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One exciting class of future genetic devices could be those deployed in microbes that join complex microbial environments in the wild. We sought to determine whether genetic parts designed for monoculture are predictable when used in co-culture by testing constitutive Anderson promoters driving the expression of chromoproteins from a plasmid. In Escherichia coli monoculture, a high copy number origin of replication causes stochastic expression regardless of promoter strength, and high constitutive Anderson promoter strength leads to selection for inactivating mutations, resulting in inconsistent chromoprotein expression. Medium- and low-strength constitutive Anderson promoters function more predictably in E. coli monoculture but experience an increase in inactivating mutations when grown in co-culture over many generations with Pseudomonas aeruginosa. Expression from regulated promoters instead of constitutive Anderson promoters can lead to stable expression in a complex wastewater culture. Overall, we show intraspecies selection for inactivating mutations due to a competitive growth advantage for E. coli that do not express the genetic device compared to their peers that retain the functional device. We show additional interspecies selection against the functional device when E. coli is co-cultured with another organism. Together, these two selection pressures create a significant barrier to genetic device function in microbial communities that we overcome by utilizing a regulated E. coli promoter. Future strategies for genetic device design in microorganisms that need to function in a complex microbial environment should focus on regulated promoters and/or strategies that give the microorganism carrying the device a selective or growth advantage. IMPORTANCE: First-generation biotechnology focused on genetic devices designed for use in monoculture conditions. One class of next-generation biotechnology devices could be designed to function in complex ecosystems with other organisms, so we sought to create conditions where the genetic device retained function when the organism carrying it is in co-culture with other organisms. We discovered that when the genetic device is a significant resource burden on the organism carrying the device, mutations will be selected for due to intraspecies and interspecies selection pressures, and the device will be rendered non-functional. Therefore, genetic device design for complex ecosystems in next-generation biotechnology needs to balance functionality of the genetic device with the need to reduce resource burden on the organism carrying it.

Phenotypic variation is common across life history and among populations occupying different environments, yet the molecular mechanisms underlying these axes of divergence remain poorly understood. Much work has focused on gene expression as a link between genetic variation, environmental variation, and phenotypes, but post-transcriptional processes such as alternative splicing—which affect how transcripts are assembled rather than how much of a transcript is produced—are increasingly recognized as additional modulators of plasticity and adaptation. Here, we examined gene expression and alternative splicing together in the wood frog (Rana sylvatica), an amphibian with a complex life cycle whose populations differ across replicated gradients of road adjacency and associated pollution. We found extensive transcriptomic differences between hatchlings and adults, with thousands of genes differentially expressed or spliced. Individuals clustered strongly by population for both expression and splicing. Differences at the habitat level were less extensive, but revealed two differentially expressed genes (HSP70 and Gpsm2) and one differentially spliced gene (Cd82) that consistently distinguished roadside and woodland populations. Overall, genetic differentiation between populations was low, suggesting that phenotypic and transcriptomic differences likely emerge in the presence of gene flow and reflect plastic responses. Together, these results highlight transcriptomic plasticity as an important mechanism shaping variation across both development and population differentiation. © 2025 The Author(s). Ecology and Evolution published by British Ecological Society and John Wiley & Sons Ltd.

Freshwater salinization is an emerging threat to aquatic ecosystems across the planet, degrading habitats and negatively impacting wild populations. Deicing practices are a leading cause of freshwater salinization, particularly in the snowbelt region of North America where a variety of salts are widely applied to roads and other surfaces to melt snow and ice. Seasonal pools near roads are considered the most severely impacted aquatic habitats. Runoff into these low water-volume ponds can generate high salinity. Impacts of salt pollution are numerous, ranging from toxicity to population decline to impaired ecosystem function. Here, we investigate a suite of physiological consequences of salinization across multiple life history stages of the wood frog (Rana sylvatica), a pool-dwelling amphibian. Previous work has shown that salinized populations have diverged from unpolluted populations for a suite of physiological, morphological, and reproductive traits, and can experience severe edema (bloating) during the breeding season. Here, we measured swim performance before and after aspirating edema in wild captured wood frogs to show that edema compromises adult aquatic locomotion during breeding. We also found that wood frog mothers from salinized ponds produce ova with inherently higher rates of water uptake compared to mothers from unpolluted pools, consistent with countergradient adaptation, but the ova are smaller. Finally, we found that exposure to road salt inhibits expansion of vitelline membranes in developing embryos and is associated with reduced embryo growth. Together, these results reveal the complexity of population level responses to freshwater salinization, highlighting that impacts occur across multiple life history stages, and that local populations might be evolving adaptations to cope with anthropogenic salinity gradients in freshwater habitats. © The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.

