G3 Genes|Genomes|Genetics

Anthropogenic copper (Cu) contamination from agriculture, mining, and industrial runoff creates environmental gradients affecting physiology and behavior in wild populations. While Cu toxicity in Drosophila melanogaster is well characterized, it remains unclear whether Cu resistance is one integrated trait or several independently evolving components. Using a subset of recombinant inbred lines (RILs) from the Drosophila Synthetic Population Resource (DSPR), we measured three components of Cu response: feeding avoidance, oviposition avoidance, and physiological tolerance (median lethal time, LT50) under sustained Cu exposure. All three traits showed substantial phenotypic variation among RILs. Feeding and oviposition avoidance were both highly heritable (H 2 ~ 0.88), and RIL identity accounted for 49.5% of the variance in LT50. However, the three traits showed no significant correlation across RILs, indicating distinct genetic architecture. We identified a single male specific quantitative trait locus (QTL) on chromosome 2R that explained 17.7% of the variation in feeding preference; the interval included candidate detoxification genes Jheh1, Jheh2, Jheh3 and sano, the latter of which is associated with olfactory behavior. No significant QTL were detected for oviposition preference, suggesting a highly polygenic structure that may difficult to detect with our limited panel size. Together, these results indicate that Cu resistance in D. melanogaster is genetically modular. Behavioral avoidance during feeding, oviposition, and physiological tolerance are heritable but architecturally distinct components, each with potential to respond to selection independently.

Tillering plasticity is a key adaptive trait in sorghum influencing resource use efficiency via a plants ability to adjust branching to neighbour density. Neighbour detection through red:far-red (R:FR) light sensing regulates this plasticity. While molecular pathways regulating tiller outgrowth are partly known, the genetic architecture underlying density-responsive tillering has not been resolved in any grass species. A sorghum diversity panel (n = 895) was evaluated over two growing seasons (2023 and 2024) with plant spacing ranging from 5 to 60 cm. A linear mixed model incorporating neighbour distance and tiller counts estimated genotype-specific response. GWAS was conducted on isolated plants (no neighbours within 60 cm) and on estimated responsiveness to neighbours. GWAS identified 52 baseline tillering QTLs and 50 for spacing responsiveness, with 10 overlapping, suggesting shared genetic control. Comparison with 41 R:FR pathway candidate genes revealed enrichment in responsiveness QTLs (5/50, 10%) versus baseline (0/52, 0%) (Fishers exact test, P = 0.025). Our model identified 40 unique density-responsive tillering QTL regions. Reducing genotype response to neighbour absence could be a selection target to develop water-efficient sorghum varieties where controlled architecture may be more valuable than natural plasticity.

