Yeast

Course-based undergraduate research experiences (CUREs) are widely recognized as a high-impact practice in biology education, yet most existing CURE frameworks treat the research organism as an interchangeable teaching prop rather than a genuine scientific contribution. We argue that place-based, community-embedded CUREs - in which students isolate, characterize, and publicly deploy a locally meaningful wild organism - constitute a qualitatively distinct model warranting broader adoption. As proof of concept, we present the Declaration of Fermentation project at Indiana University Bloomington: graduate researchers isolated a wild Saccharomyces cerevisiae strain from the bark of a campus landmark tree, confirmed its wild provenance by whole-genome sequencing and phylogenomics, and partnered with local craft breweries to produce a colonial-era inspired ale released publicly for the 250th anniversary of the Declaration of Independence. Volunteer sensory panels at two independent public tasting events (combined n = 33-34 per attribute) confirmed a fruity-funky profile consistent with wild-strain fermentation, with no significant differences between events (Mann-Whitney U, Benjamini-Hochberg-corrected p > 0.05 for all 11 attributes). We describe three design principles - genomically confirmed strain identity, mandatory community partnership, and place-based historical narrative - that distinguish this model from prior wild yeast brewing CUREs, discuss how these principles generalize to other institutions and fermentation vehicles, and identify next steps for formal learning assessment. Complete implementation protocols are provided as supplemental Appendices 1-6, and the bioinformatics pipeline is freely available at https://doi.org/10.5281/zenodo.20679384.

Automated platforms such as Chi.Bio enable simultaneous monitoring of optical density and fluorescent reporter expression in 20 ml reactor cultures with controllable pump systems. As such, they provide an appealing option for contemporary gene expression quantification, quantitative physiology, and laboratory evolution and ecology experiments. While optical density calibration for this device is well established, no equivalent calibration framework exists for fluorescence, making quantitative comparison with reference instruments unreliable. Here, we characterize Chi.Bio fluorescence capabilities using fluorescent calibration microspheres and fixed GFP-expressing S. cerevisiae and E. coli cells, compared with orthogonal plate-reader measurements. We show that microsphere fluorescence is detectable and scales linearly with concentration, whereas the GFP signal from both species falls below the device detection limit. Comparison of background-correction strategies indicates that direct subtraction of a non-fluorescent control measured within the same device yields more reliable fluorescence estimates than the commonly used on-line normalization method. Knowledge of these sensitivity boundaries of the device provides practical guidelines for experimental design of future studies.

Endosymbiosis is widespread throughout the tree of life. Understanding how the transition of a bacterial endosymbiont from facultative to host-dependent obligate life occurs is an important question for defining the origin of endosymbiosis. A novel gram-positive bacillus, Brevibacillus sp. TJ4 was isolated from the nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1, which houses several endobacteria within its cells. TJ4 can survive independently of yeast but exhibits genomic and metabolic features characteristic of an evolving endosymbiont, slowly assuming a host-dependent, obligate lifestyle. The TJ4 genome contains several incomplete pathways for carbohydrate, amino acid, vitamin, and cofactor metabolism, which is reflected in its increased reliance on host-derived nutrients and auxotrophy compared with that of other Brevibacillus spp. Comparative genomics revealed widespread genome rearrangements, loss of synteny, and multiple cross-genus and inter-kingdom horizontal gene transfer (HGT) events in TJ4 compared to other Brevibacillus spp. These HGTs include the acquisition of genes from bacteriophages and co-resident endobacteria of JGTA-S1. One such horizontally acquired gene, Type II 3-dehydroquinate dehydratase (AroQ), appears to have originated from the Rhodotorula host itself. This acquisition functionally restores the shikimate pathway in strain TJ4, as evidenced by the phylogenetic placement of AroQ from TJ4 within the clade of fungal AroQ homologs. Potential exploitation of the host JGTA-S1 appears to be a probable mode of endosymbiosis of TJ4, an evolving endosymbiont that we named Brevibacillus rhodotorulae sp. nov.

