The conserved Cdc42 GTPase is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit effectors to organize the cytoskeleton and polarize secretion. Here, we show that Cdc42...The conserved Cdc42 GTPase is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit effectors to organize the cytoskeleton and polarize secretion. Here, we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction, but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae. Notably, the cdc42-mCherrySW allele, which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherrySW and demonstrate that mating and cell fusion require higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that mitotic polarized growth responds linearly to Cdc42 protein levels, whereas mating exhibits a sharp switch-like response. We further trace this all-or-none response to pheromone-induced polarized growth. Thus, polarized growth in response to intrinsic or extrinsic cues exhibits distinct requirements to Cdc42 protein levels.
No matter where they are performed, studies with infectious agents need ironclad biorisk management. This can be promoted through robust gatekeeping of funding and publication, using a new formal reporting standard for p...No matter where they are performed, studies with infectious agents need ironclad biorisk management. This can be promoted through robust gatekeeping of funding and publication, using a new formal reporting standard for pathogen research.
Tunicates are the closest living relatives of vertebrates, but many details of their evolutionary history remain unclear. A study published in PLOS Biology explores the unique embryogenesis of Thaliacea, the tunicates th...Tunicates are the closest living relatives of vertebrates, but many details of their evolutionary history remain unclear. A study published in PLOS Biology explores the unique embryogenesis of Thaliacea, the tunicates that regained mobility from their sessile ancestor.
ERK is a key signaling mediator controlling both proliferation and lineage specification during embryo development. How ERK choreographs differentiation and proliferation to achieve balanced developmental outcomes in lin...ERK is a key signaling mediator controlling both proliferation and lineage specification during embryo development. How ERK choreographs differentiation and proliferation to achieve balanced developmental outcomes in lineages with variable ERK activities remains unclear. To investigate this, we established multiplex quantitative live-cell imaging to track human pluripotent stem cell differentiation into mesendoderm (ME), a lineage specified by gastrulation morphogens and dependent on high ERK activity. We found that distinct morphogen combinations generate varying ERK activity levels, which correlate with heterogeneous ME fate choices despite relatively uniform cell cycle dynamics. To dissect how heterogenous ERK levels directly modulate and coordinate ME differentiation and proliferation, we engineered a synthetic spectrum of titrated ERK activities. Our results showed that ERK fine-tunes ME differentiation potential and cell division speed under nonoverlapping activity ranges, enabling quantitative control of ME fate specification without major effect on cell cycle progression. Mechanistically, this uncoupling stems from differential transcriptional and translational sensitivities of ME-specifying genes versus cell cycle genes to ERK input. Together, our findings reveal how a single signaling pathway quantitatively balances differentiation and proliferation during lineage commitment and embryogenesis.
Adaptive responses to environmental stimuli are integral to the survival and virulence of microbial pathogens. The thermally dimorphic human fungal pathogen Histoplasma senses temperature to transition between a mold for...Adaptive responses to environmental stimuli are integral to the survival and virulence of microbial pathogens. The thermally dimorphic human fungal pathogen Histoplasma senses temperature to transition between a mold form in soil and a pathogenic yeast in mammalian hosts. The contributions of chromatin-modifying enzymes to the ability of Histoplasma to appropriately respond to temperature have never been explored. Through chemical inhibition and genetics, we determined that the class I histone deacetylase (HDAC) RPD3 is required for normal Histoplasma yeast morphology at 37 °C. Rpd3 regulated the expression of key morphology-specific genes, including critical virulence factors and transcription factors (TFs), was required for normal DNA-binding activity of yeast-promoting TFs, and influenced histone acetylation levels at the loci of putative pro-filamentation TFs. Furthermore, Rpd3 was required for virulence in a macrophage model of infection. Taken together, Rpd3 is a critical regulatory component that both activates the pathogenesis program and represses the filamentation program to enable thermal dimorphism in Histoplasma. This work uncovers the crucial role that chromatin regulation plays in temperature response of this ubiquitous pathogen.