Soybean, a widely cultivated leguminous crop valued for its protein, amino acids, and oil, faces the challenge of maintaining protein levels, which have an inverse correlation with yield. Reducing leaf chlorophyll levels could increase seed protein levels without compromising yield; however, this is yet to be tested. Therefore, to understand the impacts of low chlorophyll mutations on soybean yield and seed composition, we screened and compared 25 low chlorophyll soybean mutants to their 11 dark green parents. PI548210 (Lincoln mutant) demonstrates a higher concentration of protein without affecting yield compared to its dark green parent PI548362 (Lincoln), suggesting it as a good candidate for further large-scale field trials. PI547555 (Y11/y11, Clark mutant) demonstrates a lower concentration of oil without impacting yield, alongside lower gross photosynthesis, but with chlorophyll levels in the pod and seed tissues that are comparable to its dark green parent PI548533 (Clark). These findings are consistent with the oil concentration of the soybean being influenced by pod and seed photosynthesis, which is correlated with pod height and row spacing. Chlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed. SIGNIFICANCEO_LIPI548210 (Lincoln mutant), one of twenty-five low chlorophyll soybean mutants, demonstrates a higher concentration of soybean protein without affecting yield compared to its dark green parent (Figure 1 and Table 1). C_LIO_LIPI547555 (Y11/y11, Clark mutant), a low chlorophyll soybean mutant, demonstrates a reduced concentration of soybean oil without impacting yield, alongside lower gross photosynthesis in pod and seed tissues compared to its dark green parent (Figures 3 and Table 2). These findings suggest that the oil concentration of the soybean is influenced by pod and seed photosynthesis, which is in turn influenced by pod height and row spacing (Figure 2). C_LIO_LIChlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed (Figure 5-6). C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/744892v1_fig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@4282dcorg.highwire.dtl.DTLVardef@9d565forg.highwire.dtl.DTLVardef@1918292org.highwire.dtl.DTLVardef@1359b1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Two low chlorophyll mutants are as healthy as their dark green parents. Lincoln and its low chlorophyll mutant, left; Clark and its low chlorophyll mutant, known as Y11/y11, right. It can be seen by eye that the plants have low chlorophyll (light green/yellow leaves) but a similar growth habit to their dark green parents. See Supplemental Figures 1-4 for contrast, where low chlorophyll mutants are stunted in growth compared to their dark green parents. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@657ec9org.highwire.dtl.DTLVardef@166e75borg.highwire.dtl.DTLVardef@df23c7org.highwire.dtl.DTLVardef@1a60124org.highwire.dtl.DTLVardef@194ed96_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONComparison of seed yield, weight, seed composition between low chlorophyll mutants and their dark green parents. ANOVA is used with linear mixed model (random effect = block, fixed effect = variety). Least squares mean is used to compare. For yield and seed composition, N=4 blocks. For leaf chlorophyll (SPAD), N=40. Yield is average yield per plant (g). n.s. = not significant. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/744892v1_fig3.gif" ALT="Figure 3"> View larger version (26K): org.highwire.dtl.DTLVardef@7a368aorg.highwire.dtl.DTLVardef@192b8f0org.highwire.dtl.DTLVardef@1abb738org.highwire.dtl.DTLVardef@89e978_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO Light response curve of low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Rates of net and gross photosynthesis of low chlorophyll (white) and dark green parents (black) pods under field conditions. Each dot represents a value (n=4) {+/-}SE. We assumed that the seeds greatly inhibited the transmittance of light through the pod and used photosynthetic photon flux density for a single-side. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@3f0528org.highwire.dtl.DTLVardef@16ba712org.highwire.dtl.DTLVardef@a5ab2aorg.highwire.dtl.DTLVardef@889254org.highwire.dtl.DTLVardef@3efa4f_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONPod photosynthetic parameters for low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Photosynthesis was measured 1 September through 15 September 2021 at the University of Illinois Energy Farm in Urbana, IL, USA. The statistical analysis was done using ANOVA with linear mixed model (alpha=0.05). N=4 {+/-} SEM for Clark and N=3 {+/-} SEM for Y11. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/744892v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@a36c26org.highwire.dtl.DTLVardef@1116c8forg.highwire.dtl.DTLVardef@ee5e61org.highwire.dtl.DTLVardef@1766712_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533) differ in concentration of seed oil, which interacts with height of pod and row spacing. The box plots show the median (central line), the lower and upper quartiles (box) and the minimum and maximum values (whiskers). The statistical analysis was done using ANOVA with linear mixed model (n=3 blocks, alpha=0.05). Least squares mean is used to compare. N.s., non- significant in the analysis. A. Concentration of oil in low chlorophyll mutant seeds from the upper canopy decreased by 4% compared to the dark green parent (18.2% vs 19%) while there was no difference between them in the seeds from the lower canopy (20.2% vs 20.6%). B. Schematic layout of 2013 field setting showing two different row spacings. C. Concentration of oil in low chlorophyll mutant decreased by 2% in 38cm spacing (21.4% vs 22%) while there was no difference in 19cm spacing (21.3% vs 21.7%) in 2013 field. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/744892v1_fig5.gif" ALT="Figure 5"> View larger version (22K): org.highwire.dtl.DTLVardef@68e508org.highwire.dtl.DTLVardef@94a6ccorg.highwire.dtl.DTLVardef@152a187org.highwire.dtl.DTLVardef@1eae137_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO (greenhouse). Correlation between the level of leaf chlorophyll (x-axis: SPAD reading) and the level of immature pod or seed chlorophyll (y-axis, mg/g DW). Line represents the linear regression model. R-squared is a coefficient of determination, the percentage of the response variable variation that is explained by the linear model. Pod is labeled by the fresh weight of seeds it contained. A. Level of chlorophyll of 25-100mg pod (n=18). B. Level of chlorophyll of 100-200mg pod (n=17) . C. Level of chlorophyll of 25-100mg seed (n=17). D. Level of chlorophyll of 100-200mg seed (n=20). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/744892v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@167fd88org.highwire.dtl.DTLVardef@361472org.highwire.dtl.DTLVardef@786325org.highwire.dtl.DTLVardef@1b53855_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Levels of gene expression in chlorophyll synthesis pathway. A. CHL common pathway genes; Glutamyl-tRNA reductase (GluTR). Glutamate 1- semialdehyde aminotransferase (GSA-AT). ALA dehydratase (ALAD). Uroporphyrinogen III synthase (UROS). Uroporphyrinogen III decarboxylase (UROD). Protoporphyrinogen IX oxidase (PPO). B. Mg branch; Mg-chelatase (Mgch). Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC). Protochlorophyllide reductase (POR). 3,8-divinyl protochlorophyllide a 8-vinyl-reductase (4VCR). Heme pathway; Ferrochelatase (FECH). Heme oxygenase (HO). Phytochromobilin synthase (HY). Data come from Severin et al (2010). RPKM, reads per kilobase per million mapped reads. DAF, days after flowering. The source seed is experimental line A81-356022 which was generated by introgressing G. soja (PI468916) into G. max (A81-356022). C_FIG