Lipid peroxidation (LPO) is widely used as a biomarker of oxidative stress in coral bleaching research, yet its measurement remains poorly standardized across the field. A systematic review of the coral LPO literature reveals substantial variation in methodological approaches, including tissue fraction analysis, lysis protocols, assay choice, and normalization metrics, confounding cross-study comparison and obscuring the biological interpretation of results. We experimentally investigate two key sources of variation: the use of bulk holobiont vs separated host and algal symbiont fractions, and the choice of normalization metric. To do so, we used Montastraea cavernosa (n = 6 colonies) exposed to ambient (28C), heat stress (30.5C), and heat stress + artificial upwelling (AU; heat stress intermitted by daily pulses of cooler water, 30.5/27.5C) conditions in a controlled mesocosm experiment. Using a TBARS-based MDA assay with a lysis buffer optimized for coral tissue, we measured LPO separately in coral host and algal symbiont fractions across four time points throughout the day. Host MDA remained stable across all treatments and time points, consistent with either sufficient antioxidant buffering capacity or thermal acclimation over the experimental period. Algal symbiont MDA, in contrast, exhibited pronounced diel and treatment-specific dynamics, and the two fractions responses were decoupled from one another. Normalizing MDA to coral surface area instead of total protein content produced largely consistent diel and treatment patterns, but the two metrics diverged at specific time points, indicating that normalization choice is not interchangeable and can itself affect interpretation. Together, our literature review and empirical results demonstrate that host and algal symbiont LPO dynamics are not comparable when aggregated and argue for host-symbiont fraction separation and consistent, explicitly reported normalization as minimum standards for interpretable and cross-comparable coral LPO measurement.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

BackgroundThe fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors can have stressor-specific effects that may be difficult to separate from the effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA, in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This required combining multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactive. While this strain was useful, it had several limitations. ResultsHere we describe and validate a more rationally-designed strain, SISA4, that retains the features of the original SISA strain while overcoming its limitations. SISA4 is more stable genetically than SISA, easier to use in genetic crosses, and easy to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo, and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactive state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these are modest and generally do not affect interpretation of experiments. Finally, using SISA4, we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. ConclusionsSISA4 will be useful for investigating how SAPK pathway activation regulates diverse cellular processes.

Reactive nitrogen species (RNS), particularly peroxynitrite generated from the reaction of superoxide and nitric oxide, are implicated in thermally-induced oxidative stress but remain difficult to resolve in live coral cells. We optimized fluorescent dye strategies to directly quantify superoxide, nitric oxide, and peroxynitrite production in thermally stressed Pocillopora acuta cell suspensions. Thermal stress was associated with an increase in intracellular peroxynitrite concentration, but not in its precursors, nitric oxide and superoxide, highlighting challenges with the application of fluorescent probes and their controls to live coral cells. Compounds developed for mammalian systems often translate poorly to non-model systems such as corals: strong endogenous fluorescence and multiple membrane barriers within the coral symbiocyte, for instance, limited the function of the nitric oxide probe, DAF-2DA. Despite these limitations, the detection of peroxynitrite in live, thermally stressed P. acuta cells represents a step forward in understanding the mechanism of coral bleaching. We also outline strategies for improving the performance of commercial dyes in non-model systems, including media optimization with EDTA treatment to preserve both cell viability and probe performance.

John Raven pioneered the field of algae ecophysiology, advancing our understanding of cellular resource economics, carbon acquisition, and energy allocation. His work laid the foundation for investigating integrative physiology, linking growth-survival trade-offs across diverse environments. The sea ice habitat provides an excellent framework to continue the research John championed. With steep temperature-salinity gradients, algae survival requires a shift in physiology that we are only beginning to understand. We developed two small scale, reproducible icecosms to investigate physiological changes associated with incorporation into sea ice and survival potential post-melt. Fragilariopsis cylindrus and Nitzschia frigida, known for their association with the ice environment, and Porosira glacialis, known for its association with the ice edge, were used to mechanistically link physical properties with algal physiology and post-melt survival. We observe incorporation into the ice of F. cylindrus and N. frigida alongside vertical photophysiological profiles of F. cylindrus revealing inhospitable conditions in the top compared to the bottom layers of ice. N. frigida and P. glacialis remain viable within the ice and retain the capacity to seed populations following melt. Our results establish icecosms as experimental framework to investigate ecophysiological responses of sea ice algae and provide a foundation toward ecological and evolutionary questions.