Bacterial warfare is a widespread phenomenon in which bacteria deploy toxins to inhibit or kill competitors. These toxins disrupt essential cellular processes, and their diversification is driven by an evolutionary arms...Bacterial warfare is a widespread phenomenon in which bacteria deploy toxins to inhibit or kill competitors. These toxins disrupt essential cellular processes, and their diversification is driven by an evolutionary arms race involving toxin and immunity gene acquisition. Here, we used in-silico approaches to analyze genomes from the 10k Salmonella Project and identify effectors secreted via the Type VI Secretion System (T6SS). We uncovered 128 candidates distributed across diverse Salmonella serovars and other bacterial species. Among them, Tox-Act1 was selected for in-depth characterization. Tox-Act1 contains a permuted NlpC/P60 papain-like catalytic core typical of lipid-targeting enzymes. Evolutionary analysis revealed its relationship with acyltransferases. Biochemical assays and lipidomics of intoxicated cells showed that Tox-Act1 acts as a phospholipase, cleaving phosphatidylglycerol and phosphatidylethanolamine. We further demonstrate that Tox-Act1 is secreted in a T6SS-dependent manner and provides a competitive advantage during mouse gut colonization. This study broadens our understanding of toxin domain diversity and provides the first direct characterization of a lipid-targeting NlpC/P60 toxin domain.
The probiotic strain Escherichia coli Nissle 1917 (EcN), a potential member of tumor-targeting bacteria, shows great promise for cancer treatment. By leveraging engineered EcN, we can design a bacteria-assisted, tumor-ta...The probiotic strain Escherichia coli Nissle 1917 (EcN), a potential member of tumor-targeting bacteria, shows great promise for cancer treatment. By leveraging engineered EcN, we can design a bacteria-assisted, tumor-targeted therapy for the biosynthesis and targeted delivery of small-molecule anticancer agents. In this study, we aimed to use EcN as a base for synthesizing Romidepsin (FK228), an FDA-approved drug originally made by Chromobacterium violaceum No. 96. Through gene cluster reconstruction, promoter optimization, and genome modification, we created FK228-producing strains to boost anticancer efficacy. The engineered strain achieved a maximum in vitro yield of 1.5 mg/L. In 4T1 tumor-bearing BALB/c mouse xenograft models, six recombinant strains outperformed the wild-type EcN. Proteome showed that inflammatory response induced by EcN combined with intratumoral FK228 production improved treatment results. Also, targeted synthesis reduced FK228's cardiotoxicity and mortality. Engineered EcN enables drug biosynthesis and precise delivery, offering powerful anticancer activity.
Tunicates are the closest living relatives of vertebrates. Recent phylogenies place the little-studied, free-swimming thaliaceans-including salps-within sessile ascidians, highlighting a remarkable ecological transition....Tunicates are the closest living relatives of vertebrates. Recent phylogenies place the little-studied, free-swimming thaliaceans-including salps-within sessile ascidians, highlighting a remarkable ecological transition. Historical reports hinted at a parallel developmental shift. Salp embryogenesis diverges from that of ascidians and involves unique maternal cells called calymmocytes. Here, we provide foundational resources for two distantly related salp species, Salpa fusiformis and Thalia democratica. Using advanced microscopy, we generated developmental staging tables showing that while embryogenesis is stereotyped within species, it differs in cleavage patterns and blastomere positioning between them. We traced the origins of calymmocytes and confirmed their conserved role in separating blastomere clusters that form adult tissues. Apoptosis contributes to the progressive elimination of maternal calymmocytes. Finally, we show that calymmocytes express embryonic developmental regulators, suggesting co-option of an embryonic gene program. These findings provide an advanced framework for studying embryogenesis evolution in a previously underexplored chordate lineage.