Decades of research has uncovered a wealth of mechanistic information about the development of sensory bristles in Drosophila melanogaster. By studying large-effect, often loss-of-function mutations, many genes have been associated with bristle development, morphology, patterning, and number. Equally, the number of bristles present in certain areas of the fly cuticle is a classic quantitative trait, the genetic basis of which has been studied using a range of tools, from artificial selection to QTL (Quantitative Trait Locus) mapping. Such studies have often implicated well-understood bristle development genes as contributing to natural variation in bristle number. Here we contribute to the study of bristle number genetic variation in flies by executing a GWAS (genomewide association study). We generated whole genome sequences for 897 phenotyped male D. melanogaster individuals derived from a wild-derived, but lab-adapted outbred population, revealing - following quality control and filtering - over 780,000 variants with frequencies greater than 5%. Using these data we estimated the SNP (Single Nucleotide Polymorphism) heritability for ABN (abdominal bristle number) and SBN (sternopleural bristle number) as 0.28 and 0.35, respectively. These values indicate that our set of genotyped variants collectively explain a substantial fraction of the variance in phenotype in the mapping panel. Subsequently, genome scans revealed 1085 (ABN) and 211 (SBN) genomewide significant sites, and - due to extensive LD (Linkage Disequilibrium) in our panel - nearly all these sites are clustered into three locations; We find a GWAS hit for ABN in the middle of chromosome 3L, and hits for SBN at the tip of the X chromosome (where several prior mapping studies have resolved QTL for bristle number), and on 2L. Surveying existing studies that identified genes that control bristle number/development, we highlight several candidates that may segregate for causative, functional variants.

Gene model for the ortholog of Insulin-like peptide 3 (Ilp3) in the D. pseudoobscura Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

