Biology subjects

Phillips, P. C.

Publications and source records attributed to Phillips, P. C..

36 records · Page 2Linked to original sources

bioRxiv2020

Opposing directions of stage-specific body length change in a close relative of C. elegans

BackgroundBody size is a fundamental organismal trait. However, as body size and ecological contexts change across developmental time, evolutionary divergence may cause unexpected patterns of body size diversity among developmental stages. This may be particularly evident in polyphenic developmental stages specialized for dispersal. The dauer larva is such a stage in nematodes, and Caenorhabditis species disperse by traveling on invertebrate carriers. Here, we describe the morphology of the dispersal dauer larva of the nematode Caenorhabditis inopinata, whose adults can grow to be nearly twice as long as its close relative, the model organism C. elegans. ResultsWe find that the C. inopinata dauer larva is shorter and fatter than those of its close relatives C. elegans, C. briggsae, and C. tropicalis, despite its much longer adult stage. Additionally, many C. inopinata dauer larvae were ensheathed, an apparent novelty in this lineage reminiscent of the infective juveniles of parasitic nematodes. We also found abundant variation in dauer formation frequency among twenty-four wild isolates of C. inopinata, with many strains unable to produce dauer larvae under laboratory conditions. ConclusionMost Caenorhabditis species thrive on rotting plants and disperse on snails, slugs, or isopods (among others) whereas C. inopinata is ecologically divergent and thrives in fresh Ficus septica figs and disperses on their pollinating wasps. These wasps are at least an order of magnitude smaller in length than the vectors of other Caenorhabditis species. While there is some unknown factor of the fig environment that promotes elongated body size in C. inopinata adults, the smaller size of its fig wasp carrier may be driving the reduced body length of its dauer larva. Thus ecological divergence across multiple developmental stages can promote unexpected and opposing changes in body size within a single species.

evolutionary biology↗

bioRxiv2020

Rapid self-selecting and clone free integration of transgenes into engineered CRISPR safe harbor locations in Caenorhabditis elegans

Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into the model nematode Caenorhabditis elegans has focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require laborious screening processes that often result in false positives from heritable extrachromosomal arrays or rely on co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene insertion requires anti-array selection screening methods or extensive PCR genotyping respectively. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods, and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native homology-based formation of extra-chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

genetics↗

bioRxiv2020

Sexually Antagonistic Selection on Genetic Variation is Rare in Humans

Sex and sexual differentiation are ubiquitous across the tree of life. Because females and males often have substantially different functional requirements, we expect selection to differ between the sexes. Recent studies in diverse species, including humans, suggest sexually antagonistic viability selection creates allele frequency differences between the sexes at many different loci. However, theory and population-level simulations indicate that sex-specific differences in viability would need to be very extreme in order to produce and maintain reported levels of between-sex allelic differentiation. We address this paradox between theoretical predictions and empirical observations by evaluating evidence for sexually antagonistic viability selection on autosomal loci in humans using the largest cohort to date (UK Biobank, n=438,427) along with a second large, independent cohort (BioVU, n=93,864). We performed association tests between genetically ascertained sex and genotypes. Although we found dozens of genome-wide significant associations, none replicated across samples. Moreover, closer inspection revealed that all associations are likely due to cross-hybridization with sex chromosome regions during genotyping. We report loci with potential for mis-hybridization found on commonly used genotyping platforms that should be carefully considered in future genetic studies of sex-specific differences. Despite being well-powered to detect allele frequency differences of up to 0.8% between the sexes, we do not detect evidence for this signature of sexually antagonistic viability selection on autosomal variation. These findings suggest a lack of strong ongoing sexually antagonistic viability selection acting on single locus autosomal variation in humans.

evolutionary biology↗

bioRxiv2020

Chromosome-level assembly of the Caenorhabditis remanei genome reveals conserved patterns of nematode genome organization