Background: We propose the biological drift framework for the interpretation of biological test results: a z-score-like framework based on optimized and personalized reference populations and a distance-to-optimum drift metric for longitudinal interpretation relative to an estimated individual optimum. We benchmarked biological drifts against Reference Change Values (RCVs), which are used to interpret serial laboratory results by defining the minimum change expected to exceed normal within-subject biological variation CVi. Objectives: To benchmark biological drifts against the classical biological-variation framework and assess their consistency with RCV thresholds across routine biomarkers. Methods: For 62 routine biomarkers, biological drift levels were compared with RCVs after transformation to test the consistency between the two frameworks. Results: Severe biological drifts mostly exceeded the 95% RCV threshold, indicating changes unlikely to be explained by short-term biological variation alone. In contrast, moderate drifts reached the 95% RCV threshold for approximately one in two biomarkers, suggesting that many moderate distance-to-optimum deviations may remain within expected variability, particularly for biomarkers with large within-subject variation CVi. Results are particularly interesting for the follow-up of people with diabetes and for the management of thyroid and hepatic disorders. Conclusions: Biological drifts derived from optimized personalized reference populations are broadly consistent with the RCV framework for identifying biologically meaningful deviations from the optimum and may therefore be relevant for the monitoring of certain biomarkers across several medical conditions in clinical practice.

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a {beta}-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 {micro}M and maximum binding capacity (Bmax) of 144 {micro}mol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 {degrees}C for ONPG and 50 {degrees}C for lactose, and CBE retained >80% activity after 390 min at 45 {degrees}C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 x106 M-1{middle dot}s-1 for ONPG and 4.40 x102 M-1{middle dot}s-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca2+ and Fe2+. These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.

Anaerocellum (formerly Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C) lignocellulolytic bacterium, is a promising chassis for metabolic engineering and next-generation bioprocessing. Yet, a lack of well-characterized genetic parts in A. bescii has hampered metabolic engineering efforts. Here, using a previously developed hyperthermophilic {beta}-galactosidase reporter system, we screened a diverse panel of putative A. bescii promoter sequences, identifying promoters that drove reporter output across a broad range. For a select subset, we mapped their transcriptional start sites (TSSs) and evaluated ribosome binding site (RBS) regions using chimeric promoter constructs. By constructing truncated promoter variants, we defined functional regions within the widely used, high-expression S-layer protein promoter (Pslp) and engineered a compact 99 bp variant that retained substantial reporter activity. Finally, we demonstrated that these new promoters can be used for metabolic engineering by using two newly characterized promoters to express an established thermostable alcohol dehydrogenase from Thermoclostridium stercorarium to drive ethanol production in A. bescii. Together, this work expands and diversifies the A. bescii genetic toolkit, opening doors to future metabolic engineering efforts in this species.

Saturated cultures of the pathogenic yeast, Cryptococcus neoformans, arrest as unbudded cells in the G2 phase of the cell cycle. As cells divided and cultures saturated, we found that oxygen levels in the culture medium dropped nearly tenfold. When saturation-arrested cultures were re-oxygenated without adding fresh growth medium, cells immediately formed a bud and then underwent mitosis. Thus, the arrest is due to low oxygen concentration rather than nutrient depletion. Because the G2 arrest was associated with unbudded cells, we asked whether C. neoformans cells have a morphogenesis checkpoint that blocks mitosis until cells can form a bud. Inhibition of budding by treatment with Latrunculin A also led to G2 arrest, and we determined that this arrest is dependent on the CDK inhibitory kinase, Swe1. This finding suggests that C. neoformans possesses a morphogenesis checkpoint analogous to that in the distantly related Saccharomyces cerevisiae. We also demonstrated that Swe1 is required to enforce the hypoxia-induced G2 arrest. We propose that hypoxia inhibits budding in C. neoformans, which in turn triggers a morphogenesis checkpoint to arrest cells in G2 even when nutrients are plentiful.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

