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Most bacteria in nature live in surface-associated communities rather than planktonic populations. Nonetheless, how surface-associated environments shape bacterial evolutionary adaptation remains poorly understood. Here, we show that subjecting Pseudomonas aeruginosa to repeated rounds of swarming, a collective form of surface migration, drives remarkable parallel evolution toward a hyperswarmer phenotype. In all independently evolved hyperswarmers, the reproducible hyperswarming phenotype is caused by parallel point mutations in a flagellar synthesis regulator, FleN, which locks the naturally monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although hyperswarmers outcompete the ancestral strain in swarming competitions, they are strongly outcompeted in biofilm formation, which is an essential trait for P. aeruginosa in environmental and clinical settings. The finding that evolution in swarming colonies reliably produces evolution of poor biofilm formers supports the existence of an evolutionary trade-off between motility and biofilm formation.

Pathogenic bacteria require iron for replication within their host. Klebsiella pneumoniae and other Gram-negative pathogens produce the prototypical siderophore enterobactin (Ent) to scavenge iron in vivo . In response, mucosal surfaces secrete lipocalin 2 (Lcn2), an innate immune protein that binds Ent to disrupt bacterial iron acquisition and promote acute inflammation during colonization. A subset of K. pneumoniae isolates attempt to evade Lcn2 by producing glycosylated Ent (Gly-Ent, salmochelin) or the alternative siderophore yersiniabactin (Ybt). However, these siderophores are not functionally equivalent and differ in their abilities to promote growth in the upper respiratory tract, lungs, and serum. To understand how Lcn2 exploits functional differences between siderophores, isogenic mutants of an Ent + Gly-Ent + Ybt + K. pneumoniae strain were inoculated into Lcn2 +/+ and Lcn2 −/− mice, and the pattern of pneumonia was examined. Lcn2 effectively protected against the iroA ybtS mutant (Ent + Gly-Ent − Ybt − ). Lcn2 +/+ mice had small foci of pneumonia, whereas Lcn2 −/− mice had many bacteria in the perivascular space. The entB mutant (Ent − Ybt + Gly-Ent − ) caused moderate bronchopneumonia but did not invade the transferrin-containing perivascular space. Accordingly, transferrin blocked Ybt-dependent growth in vitro . The wild type and the iroA mutant, which both produce Ent and Ybt, had a mixed phenotype, causing a moderate bronchopneumonia in Lcn2 +/+ mice and perivascular overgrowth in Lcn2 −/− mice. Together, these data indicate that Lcn2, in combination with transferrin, confines K. pneumoniae to the airways and prevents invasion into tissue containing the pulmonary vasculature. IMPORTANCE Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung. Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung.

With the decline of bee populations worldwide, studies determining current wild bee distributions and diversity are increasingly important. Wild bee identification is often completed by experienced taxonomists or by genetic analysis. The current study was designed to compare two methods of identification including: (1) morphological identification by experienced taxonomists using images of field-collected wild bees and (2) genetic analysis of composite bee legs (multiple taxa) using metabarcoding. Bees were collected from conservation grasslands in eastern Iowa in summer 2019 and identified to the lowest taxonomic unit using both methods. Sanger sequencing of individual wild bee legs was used as a positive control for metabarcoding. Morphological identification of bees using images resulted in 36 unique taxa among 22 genera, and >80% of Bombus specimens were identified to species. Metabarcoding was limited to genus-level assignments among 18 genera but resolved some morphologically similar genera. Metabarcoding did not consistently detect all genera in the composite samples, including kleptoparasitic bees. Sanger sequencing showed similar presence or absence detection results as metabarcoding but provided species-level identifications for cryptic species (i.e., Lasioglossum ). Genus-specific detections were more frequent with morphological identification than metabarcoding, but certain genera such as Ceratina and Halictus were identified equally well with metabarcoding and morphology. Genera with proportionately less tissue in a composite sample were less likely to be detected using metabarcoding. Image-based methods were limited by image quality and visible morphological features, while genetic methods were limited by databases, primers, and amplification at target loci. This study shows how an image-based identification method compares with genetic techniques, and how in combination, the methods provide valuable genus- and species-level information for wild bees while preserving tissue for other analyses. These methods could be improved and transferred to a field setting to advance our understanding of wild bee distributions and to expedite conservation research.