Numerous studies across species emphasize the importance of theta oscillations within medial temporal lobe (MTL) regions, such as the hippocampus, in relation to memory. In rodents, physical movement strongly influences...Numerous studies across species emphasize the importance of theta oscillations within medial temporal lobe (MTL) regions, such as the hippocampus, in relation to memory. In rodents, physical movement strongly influences theta activity, while this relationship remains more ambiguous in primates. This disparity could stem from the increased reliance on visual search in primates during navigation. To explore this, we analyzed intracranial electroencephalographic (iEEG) activity from the human MTL recorded simultaneously with body and eye movements during ambulatory navigation. We found that MTL theta power was significantly higher during periods when saccadic eye movements were taking place, and this effect was observed only during periods with overt memory demands. The largest increases occurred during saccades with more variable and exploratory gaze patterns, on trials with better memory performance, and during the early planning period of each route. The modulation was also amplified near environmental boundaries, spatial features known to anchor memory representations and guide navigation. During memory-guided navigation, theta power further tended to increase during both locomotion and stationary periods, consistent with broad engagement during active information gathering. In addition to these memory-specific effects, theta aligned its phase to saccade onset during both memory-guided and visually-guided navigation, suggesting that eye movements impose a consistent temporal structure on ongoing MTL activity. Together, these findings reveal that memory-related theta dynamics in the human MTL are tightly coupled to exploratory visual search and prospective planning during memory-guided navigation, revealing a mechanism by which saccades may help organize mnemonic computations in naturalistic settings.
In the auditory cortex, neural responses to stimuli inside receptive fields (RFs) can be further facilitated by behavioral demands, such as attending to a spatial location. It is less clear how off-RF stimuli modulate ne...In the auditory cortex, neural responses to stimuli inside receptive fields (RFs) can be further facilitated by behavioral demands, such as attending to a spatial location. It is less clear how off-RF stimuli modulate neural responses and contribute to behavioral tasks. Our recent study revealed a particular form of location-specific facilitation evoked by repeated stimulation from an off-RF location, suggesting behavioral modulation of spatial RFs. To further explore this question, we trained marmosets to attend to sound locations that were either inside or outside the RFs of auditory cortical neurons. The majority of neurons showed increased firing rates at target locations inside their RFs. Interestingly, this increase also occurred outside the RFs, sometimes exceeding the responses at the RF center during passive listening. This task-related off-RF facilitation was much more common in the caudal area than in the rostral area and the primary auditory cortex. A normalization model reproduced the off-RF facilitation using widespread suppression. The model's prediction was confirmed by experimental observations of widespread reductions in firing rate and hyperpolarized membrane potentials for off-RF stimuli. These results suggest that behavioral task demands recruit a broader range of neurons than those that are responsive to a target sound in the passive state.
Cytoplasmic receptors of the RRNPPA superfamily mediate peptide-based quorum sensing in Gram-positive bacteria and are thought to be activated exclusively by short, unmodified pheromones. Here, we show that the RRNPPA re...Cytoplasmic receptors of the RRNPPA superfamily mediate peptide-based quorum sensing in Gram-positive bacteria and are thought to be activated exclusively by short, unmodified pheromones. Here, we show that the RRNPPA regulator ComR in the human commensal Streptococcus salivarius can also be activated by a distinct class of non-peptide metabolites. A screen of ~200 organic compounds identified hydroxyphenylacetic acid (HPAA)-a microbial dysbiosis-associated catabolite-as a potent activator of ComR. Using biochemical and genetic approaches, we demonstrate that HPAA and related aromatic carboxylic acids bind the canonical pheromone pocket and induce sustained expression of predatory bacteriocins, while bypassing the competence program triggered by the native peptide signal (XIP). We further show that the oral pathogen Porphyromonas gingivalis produces physiologically relevant amounts of (H)PAA, enabling metabolite-driven activation of predation in S. salivarius. These findings reveal an unexpected capacity of RRNPPA receptors to sense both peptide and metabolite cues, uncovering a chemical mode of interspecies communication that links dysbiosis to predatory behavior in the oral microbiome.