O_LIFloral nectar plays a critical role in shaping the ecology and evolution of plant-pollinator interactions. Effective and efficient methods that allow for broad-scale sampling of nectar volume and sugar concentration across a diversity of taxa are needed to improve our understanding of many dimensions of mutualistic plant-pollinator interactions--including their basic ecology and evolution, their responses to environmental change, and their conservation and restoration. C_LIO_LIDespite the key importance of nectar for mediating plant-pollinator interactions, quantifying floral nectar in the field from many different plant species is challenging because there is often no one-size-fits-all sampling method that is effective across a diversity of floral structures and nectar traits. Different methods require different preparation, and sampling from many species involves a variety of logistical challenges. C_LIO_LIHere we provide a methodological roadmap for sampling floral nectar in the field from many different plant species. We describe our nectar collection methods in detail, including necessary equipment, calculations, and approaches appropriate for different floral morphologies. We also provide a troubleshooting guide for common problems encountered while collecting nectar in the field. To demonstrate the utility and effectiveness of our methods for collecting nectar from many different species, we present results on nectar trait variation from 53 species in an ecosystem. C_LIO_LIOur method illustrates that nectar traits vary considerably within and among plant species, indicating that large-scale nectar sampling projects are an important consideration for many basic and applied questions in pollination ecology and evolution. We hope that across many plant communities and ecosystems, our paper provides a practical roadmap for how to navigate the complexities of quantifying floral nectar traits. C_LI

Sulfolobus acidocaldarius is a thermoacidophilic archaeon used as a model system for studying fundamental cellular processes and for emerging biotechnological applications. However, the limited availability of selectable markers restricts advanced genetic manipulation in this organism. Here, we report the development of a tryptophan auxotrophy-based selection system in S. acidocaldarius. A {Delta}trpBA mutant was constructed in the {Delta}pyrE background strain using a classical pop-in/pop-out recombination strategy. The resulting mutant exhibited little growth defects in rich medium, likely due to exogenous tryptophan supplied by complex nutrients, but failed to grow in a newly developed defined Brock-based amino acid dropout medium lacking tryptophan. Exploiting both uracil and tryptophan auxotrophies, we achieved dual-plasmid co-transformation and co-expression of the surface layer proteins and a dominant-negative mutant of the AAA-ATPase Vps4, revealing that the accumulation of surface layer lattice forming protein SlaA at the midzone of division-arrested cells together with its membrane anchor SlaB. Together, these results provide evidence for spatial regulation of S-layer assembly during archaeal cytokinesis while expanding the genetic toolkit available for S. acidocaldarius. ImportanceSulfolobus acidocaldarius is a key archaeal model organism for studying cellular processes shared with more complex life and is increasingly used for biotechnological applications. Here, we establish tryptophan auxotrophy as a new selectable marker in S. acidocaldarius, expanding the range of genetic selection systems available in this organism. By developing a defined Brock-based dropout medium, we enable stringent amino acid auxotrophy selection and precise control over nutrient composition. This system can be combined with existing uracil-based selection to support dual auxotrophy workflows, enabling co-transformation, simultaneous expression of multiple proteins, and more sophisticated genetic manipulation strategies. Using both markers, we show that S-layer proteins are localised to the division bridge in cytokinesis-arrested cells. This exemplifies ways in which the expanding molecular genetic tool kit available for Sulfolobus acidocaldarius is furthering our understanding of archaeal cell biology.

In many tropical countries, fermentation of palm sap into palm wine is an important fermented beverage contributing to local economies, tradition, and culture. Traditionally made in villages and families, palm sap is not inoculated with starter cultures and fermentation commences spontaneously. It is therefore possible that fermentation is influenced by multiple ecological factors, which affect microbial dynamics and thus flavour outcomes. Here, we studied microbial communities during fermentation of palm sap from three different palm tree species (palmyra, coconut, and sugar palm) on the island of Bali, Indonesia in both the wet and dry seasons. Our results suggest that season of collection has a strong influence on microbial dynamics and succession, and these changes positively correlate to metabolite concentration. The change of the season from the dry to wet season led to the loss of microbial diversity with lower richness in the dry season. The dominance of Saccharomyces cerevisiae was not affected by season and fermentation time and was dominant in all samples. Potential spoilage species, such as Candida tropicalis were negatively correlated to ester production and more abundant in the dry season. As microbial species varied in incidence and thus biochemical activity, the chemical groups of esters from their metabolism related to the change of season and fermentation time, while volatile compounds and small molecules were highly discriminated by season in the resultant wines. Ethyl octanoate was consistently different across all variables through comparison by three-way ANOVA and is proposed as a biomarker of seasonal variation in palm sap fermentation. These findings improve our understanding of microbial dynamics in palm sap fermentation, revealing flavour differentiation within season and suggests that strategies for microbial management, product development and quality assurance will elevate this traditional product into the future.