Characterizing genetic structure across a species’ range is relevant for management and conservation as it can be used to define population boundaries and quantify connectivity. Wide-ranging species residing in continuously distributed habitat pose substantial challenges for the characterization of genetic structure as many analytical methods used are less effective when isolation by distance is an underlying biological pattern. Here, we illustrate strategies for overcoming these challenges using a species of significant conservation concern, the Greater Sage-grouse ( Centrocercus urophasianus ), providing a new method to identify centers of genetic differentiation and combining multiple methods to help inform management and conservation strategies for this and other such species. Our objectives were to (1) describe large-scale patterns of population genetic structure and gene flow and (2) to characterize genetic subpopulation centers across the range of Greater Sage-grouse. Samples from 2,134 individuals were genotyped at 15 microsatellite loci. Using standard STRUCTURE and spatial principal components analyses, we found evidence for four or six areas of large-scale genetic differentiation and, following our novel method, 12 subpopulation centers of differentiation. Gene flow was greater, and differentiation reduced in areas of contiguous habitat (eastern Montana, most of Wyoming, much of Oregon, Nevada, and parts of Idaho). As expected, areas of fragmented habitat such as in Utah (with 6 subpopulation centers) exhibited the greatest genetic differentiation and lowest effective migration. The subpopulation centers defined here could be monitored to maintain genetic diversity and connectivity with other subpopulation centers. Many areas outside subpopulation centers are contact zones where different genetic groups converge and could be priorities for maintaining overall connectivity. Our novel method and process of leveraging multiple different analyses to find common genetic patterns provides a path forward to characterizing genetic structure in wide-ranging, continuously distributed species.

Sex ratio, and the extent to which it varies over time, is an important factor in the demography, management, and conservation of wildlife populations. Greater sage‐grouse Centrocercus urophasianus populations in western North America are monitored using counts of males at leks in spring. Population estimates derived from lek‐count data typically assume a constant, female‐biased sex ratio, yet few rigorous, empirically derived estimates of sex ratio are available to test that assumption. We estimated pre‐breeding sex ratio of greater sage‐grouse in a peripheral, geographically isolated population in northwestern Colorado during two consecutive winters using closed‐population, robust‐design, multi‐state, genetic mark–recapture models in program MARK. Sex ratio varied markedly between years, with estimates of 3.29 (95% CI: 2.36–4.59) females per male in winter 2012–2013 and 1.54 (95% CI: 1.22–1.95) females per male in winter 2013–2014. Rather than assuming a constant sex ratio, biologists should consider the potential for large annual variation in sex ratio of greater sage‐grouse populations when estimating population size or trend from male lek‐count data.

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.

Background Dentists and oral surgeons are leading prescribers of opioids to adolescents and young adults (AYA), who are at high risk for developing problematic opioid use after an initial exposure. Most opioids are prescribed after tooth extraction, but non-opioid analgesics provide similar analgesia and are recommended by multiple professional organizations. Methods This multi-site stepped wedge cluster-randomized trial will assess whether a multicomponent behavioral intervention can influence opioid prescribing behavior among dentists and oral surgeons compared to usual practice. Across up to 12 clinical practices (clusters), up to 33 dentists/oral surgeons (provider participants) who perform tooth extractions for individuals 12–25 years old will be enrolled. After enrollment, all provider participants will receive the intervention at a time based on the sequence to which their cluster is randomized. The intervention consists of prescriber education via academic detailing plus provision of standardized patient post-extraction instructions and blister packs of acetaminophen and ibuprofen. Provider participants will dispense the blister packs and distribute the patient instructions at their discretion to AYA undergoing tooth extraction, with or without additional analgesics. The primary outcome is a binary, patient-level indicator of electronic post-extraction opioid prescription. Data for the primary outcome will be collected from the provider participant’s electronic health records quarterly throughout the study. Provider participants will complete a survey before and approximately 3 months after transitioning into the intervention condition to assess implementation outcomes. AYA patients undergoing tooth extraction will be offered a survey to assess pain control and satisfaction with pain management in the week after their extraction. Primary analyses will use generalized estimating equations to compare the binary patient-level indicator of being prescribed a post-extraction opioid in the intervention condition compared to usual practice. Secondary analyses will assess provider participants’ perceptions of feasibility and appropriateness of the intervention, and patient-reported pain control and satisfaction with pain management. Analyses will adjust for patient-level factors (e.g., sex, number of teeth extracted, etc.). Discussion This real-world study will address an important need, providing information on the effectiveness of a multicomponent intervention at modifying dental prescribing behavior and reducing opioid prescriptions to AYA. ClinicalTrials.gov NCT06275191.