Overexpression of -synuclein (-syn), an inherently disordered protein, triggers chronic activation of the mitochondrial unfolded protein response (UPRmt) pathway in Caenorhabditis elegans with enhanced dopaminergic (DAergic) neurodegeneration. Introduction of a loss-of-function(lf) mutation in atfs-1, the main transcriptional regulator of the UPRmt, into -syn nematodes results in significant neuroprotection from -syn-induced DA neuron loss. Using this sensitized neuroprotective background, we performed a F3 forward genetic screen in C. elegans atfs-1(lf) mutants to identify molecular components associated with the modulation of neurodegeneration in -syn-expressing DA neurons. Homozygous mutant animals were examined for enhanced neurodegeneration; multiple independent alleles were uncovered. Among these, we identified new nonsense alleles encoding the histone lysine demethylases (H3K27me3), jmjd-1.2 (orthologous to human KDM7A, PHF2, and PHF8) and jmjd-3.1 (homologous to yeast CYC8). Another line carried a nonsense allele of twk-14. This gene encodes a conserved protein termed KCNK12 in mammals that facilitates passive background K+ leak currents to set and stabilize resting membrane potential. To further examine the association of these gene products with DA neurodegeneration, we used neuron-targeted RNA interference, mutants, or both. DA neurodegeneration was observed in the -syn + atfs-1(lf) background when jmjd-1.2, jmjd-3.1, or twk-14 were individually depleted. These results provide evidence that jmjd-1.2 and jmjd-3.1, which encode previously characterized H3K27me3 demethylases, and the uncharacterized twk-14 gene product, orthologous to human KCNK12, naturally confer protection from -syn-induced neurotoxicity.

The tunicate Ciona robusta offers a tractable non-vertebrate chordate model for probing gene function via tissue-specific, CRISPR/Cas9-mediated mutagenesis in F0. Building on Arcadia Sciences Zoogle platform, which identifies and ranks orthologs of human genes from various non-traditional model organisms, we carried out a pilot project to probe the developmental roles of three notochord- and endoderm-expressed candidate orthologs of human disease genes (Fcho, Pgm3, and Nckap1) alongside a fourth gene (Plastin) implicated in papilla cell elongation. This preprint compiles and updates a series of research project milestones previously posted episodically on Zenodo. Here we summarize the full results and our conclusion about this pilot project. Using CRISPR/Cas9, we found that tissue-specific knockout of Pgm3 and, to a lesser extent, Fcho caused significant defects in larval tail elongation. Separately, CRISPR knockout of Plastin, an actin-bundling gene expressed throughout the sensory-adhesive papillae of the larva, caused a subtle reduction in papilla cell elongation when combined as a duoble knockout with another actin-bundling protein-encoding gene, Villin. These results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.

Copper is an essential micronutrient in most organisms that becomes toxic in large quantities. Repeated or prolonged sub-lethal exposure can lead to evolved resistance to copper toxicity over many generations, which may result in trade-offs between energetically expensive detoxification mechanisms and fitness. Alternatively, evolved resistance to chemical stressors may lead to correlated changes in other traits. This study focuses on a population of flies for which artificial selection for copper resistance led to an increase in both copper resistance and longevity. The apparent off-target benefit of copper selection on one component of fitness led us to investigate differences in fecundity and developmental viability in copper resistant and copper sensitive, non-selected populations. We assessed the effect of copper selection and copper exposure on multiple aspects of fecundity over the lifespans of females from the non-selected and copper-selected populations. Our study corroborated previously observed increased longevity in copper-selected flies. Controlling for variation in lifespan, copper-resistant females had comparable age-matched fecundity to copper-sensitive females and benefitted from increased longevity with higher lifetime fecundity. Overall, copper exposure negatively affected egg quality, but we found no difference in this trait between the copper-resistant and sensitive populations. Further, we found developmental viability under copper stress was significantly higher for eggs laid by copper-resistant females. Overall, we determined that copper resistant flies experienced a fitness benefit through both lifespan and fecundity. Costs of maintaining copper resistance may be associated with energetic costs, but these trade-offs may not always manifest in reproductive or lifespan fitness costs.