Luminescence is a powerful method for detecting trace analytes and monitoring biological processes. However, most bioluminescence reagents, including luciferase and its substrates, are sensitive to temperature, limiting their useable shelf lives, and resulting in inconsistent performance. Enhancing the stability of these reagents could improve data quality, simplify workflows, and address cold chain storage issues. In this study, we demonstrate the application of the platform stabilization technology, capillary-assisted vitrification (CAV), as a tool to stabilize different luciferases and their substrates, and the application of the stabilized reagents in both in vitro and in vivo bioluminescent assays. We demonstrate that CAV-stabilized reagents can be stored and shipped ambiently, maintain consistent performance over time, and are suitable for use in cell viability quantification, tumor monitoring, in vivo imaging, microbial detection, and immunoassays. Additionally, different reagents can be co-formulated to make ready-to-use assay kits that can also be shipped and stored ambiently. Our results demonstrate that CAV stabilization is a viable alternative to traditional storage methods, with broad potential to improve bioluminescence workflows.

As the vast majority of archival data is short-read based, reliable strategies for detecting and classifying plasmids out of these data are still needed. A widely adopted solution for binning plasmids with rich contextual data is MOB-Suite. However, a large portion of the available data has low coverage and results in fragmented assemblies. Under these circumstances, MOB-Suite delivers a high number of false positives, leading to wasteful follow-up experiments. To improve the reliability of MOB-Suite, while retaining and enriching its biological contextual output, we have developed PLAGUE. PLAGUE is a fast and low-memory consumption post-processing tool for MOB-Suite outputs, relying on checks of circularity, reads spanning the gap between the 3 and 5 ends and total coverage of the plasmid candidate. PLAGUE can reduce the number of false positives in MOB-Suite outputs by over 30%, while retaining almost full sensitivity. It is fully containerized and easy to use in clusters as well as locally.

Untranslated regions (UTRs) flanking the coding sequence govern mRNA translation, localisation, stability, and decay, making accurate UTR boundaries essential for quantitative RNA sequencing and the study of post-transcriptional control in Saccharomyces cerevisiae and beyond. Reference transcriptomes built from short-read sequencing have been invaluable to the yeast community, yet in a genome as gene-dense as that of S. cerevisiae, short reads frequently cannot be assigned to a single transcript of origin, leaving roughly one quarter of transcripts without a confidently defined UTR. Here we use Oxford Nanopore Direct RNA Sequencing (DRS), in which each full-length polyadenylated molecule is read end to end, to resolve this ambiguity and deliver two complementary resources. First, an updated, ready-to-use S. cerevisiae S288C reference: change-point segmentation of per-gene DRS coverage defined boundaries for 5,416 of the 6,695 annotated genes, and a merge-max rule retaining the longer UTR from each source ensures no gene loses existing annotation. The result adds previously absent UTRs to 927 (5') and 896 (3') genes and extends 29.4% of 5' and 26.1% of 3' boundaries among comparable genes. Second, the complete, documented pipeline so that any laboratory can rebuild or update a transcriptome from its own DRS data. Validation on two independent datasets shows improved mapping rates, reduced soft-clipping, and metagene profiles consistent with genuine transcript signal.

Artificial light at night (ALAN) can alter the movement of nocturnal pollinators such as moths, with potential consequences for pollination services. However, most studies have focused on the presence or properties of lights, while the role of spatial lighting configuration remains poorly understood. We conducted a small-scale field experiment to test how different lighting configurations affect pollination success in moth-pollinated plants, using low-intensity LED garden lights. Potted phytometer plants of three moth-pollinated species were exposed to one of three treatments: no-light control, isolated LED point lights, or 25 m linear arrays of multiple LED lights. We quantified pollination success as both the probability of seed set and reproductive output through seed and capsule production. Experimental lighting affected plant reproductive success, with light arrays increasing seed-set probability, seed and seed capsule number relative to unlit controls, whereas point lights showed no clear effect. These results provide preliminary evidence that the spatial configuration of artificial lights may influence nocturnal pollination outcomes. Future work combining phytometer assays with direct tracking of moth movement is needed to assess whether light arrays facilitate or redirect the dispersal of nocturnal pollinators.