Nearly a century of road salt use in the snowbelt region of North America has led to substantial increases in salinity levels in freshwater habitats. Salt pollution in lakes and rivers is well characterized. Lacking are broad insights for seasonal ponds. As critical habitats for many endemic species, these small and often poorly flushed surface waters are especially vulnerable to accumulating high levels of salts and other pollutants. Here, we measured salinity in 165 seasonal ponds, characterizing salt pollution patterns across space, through time, and over depth within ponds. We found that 70% of ponds within 37 m of a road contained salinity levels exceeding Canadian federal guidelines. 54% of ponds within 25 m exceeded less conservative US federal guidelines. Within ponds, the water column was stratified due to the combined density effects of salt and temperature. Bottom waters of polluted ponds were about 57% saltier than near-surface waters, though many were much saltier than this. Compared to lakes and rivers, far more seasonal ponds appear to be compromised by deicing salt, and overall, the concentration of salt appears to be substantially higher. Among aquatic habitats, seasonal ponds are experiencing the most severe impacts of freshwater salinization, with consequent impacts on sensitive aquatic organisms.

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

Theropods are obligate bipedal dinosaurs that appeared 230 Ma and are still extant as birds. Their history is characterized by extreme variations in body mass, with gigantism evolving convergently between many lineages. However, no quantification of hindlimb functional morphology has shown whether these body mass increases led to similar specializations between distinct lineages. Here we studied femoral shape variation across 41 species of theropods (n = 68 specimens) using a high-density 3D geometric morphometric approach. We demonstrated that the heaviest theropods evolved wider epiphyses and a more distally located fourth trochanter, as previously demonstrated in early archosaurs, along with an upturned femoral head and a mediodistal crest that extended proximally along the shaft. Phylogenetically informed analyses highlighted that these traits evolved convergently within six major theropod lineages, regardless of their maximum body mass. Conversely, the most gracile femora were distinct from the rest of the dataset, which we interpret as a femoral specialization to “miniaturization” evolving close to Avialae (bird lineage). Our results support a gradual evolution of known “avian” features, such as the fusion between lesser and greater trochanters and a reduction of the epiphyseal offset, independent from body mass variations, which may relate to a more “avian” type of locomotion (more knee than hip driven). The distinction between body mass variations and a more “avian” locomotion is represented by a decoupling in the mediodistal crest morphology, whose biomechanical nature should be studied to better understand the importance of its functional role in gigantism, miniaturization, and higher parasagittal abilities.

Human impacts on wild populations are numerous and extensive, degrading habitats and causing population declines across taxa. Though these impacts are often studied individually, wild populations typically face suites of stressors acting concomitantly, compromising the fitness of individuals and populations in ways poorly understood and not easily predicted by the effects of any single stressor. Developing understanding of the effects of multiple stressors and their potential interactions remains a critical challenge in environmental biology. Here, we focus on assessing the impacts of two prominent stressors associated with anthropogenic activities that affect many organisms across the planet – elevated salinity (e.g., from road de-icing salt) and temperature (e.g. from climate change). We examined a suite of physiological traits and components of fitness across populations of wood frogs originating from ponds that differ in their proximity to roads and thus their legacy of exposure to pollution from road salt. When experimentally exposed to road salt, wood frogs showed reduced survival (especially those from ponds adjacent to roads), divergent developmental rates, and reduced longevity. Family-level effects mediated these outcomes, but high salinity generally eroded family-level variance. When combined, exposure to both temperature and salt resulted in very low survival, and this effect was strongest in roadside populations. Taken together, these results suggest that temperature is an important stressor capable of exacerbating impacts from a prominent contaminant confronting many freshwater organisms in salinized habitats. More broadly, it appears likely that toxicity might often be underestimated in the absence of multi-stressor approaches. © 2024 Elsevier Ltd

Objective Florida manatee feeding ecology is critical to species survival, but the role of dental pads in feeding has received limited attention. This study characterized the gross and microscopic anatomy of the manatee’s dorsal and ventral dental pad in relation to these structures’ importance in mastication, which furthers our understanding of manatee feeding and health. Design Whole heads from 6 animals (4 male and 2 female) of varying sizes were examined grossly. Sections (5 µm) from throughout the dorsal and ventral dental pads were stained with Hematoxylin and Eosin to document microanatomy. The thickness of the epithelium and stratum corneum were measured. Results The ventral dental pad epidermal (1129–3391 µm) and stratum corneum (331–1848 µm) thickness increased with increased body size. The dorsal dental pad epidermal (690–1988 µm) and stratum corneum (121–974 µm) thickness varied relative to size. The dental pad anatomy, including the thickened stratum corneum, indicates an importance similar to molars in grinding and physically breaking up plant material. Extensive appendages including filiform-like papillae and well-developed rete were observed and likely provide physical support for mastication. Conclusion While the sample size limits specific conclusions based on sex or age, it provides a good overview of the anatomy of the dental pads. The manatee is the only mammal known to have a ventral dental pad and the well-developed grinding surfaces demonstrates a crucial role in mastication for these structures. These dental pads should be evaluated during health checks and necropsies and considered in future research on manatee’s feeding mechanisms.