Inhibition plays an important role in controlling the flow and processing of auditory information throughout the central auditory pathway, yet how inhibitory circuits shape auditory processing in the medial geniculate bo...Inhibition plays an important role in controlling the flow and processing of auditory information throughout the central auditory pathway, yet how inhibitory circuits shape auditory processing in the medial geniculate body (MGB), the key region in the auditory thalamus, is poorly understood. The MGB gates the flow of auditory information to the auditory cortex, and it is inhibited largely by the thalamic reticular nucleus (TRN). The TRN contains two major classes of inhibitory neurons: parvalbumin (PVTRN)-positive and somatostatin (SSTTRN)-positive neurons. PV and SST neurons have been shown to play differential roles in controlling sound responses in auditory cortex. In the somatosensory and visual subregions of the TRN, PVTRN and SSTTRN neurons exhibit anatomical and functional differences. However, it remains unknown whether and how PVTRN and SSTTRN neurons differ in their anatomical projections from the TRN to the auditory thalamus, and whether and how they differentially modulate activity in the MGB. Here, we investigated virally labeled projections of PVTRN or SSTTRN neurons, and recorded neuronal responses in the MGB of awake, head-fixed mice while presenting sound stimuli and selectivity suppressing PVTRN or SSTTRN neurons on a subset of trials. We find that PVTRN and SSTTRN neurons exhibit differential projection patterns within the auditory thalamus: PVTRN neurons predominantly project to ventral MGB, whereas SSTTRN neurons project to the dorso-medial regions of MGB. Optogenetic inactivation of PVTRN neurons bidirectionally modulated sound-evoked activity in MGB, increasing firing in 29% of MGB neurons, while suppressing firing in 41%. In contrast, inactivating SSTTRN neurons largely suppressed tone-evoked activity in MGB neurons. Cell type-specific computational models identified candidate circuit mechanisms for generating the differential effects of TRN inactivation on MGB sound responses. These distinct inhibitory pathways within the auditory thalamus reveal cell type-specific organization of thalamic inhibition in auditory computation.
Cancer cells reprogram the metabolic pathways to adapt to nutrient deficiency, while the underlying mechanism has not been fully understood. Phosphofructokinase 1 muscle type (PFKM) is the second rate-limiting step of gl...Cancer cells reprogram the metabolic pathways to adapt to nutrient deficiency, while the underlying mechanism has not been fully understood. Phosphofructokinase 1 muscle type (PFKM) is the second rate-limiting step of glycolysis, catalyzing the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Here we show, using an orthotopic xenograft glioma mouse model, that PFKM is deubiquitinated and translocated into nucleus upon glucose deficiency, thereby activating fatty acid oxidation (FAO), which sustains tumor cell survival and ultimately promotes glioblastoma (GBM) development. Mechanistically, the levels of fructose-2,6-bisphosphate (F-2,6-BP) are decreased in tumor cells upon glucose deficiency, which enhances the interaction between ubiquitin carboxyl-terminal hydrolase 7 (USP7) and PFKM. USP7 removes the monoubiquitination of PFKM at lysine (K) 615, thereby promoting PFKM's translocation into the nucleus. Nuclear PFKM interacts with c-MYC, which upregulates the expression of carnitine o-palmitoyltransferase 1 muscle isoform (CPT1B) to activate FAO, thereby sustaining tumor cell survival upon glucose deficiency. Notably, USP7 inhibitor effectively dampens GBM development and extends the survival duration of the mice. The levels of nuclear PFKM correlate with the malignancy and prognosis of human GBM patients. Our findings reveal a novel mechanism through which USP7 senses fructose-2,6-bisphosphate levels to promote PFKM nuclear translocation, thereby sustaining tumor cell survival under nutrient deficiency by activating FAO. This establishes the critical role of USP7 in brain tumor development and suggests the therapeutic potential of USP7 inhibitors for treating GBM.
Acute stress triggers the release of cortisol, which broadly affects cognitive processes. Path integration, a specific navigational process, relies heavily on grid cells in the entorhinal cortex. The entorhinal cortex co...Acute stress triggers the release of cortisol, which broadly affects cognitive processes. Path integration, a specific navigational process, relies heavily on grid cells in the entorhinal cortex. The entorhinal cortex contains glucocorticoid receptors and is therefore likely to be influenced by cortisol, though little is known about this relationship. Given the role of the entorhinal cortex in neurological diseases such as Alzheimer's Disease, investigating the effects of cortisol on this brain region may offer insights into how stress affects these diseases. In this study, we examined the effects of cortisol on human path integration in 39 healthy men across two sessions. On each day, they received either 20 mg cortisol or a placebo and performed a virtual homing task during functional magnetic resonance imaging (fMRI). Cortisol markedly impaired path integration performance, independent of incoming distance or the presence of spatial cues, but did not affect navigational pattern as measured by proximity to the landmark. fMRI results showed that cortisol increased the activation of right caudate nucleus in the presence of landmarks. Using a representational similarity analysis, we observed grid-like representations in the right entorhinal cortex specifically on day one under placebo, but these were diminished by cortisol. Grid-like representations were associated with PI performance dependent on the availability of spatial cues and cortisol administration, suggesting that cortisol may interfere with the typical relationship of grid cells and PI. Overall, the study indicates that cortisol-induced disruption in grid cell function in the entorhinal cortex may underly stress effects on path integration.