Development requires the complex coordination of gene regulatory networks that must remain robust in the face of variable environmental cues. In Caenorhabditis elegans, the nuclear hormone receptor DAF-12 integrates metabolic cues and hormonal signals to control important life history decisions, including development, reproduction, and the rate of aging. Here, we tested the involvement of DAF-12 germline-to-soma signaling in two transgenerational longevity mutants, wdr-5 and jhdm-1. We have previously shown that both mutant populations gradually accumulate repressive H3K9me2 over multiple generations, which is necessary and sufficient for their lifespan extension. We find that daf-12 activity was required for the epigenetic establishment of longevity in both mutant populations, but was only necessary for maintaining longevity in a wdr-5 mutant background. Because DAF-12 also functions as a key regulator of dauer diapause, an alternative developmental stage triggered by environmental stress, we also tested the genetic relationship at earlier points in development. Surprisingly, mutations in either wdr-5 or jhdm-1 rescued the dauer defect of daf-12 mutants, and we found a synergistic effect on unchallenged larval development in wdr-5; daf-12 double mutants. These differing epistatic relationships indicate that, although the acquisition of longevity in both wdr-5 and jhdm-1 mutant populations shares a common mechanism, the impacts on somatic phenotypes (including lifespan extension) proceed via distinct pathways. Together, these results show how heritable chromatin states can co-opt existing developmental programs to influence key developmental decisions. ARTICLE SUMMARYHow do early experiences influence development and aging? In this study, we explore this question by testing the genetic interaction between the DAF-12 signaling pathway and heritable chromatin landscapes. Previously, we showed that two C. elegans mutants can accumulate heterochromatin over multiple generations to acquire longevity. We find that DAF-12 is required to establish this epigenetic trait but is not necessary to maintain it. We also find that chromatin landscapes bypass DAF-12s role earlier in development, including during the decision to enter dauer diapause. Overall, this study shows how chromatin states co-opt existing developmental programs to influence key life history decisions.

Exogenous and endogenous mutagens generate a wide variety of DNA lesions, including bulky adducts, chemical modifications, and single- or double-stranded breaks. A phenomenon called "lesion segregation," in which lesions evade repair and persist for multiple cell divisions, has recently been documented in tumors and healthy somatic tissues from mice and humans, respectively. Persistent lesions can generate multi-allelic variants (MAVs) by serving as templates for multiple rounds of error-prone replication. By reanalyzing data from a large C. elegans mutagenesis experiment, we observed robust evidence for MAVs at a small fraction (~0.2%) of mutated sites in the offspring of strains treated with alkylating agents. Because these sequencing data were derived from the progeny of a single F1 animal -- itself the offspring of a mutagenized P0 -- all mutations should be biallelic. The presence of multi-allelic variation implies that some DNA lesions are transmitted to the F1 zygote, evade repair, and are repeatedly bypassed by error-prone polymerases during embryogenesis. We suspect that many more lesions are inherited than is suggested by MAV prevalence, and that a large fraction of biallelic mutations are also caused by inherited lesions. Our results demonstrate that DNA lesions serve as durable, transgenerational templates for mutagenesis in C. elegans . We speculate that lesion segregation in the early embryo may be a source of mosaicism and genetic diversity in humans, as well.

Parental qualities can influence the development and fitness of their offspring via genetic and non-genetic effects. Although these effects are often linked to parental phenotypes, the effect of parental genetic variation linked with relevant phenotypes is less well understood. We performed full factorial crosses based on parental genotypes for an age-at-maturity-related gene, vgll3, to investigate how the parental genotypes influence Atlantic salmon (Salmo salar) offspring survival, growth, and development in their early life. Beyond the connection with age at maturity, the additional association between vgll3 and body condition in Atlantic salmon offers a potential pathway by which the maternal vgll3 genotype could influence offspring early life fitness. Combined with measurements of maternal phenotype and egg characteristics, the crossing design therefore allowed us to disentangle the maternal and paternal genetic and non-genetic contributions to variation in offspring survival and phenotypic traits. The phenotypic traits measured were hatching length and yolk sac area, growth, and yolk sac consumption and conversion efficiency. Parental vgll3 genotype did not influence the majority of our measured egg traits or alevin traits except for a genetic effect of paternal vgll3 genotype on offspring survival, whereby the paternal late maturation allele was associated with higher survival. Maternal effects were strongest for survival and for traits associated with hatching and weaker for alevin growth and yolk sac usage. Paternal effects on the measured alevin traits were negligible. The results from our study demonstrate that both maternal and paternal effects have the potential to influence offspring early life fitness traits.