Climate change is driving a shift in the distribution of global kelp forests, with the contraction of kelp habitats occurring at warm range edges. Declining kelps often have been replaced by novel algal turf assemblages, which are reinforced by ecological feedback mechanisms and provide fewer ecosystem services. Trophic interactions among marine herbivores, algal turfs, and kelps on algal turf-dominated reefs remain poorly resolved but could have important implications for the stability of algal turf reefs and the potential for kelp forest recovery. Here, we examine herbivory by the Atlantic purple sea urchin, Arbacia punctulata, in a degraded kelp forest ecosystem dominated by algal turf in southern New England, USA. In a localized field survey, we observed lower algal turf cover on reef areas containing A. punctulata (mean ± SE: 62 ± 12% turf cover) as compared to areas with no sea urchins present (92 ± 4% turf cover). Reef areas with and without sea urchins had similarly low cover of the previously dominant kelp, Saccharina latissima (6–8% kelp cover). In laboratory and field experiments, individuals or groups of A. punctulata enclosed with a diet choice of algal turf versus kelp had higher grazing rates on the algal turf. A. punctulata in the laboratory also exhibited greater attraction to algal turf over kelp, physically moving towards this food source. In combination, the results provide evidence that A. punctulata has a feeding preference for algal turf over kelp in southern New England. Future research is warranted to further examine the grazing ecology of A. punctulata, particularly in the context of ongoing kelp forest restoration efforts in this region.

Human impacts on wild populations are numerous and extensive, degrading habitats and causing population declines across taxa. Though these impacts are often studied individually, wild populations typically face suites of stressors acting concomitantly, compromising the fitness of individuals and populations in ways poorly understood and not easily predicted by the effects of any single stressor. Developing understanding of the effects of multiple stressors and their potential interactions remains a critical challenge in environmental biology. Here, we focus on assessing the impacts of two prominent stressors affecting many organisms across the planet – elevated salinity (an increasingly common pollutant in freshwater habitats) and elevated temperature. We examined a suite of physiological traits and components of fitness across populations of wood frogs originating from ponds that differ in their proximity to roads and thus their legacy of exposure to road salt pollution. When experimentally exposed to road salt, wood frogs showed reduced survival, especially those from ponds adjacent to roads, and delayed time to metamorphosis. Family level effects mediated these outcomes, but high salinity generally eroded family level variance. When combined, exposure to both temperature and salt resulted in very low survival, and this effect was strongest in roadside populations. Taken together, these results suggest that temperature is an important stressor capable of exacerbating impacts from a prominent contaminant confronting many freshwater organisms in salinized habitats. More broadly, it appears likely that toxicity might often be underestimated in the absence of multi-stressor approaches.

Facultatively symbiotic corals provide important experimental models to explore the establishment, maintenance, and breakdown of the mutualism between corals and members of the algal family Symbiodiniaceae. The temperate coral Astrangia poculata is one such model as it is not only facultatively symbiotic, but also occurs across a broad temperature and latitudinal gradient. Here, we report the de novo chromosome-scale assembly and annotation of the A. poculata genome. Though widespread segmental/tandem duplications of genomic regions were detected, we did not find strong evidence of a whole genome duplication (WGD) event. Comparison of the gene arrangement between A. poculata and the tropical coral Acropora millepora revealed 56.38% of the orthologous genes were conserved in syntenic blocks despite ∼415 million years of divergence. Gene families related to sperm hyperactivation and innate immunity, including lectins, were found to contain more genes in A. millepora relative to A. poculata. Sperm hyperactivation in A. millepora is expected given the extreme requirements of gamete competition during mass spawning events in tropical corals, while lectins are important in the establishment of coral-algal symbiosis. By contrast, gene families involved in sleep promotion, feeding suppression, and circadian sleep/wake cycle processes were expanded in A. poculata. These expanded gene families may play a role in A. poculata’s ability to enter a dormancy-like state (“winter quiescence”) to survive freezing temperatures at the northern edges of the species’ range.

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education.

Petroleum derived plastics are a major contributor to global pollution. There is an urgent need for biodegradable, sustainable plastic alternatives. Cyanobacteria have been studied extensively for photosynthetic production of biofuel precursors including alkanes and free fatty acids. However, large scale production has been slow to emerge from these technologies. Here, we wished to evaluate alternative uses for engineered strains of the cyanobacteria Synechocystis PCC 6803 (6803). We investigated the feasibility of using wild type and fatty acid secreting strains of 6803 to support the growth of Ralstonia eutropha. This organism is capable of producing polyhydroxyalkanoates (PHAs), which can be used in bioplastic production. Traditional feedstocks for R. eutropha include palm oil and other biological precursors that compete with cultivatable land, pitting potential bioplastic production against agricultural demands. Since PHAs are of great interest as plastic alternatives, we co-cultured R. eutropha and 6803 strains in the minimal medium BG-11 in an attempt to create carbon neutral PHA from R. eutropha. Surprisingly, we observed inhibition of R. eutropha growth in co-culture with Synechocystis but not another cyanobacterium suggesting further modification of Synechocystis is necessary for use as a feedstock.