Thapa M, Kumari A, Chin CY
… +13 more, Choby JE, Akbari E, Bogati B, Jin F, Furr E, Chopyk DM, Koduri N, Pahnke A, Burns TL, Elrod EJ, Burd EM, Weiss DS, Grakoui A
Recent advances suggest a correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that bacteria can translocate directly f...Recent advances suggest a correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that bacteria can translocate directly from the gut to the brain in small numbers when mice are fed an atherogenic, high-fat diet (Paigen diet) that causes alterations in gut microbiome composition and gut barrier permeability. The bacteria were not found in other systemic sites or the blood, but were detected in the vagus nerve. Right cervical vagotomy reduced bacterial burden in the brain, implicating the vagus nerve as a conduit for bacterial translocation from the gut to the brain. Antibiotic treatment perturbed the composition of the gut microbiome and correspondingly changed the bacteria that localized to the brain in the setting of Paigen diet feeding. To further establish the gut as the origin of bacterial translocation to the brain, we gavaged exogenous Enterobacter cloacae into Paigen diet-fed mice, subsequently detecting the E. cloacae in the gut and brain. In addition, we monocolonized germ-free mice with E. cloacae and only cultured the bacteria from the brains of mice fed Paigen diet, but not those fed standard diet. Localization of bacteria to the brain in Paigen diet-fed mice was reversible with return to normal diet. Bacteria were also detected in the brain of murine models of Alzheimer's, Parkinson's, and autism spectrum disorder fed standard diet. These data reveal a bacterial translocation axis from the gut to the brain, impacted by environmental (diet) and genetic factors, and warrant further investigation to determine if this phenomenon also occurs in humans and to elucidate whether it may play a role in diverse neurological conditions.
The genetic structure of bacterial species is most often interpreted in terms of demographic processes such as clonal descent, but can also reflect natural selection and hence give functional and ecological insight. Kleb...The genetic structure of bacterial species is most often interpreted in terms of demographic processes such as clonal descent, but can also reflect natural selection and hence give functional and ecological insight. Klebsiella pneumoniae (KP) disperses effectively around the world and has high recombination rates, which should result in the species having a well-mixed gene pool. Nevertheless, phylogenies based on diverse KP strains contain a "backbone." This structure reflects a component of variation where the first component in Principal Components Analysis (PCA), PC1, explains 16.8% of the total variation. We propose that the component reflects a "bacterial ecocline" generated by diversifying selection on a quantitative genetic trait. We simulated the evolution of a bacterial population with a polygenic quantitative trait, where strains with the most extreme trait values have a small advantage. These simulations can recapitulate our KP PCA results and other features of its genetic diversity. As well as providing an explanation for the phylogenetic backbone, our results provide insight into how species such as KP can speciate, via stronger selection on the trait or a reduction in gene flow. Our hypothesis that there is a bacterial ecocline in KP raises two questions, namely what the trait is underlying it and why is the trait under diversifying selection? The genes that are most strongly associated with PC1 provide some hints, with the top locus encoding Kpa fimbriae. Identification of the trait, if it exists, should facilitate insight into selection on quantitative genetic traits in natural bacterial populations, which have largely been unstudied in microbiology, except in the atypical context of antibiotic resistance.