Stalk lodging causes severe yield losses in maize (Zea mays L.) worldwide, worsening food and feed security. Stalk lodging resistance is influenced by multiple traits at various levels of biological organization, collectively referred to as intermediate traits, but their identities, genetic bases, and interrelationships remain poorly resolved. Here, evaluation of multiple geometric and structural intermediate traits in a maize diversity panel across four environments showed that macroenvironmental variation is the predominant driver of phenotype plasticity and that plasticity varies with internode position along the stalk, consistent with height-dependent mechanosensing. Major and minor diameters, moment of inertia, and rind penetration resistance, were genetically tractable and showed strong genetic correlations with stalk flexural stiffness. Multivariate analyses revealed two distinct but complementary mechanistic pathways, represented by cross-sectional geometry and rind architecture, that contribute to stalk mechanical performance. Association analyses using whole-genome resequencing data identified 705 SNPs associated with intermediate traits, fewer than 20% of which overlapped genic regions, indicating that most associated variation resides outside annotated genes. Interestingly, about 22% of SNPs were shared between at least two traits, indicating substantial shared genetic control among intermediate traits. Candidate gene analyses highlighted novel promising candidate loci associated with intermediate traits while recovering genes previously implicated in stalk lodging resistance. The predominance of noncoding associations further suggests that regulatory variation may contribute substantially to natural variation in intermediate traits underlying stalk lodging resistance.

Insects of the subfamily Triatominae, commonly known as kissing bugs, are obligate blood-feeding vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Rhodnius prolixus is among the most epidemiologically important vectors in Latin America, whereas Triatoma rubida frequently invades homes and is a potential vector in the southern United States and northern Mexico. Triatomines likely evolved from predatory reduviid assassin bugs through a transition from feeding on arthropods associated with vertebrate hosts to feeding directly on vertebrate blood. To investigate the genomic basis of this ecological and dietary shift, we generated highly contiguous, near chromosome-level genome assemblies and structural gene annotations for R. prolixus and T. rubida. The new R. prolixus assembly improves scaffold N50 more than 40-fold over the current reference genome, from 1.1 to 43.9 Mb, while reducing assembly gaps by several orders of magnitude. Both assemblies exceed 97% BUSCO completeness. Comparative analyses with representative hemipteran genomes revealed expansions of gene families associated with chemosensation and metabolism, including detoxification, protein degradation, and digestion, together with signatures of positive selection in genes involved in digestive and sensory functions. These assemblies represent the most contiguous and complete genomic resources available for Triatominae and provide a robust foundation for investigating vector biology, host adaptation, and the evolutionary origins of blood feeding within Reduviidae. Interpretive summaryKissing bugs are insects that are known for feeding on blood. They can spread a disease called Chagas disease because they transmit a parasite called Trypanosoma cruzi. To understand how kissing bugs evolved and which genes facilitate blood feeding of vertebrates, a collaboration between scientists at USDA-ARS, University of Georgia, and University of South Bohemia sequenced the genome of two kissing bugs: Rhodnius prolixus and Triatoma rubida. By comparing the genes with those of other insects in the order Hemiptera, scientists discovered that kissing bugs have more genes involved with detecting environmental chemical stimuli and metabolism as well as positive selection for genes involved with digestion and sensory-related proteins. These genome assemblies will help scientists learn more about how these insects evolved, and this research is important for understanding insect feeding biology which can be used to develop methods to control the kissing bugs and the spread of Chagas disease.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Insulin/IGF signaling (IIS) inhibits the nuclear localization of the DAF-16/FOXO transcription factor to regulate longevity and stress resistance in C. elegans. In the intestine, IIS promotes DAF-16 localization to endosomes and loss of TBC-2, a RAB-5 GAP, results in increased endomembrane localization of DAF-16 at the expense of nuclear localization, decreased DAF-16 target gene expression, longevity and stress resistance. Here we found that TBC-2 differentially regulates the localization of the IIS-regulated transcription factors PQM-1 and HLH-30/TFEB. Our results suggest a broader role for TBC-2 in negatively regulating IIS and that TBC-2 likely functions at an upstream point in the IIS pathway.