Use of environmental DNA (eDNA) to assess distributions of aquatic and semi-aquatic macroorganisms is promising, but sampling schemes may need to be tailored to specific objectives. Given the potentially high variance in aquatic eDNA among replicate grab samples, compositing smaller water volumes collected over a period of time may be more effective for some applications. In this study, we compared eDNA profiles from composite water samples aggregated over three hours with grab water samples. Both sampling patterns were performed with identical autosamplers paired at two different sites in a headwater stream environment, augmented with exogenous fish eDNA from an upstream rearing facility. Samples were filtered through 0.8 μm cellulose nitrate filters and DNA was extracted with a cetyl trimethylammonium bromide procedure. Eukaryotic and bacterial community profiles were derived by amplicon sequencing of 12S ribosomal, 16S ribosomal, and cytochrome oxidase I loci. Operational taxa were assigned to genus with a lowest common ancestor approach for eukaryotes and to family with the RDP Classifier software for prokaryotes. Eukaryotic community profiles were more consistent with composite sampling than grab sampling. Downstream, rarefaction curves suggested faster taxon accumulation for composite samples, and estimated richness was higher for composite samples as a set than for grab samples. Upstream, composite sampling produced lower estimated richness than grab samples, but with overlapping standard errors. Furthermore, a bimodal pattern of richness as a function of sequence counts suggested the impact of clumped particles on upstream samples. Bacterial profiles were insensitive to sample method, consistent with the more even dispersion expected for bacteria compared with eukaryotic eDNA. Overall, samples composited over 3 h performed equal to or better than triplicate grab sampling for quantitative community metrics, despite the higher total sequencing effort provided to grab replicates. On the other hand, taxon-specific detection rates did not differ appreciably and the two methods gave similar estimates of the ratio of the common fish genera Salmo and Coregonus at each site. Unexpectedly, Salmo eDNA dropped out substantially faster than Coregonus eDNA between the two sites regardless of sampling method, suggesting that differential settling affects the estimation of relative abundance. We identified bacterial patterns that were associated with eukaryotic diversity, suggesting potential roles as biomarkers of sample representativeness.

Objectives Meditative practices have grown in popularity, but the results of meditative intervention studies on immune functioning have been inconsistent. Although prior reviews have been conducted, the present meta-analysis provides a more comprehensive and updated examination. Methods One hundred and five eligible studies, including mindfulness-based, movement-based, and meditation-focused, meditative interventions with biological markers of immune functioning were analyzed. The current work (a) incorporates a greater number of studies available for review, (b) examines the overall magnitude of the effect of meditative interventions on immune functioning, (c) examines the effects of health on some individual level biomarkers (i.e., NF-κB, IgA, and IL-6), (d) compares different types of meditative interventions and (e) reveals the effect of various theoretical (e.g., the health of participants) and methodological (e.g., delivery of interventions) moderators. Results The meta-analysis indicated that meditative interventions, including formal meditation, mindfulness-based, and movement-based, have a small but significant effect on immune functioning ( g  = 0.181, k  = 105, p  < 0.001) as compared to controls ( g  = -0.001, p  = 0.982). Furthermore, the results indicated that the effect of meditative interventions on immune functioning remained robust, regardless of the type of control condition. Conclusions The present meta-analysis suggests a small and significant effect of meditative interventions on immune functioning and serves to clarify inconsistent results in the literature. Further, it provides insight into both theoretical and methodological moderators for future research. Meditative interventions could be implemented in various formats and modalities, especially among those with physiological and psychological disorders. Preregistration The present meta-analysis was not preregistered.

The Arizona Toad (Anaxyrus microscaphus) is restricted to riverine corridors and adjacent uplands in the arid southwestern United States. As with numerous amphibians worldwide, populations are declining and face various known or suspected threats, from disease to habitat modification resulting from climate change. The Arizona Toad has been petitioned to be listed under the U.S. Endangered Species Act and was considered “warranted but precluded” citing the need for additional information – particularly regarding natural history (e.g., connectivity and dispersal ability). The objectives of this study were to characterize population structure and genetic diversity across the species’ range. We used reduced-representation genomic sequencing to genotype 3,601 single nucleotide polymorphisms in 99 Arizona Toads from ten drainages across its range. Multiple analytical methods revealed two distinct genetic groups bisected by the Colorado River; one in the northwestern portion of the range in southwestern Utah and eastern Nevada and the other in the southeastern portion of the range in central and eastern Arizona and New Mexico. We also found subtle substructure within both groups, particularly in central Arizona where toads at lower elevations were less connected than those at higher elevations. The northern and southern parts of the Arizona Toad range are not well connected genetically and could be managed as separate units. Further, these data could be used to identify source populations for assisted migration or translocations to support small or potentially declining populations.