Motor learning induces alterations in neural activity that can persist long after the effects of such learning have faded. These persistent neural alterations are thought to manifest behaviorally as "savings," or faster...Motor learning induces alterations in neural activity that can persist long after the effects of such learning have faded. These persistent neural alterations are thought to manifest behaviorally as "savings," or faster relearning, via access to a latent motor memory. How the human brain forms and retrieves these latent memories, and the specific neural systems involved, remains unresolved. Here, using human functional MRI and a two-day sensorimotor adaptation paradigm, we show that savings are associated with the reinstatement of a large-scale cortical manifold structure formed during initial learning. Notably, this neural reinstatement effect was not observed across sensorimotor systems but was localized to regions of the default mode network (DMN). Moreover, the specific dynamics of DMN activity were linked to inter-subject differences in patterns of learning and relearning across days. These results suggest that motor savings arises from the re-expression of DMN activity patterns associated with initial learning, establishing a key role for this network in motor memory formation and retrieval. This finding, paralleling reinstatement principles from other memory domains (episodic memory, fear conditioning) and anticipated by recent computational models of motor learning, suggests a common mechanism for the flexible recall and reuse of stored memories across diverse behavioral contexts.
Recessive lethal mutations are widespread across studied species, with estimates suggesting that each individual carries at least one. Numerous lethal alleles persist in wild populations at higher frequencies than expect...Recessive lethal mutations are widespread across studied species, with estimates suggesting that each individual carries at least one. Numerous lethal alleles persist in wild populations at higher frequencies than expected given their extreme deleterious nature. Though these findings spurred historical debate whether classical balancing selection maintains some lethal alleles at elevated frequencies (versus mutation-selection balance acting alone), we propose the question remained unanswered, especially given that the genetic basis of most naturally occurring lethal effects is still unknown. Given current genome-wide point mutation rate estimates, mutation-selection balance alone cannot explain some of this lethal variation in nature. However, evolutionary biologists have historically studied genetic variation through a lens of single-nucleotide variants, when in fact the spectrum of mutational changes is far broader than point mutations alone, including indels, structural variants, short tandem repeats, and transposable element insertions. We uncover the genetic basis of lethality in nature and provide insight on the possible evolutionary forces allowing some to persist at higher frequencies. By locating hundreds of recessive lethal mutations in Drosophila melanogaster via complementation testing, fine-mapping, and sequencing a subset, we determine candidate lethal mutations in specific genes. We discover that many lethal disruptions are likely caused by transposable element insertions. The most common transposable elements in our data, Transib1 and Kuruka, are both estimated to have recently invaded D. melanogaster, each from a different Drosophila species (between 2013-2016 and 2017-2021, respectively). This finding demonstrates that the many lethal alleles studied in D. melanogaster in the last century had a distinct genetic basis. Hence, we propose a model that could explain lethal variation in natural populations of D. melanogaster: lethal mutation frequencies are driven by invasions of new transposable elements and as time passes after each invasion, those frequencies decline as D. melanogaster evolves suppression mechanisms, allowing for natural selection to more efficiently remove lethal insertions. Upon the invasion of a new TE, the cycle repeats. The ubiquity of lethal alleles in natural populations is a classic conundrum for evolutionary geneticists for over a century, and this study utilized modern tools and sequencing technology to provide novel insight into this age-old mystery.
Parental age and inbreeding have both been shown to have substantial fitness effects in laboratory experiments and in observations of wild animals. These demographic effects are likely to be strongly impacted by habitat...Parental age and inbreeding have both been shown to have substantial fitness effects in laboratory experiments and in observations of wild animals. These demographic effects are likely to be strongly impacted by habitat fragmentation and warming temperatures, so understanding them is a priority. In insects and other ectotherms, some processes implicated in senescence are dependent on temperature. Anticipated changes in climate may therefore have direct effects on senescence in insects, or indirect effects via parental age. Similarly, although effects of inbreeding are well studied in wild vertebrates, information about how matings between relatives affect fitness in invertebrates comes almost exclusively from laboratory studies. To bridge the divide between field studies of vertebrates and laboratory studies of insects, we conducted an experiment using wild field crickets, Gryllus campestris. We experimentally manipulated the relatedness of parents, their age at reproduction and the temperature they experience as they aged. We then released the offspring of these parents into a natural meadow and used a network of video cameras to monitor their adult behavior and life history throughout the course of their breeding season. We found no effect of parental age on their offspring. There were effects of inbreeding, but they were restricted to more inbred females mating to fewer males, and more inbred males being slightly smaller than outbred males. Our study suggests that effects that can be detected in laboratory studies may have relatively modest effects on fitness in nature.