prenatal ethanol exposure also altered the density and morphology of spines along the apical dendrites of pyramidal neurons. Thus, while both presynaptic and postsynaptic mechanisms are affected following prenatal exposure to ethanol, there is a prominent presynaptic component that contributes to altered inhibitory and excitatory synaptic transmission in the somatosensory cortex.