Drosophila connectomic datasets provide increasingly comprehensive maps of neuronal morphology and synaptic connectivity, offering an unprecedented opportunity to explore the structural organization of its neural circuits. This calls for designing automated tools to interact with connectomic datasets at scale for efficiently exploring structural features embedded in the vast amount of data. Yet the central challenge remains the understanding of the functional logic of neural circuits. In order to understand how elements of the functional logic may emerge from this structural organization, it is critical to (i) characterize the objects in the natural environment in which brain circuits operate, and (ii) formulate how brain circuits represent and process the defined objects in the natural environment. To develop and demonstrate a methodology for these requirements, we focus on the Drosophila looming-evoked escape pathway. We modeled the trajectory of looming objects that are on a collision course (direct-hits) or pass-by the fly (near-misses): their projected images on the retina can be characterized by the solid angle (angular size) and elevation. We then analyzed the pathway's morphology across the OpticLobe, Hemibrain, and FlyWire connectome datasets. By abstracting their sub-neuronal structure and retinotopic organization, we constructed an executable circuit model that maps each structural element to a processing block. We demonstrate that this model separates direct hits from near misses well before the angular size could tell them apart. To accelerate the connectomic analysis step, we developed a Python toolset with an agentic, code-free workspace interface called NeuroGraphBench (NGB). NGB provides four composable morphology-analysis primitives and an AI agent that composes them to interactively respond to natural-language queries aided by visualization on an interactive 3D canvas. Thus, NGB automates tedious and repetitive tasks to enable faster and scalable connectomic exploration, keeping human reasoning, instead of writing code, at the center of an open-ended research inquiry.

Genotype-environment association (GEA) analyses are widely used to identify loci underlying local adaptation by examining correlations between allele frequencies and environmental variables across a species' range. A major challenge for this approach is distinguishing true adaptive signals from spurious associations arising from population structure. Several methods have been developed to account for population structure, but these methods can suffer from reduced statistical power or increased false positives under some conditions. To address this, we introduce a new GEA method, termed SimGEA. In essence, SimGEA infers a neutral evolutionary model that reproduces the population structure observed in empirical data and uses this model to simulate neutral alleles. By applying the same GEA statistic to both the empirical and simulated data, SimGEA evaluates the significance of observed associations against neutral expectations that account for population structure. We compared the performance of SimGEA with that of existing GEA methods, including LFMM2 and BayPass, using simulations of local adaptation in two-dimensional space. We found that SimGEA consistently controlled the false discovery rate without substantially sacrificing statistical power across the scenarios examined. These results suggest that calibrating statistics using neutral simulations provides a robust and flexible approach for accounting for population structure in GEA analyses.

The physiology of ectotherms can change substantially during acclimation to changing environmental temperature. The role of transcription in acclimation responses has been well-established, but it remains unclear whether transcriptional regulation generally reflects abrupt changes after surpassing temperature thresholds, or whether transcript abundance is a relatively monotonic, continuous function of acclimation temperature. In this study we exposed adult male Drosophila subobscura flies to four different 96-hour acclimation treatments at temperatures that were not acutely stressful but ranged from relatively cold (10{degrees}C) to relatively warm (27{degrees}C) with respect to standard rearing conditions. Transcriptome sequencing of whole-body homogenates (mRNAseq) revealed a massive, transcriptome-wide response across acclimation temperatures, with a marked overrepresentation of genes that were continuously and monotonically up- and down-regulated in response to increasing acclimation temperature. Though some genes showed more complex relationships consistent with putative threshold responses, a high percentage of the differentially expressed transcriptome (42%) showed continuous and strictly monotonic relationships. Functional enrichment suggested continuous up-regulation of spermatogenesis-related transcripts with increasing temperature and continuous up-regulation of oxidative phosphorylation-related transcripts with decreasing temperature, illustrating contrasting patterns consistent with previous studies of thermal sensitivity of male reproduction and metabolic compensation in the cold. Thus, continuous thermal sensitivity of transcription is a hallmark of acclimation in D. subobscura that likely underlies the continuous thermal sensitivity of downstream physiological processes. We also provide evidence for shared transcriptomic responses across short-term acclimation (this study) vs. published results for long-term, developmental acclimation.