The nematode Caenorhabditis elegans is one of the key model systems in biology, including possessing the first fully assembled animal genome. Whereas C. elegans is a self-reproducing hermaphrodite with fairly limited within-population variation, its relative C. remanei is an outcrossing species with much more extensive genetic variation, making it an ideal parallel model system for evolutionary genetic investigations. Here, we greatly improve on previous assemblies by generating a chromosome-level assembly of the entire C. remanei genome (124.8 Mb of total size) using long-read sequencing and chromatin conformation capture data. Like other fully assembled genomes in the genus, we find that the C. remanei genome displays a high degree of synteny with C. elegans despite multiple within-chromosome rearrangements. Both genomes have high gene density in central regions of chromosomes relative to chromosome ends and the opposite pattern for the accumulation of repetitive elements. C. elegans and C. remanei also show similar patterns of inter-chromosome interactions, with the central regions of chromosomes appearing to interact with one another more than the distal ends. The new C. remanei genome presented here greatly augments the use of the Caenorhabditis as a platform for comparative genomics and serves as a basis for molecular population genetics within this highly diverse species.

genomics↗

bioRxiv2019

A naturally segregating polymorphism balancing semelparous reproduction versus reproductive diapause revealed via microfluidic assessment of starvation stress in Caenorhabditis elegans.

Caenorhabditis elegans typically feeds on rotting fruit and plant material in a fluctuating natural habitat, a boom-and-bust lifestyle. Moreover, stage specific developmental responses to low food concentration suggest that starvation-like conditions are a regular occurrence. In order to assess variation in the C. elegans starvation response under precisely controlled conditions and simultaneously phenotype a large number of individuals with high precision, we have developed a microfluidic device that, when combined with image scanning technology, allows for high-throughput assessment at a temporal resolution not previously feasible and applied this to a large mapping panel of fully sequenced intercross lines. Under these conditions worms exhibit a markedly reduced adult lifespan with strain-dependent variation in starvation resistance, ranging from <24 hours to [~]120 hours. Genome-wide mapping of the responses of more than 7,855 individuals identified four quantitative trait loci (QTL) of large effects. Three of these loci are associated with single genes (ash-2, exc-6, and dpy-28) and the fourth is a [~]26 KB region on Chromosome V encompassing several genes. Backcross with selection confirmed the effect of the Chromosome V locus. Segregating natural variation for starvation response in this species suggests that different isolates may use different strategies (facultative vivipary versus reproductive diapause) for dealing with extreme food deprivation.

evolutionary biology↗

bioRxiv2019

Slow recovery from inbreeding depression generated by the complex genetic architecture of segregating deleterious mutations

The deleterious effects of inbreeding have been of extreme importance to evolutionary biology, but it has been difficult to characterize the complex interactions between genetic constraints and selection that lead to fitness loss and recovery after inbreeding. Viruses, bacteria, and the selfing nematode Caenorhabditis elegans have been shown to be capable of rapid recovery from the fixation of novel deleterious mutation, however the potential for fitness recovery from fixation of segregating variation under inbreeding in outcrossing organisms is poorly understood. C. remanei is an outcrossing relative of C. elegans with high polymorphic variation and extreme inbreeding depression. Here we sought to characterize changes C. remanei in patterns of genomic diversity after [~]30 generations of inbreeding via brother-sister mating followed by several hundred generations of recovery at large population size. As expected, inbreeding led to a large decline in reproductive fitness, but unlike results from mutation accumulation experiments, recovery from inbreeding at large populations sizes generated only very moderate recovery in fitness after 300 generations. At the genomic level, we found that while 66% of ancestral segregating SNPs were fixed in the inbred population, this was far fewer than expected under neutral processes. Under recovery, 36 SNPs across 30 genes involved in alimentary, muscular, nervous and reproductive systems changed reproducibly across all replicates, indicating that strong selection for fitness recovery does exist but is likely mutationally limited due to the large number of potential targets. Our results indicate that recovery from inbreeding depression via new compensatory mutations is likely to be constrained by the large number of segregating deleterious variants present in natural populations, limiting the capacity for rapid evolutionary rescue of small populations. Impact SummaryInbreeding is defined as mating between close relatives and can have a large effect on the genetic diversity and fitness of populations. This has been recognized for over 100 years of study in evolutionary biology, but the specific genomic changes that accompany inbreeding and the loss of fitness are still not known. Evolutionary theory predicts that inbred populations lose fitness through the fixation of many deleterious alleles and it is not known if populations can recover fitness after prolonged periods of inbreeding and deleterious fixations, or how long recovery may take. These questions are particularly important for wild populations experiencing declines. In this study we use laboratory populations of the nematode worm Caenorhabditis remanei to analyze the loss of fitness and genomic changes that accompany inbreeding via brother-sister mating, and to track the populations as they recover from inbreeding at large population size over 300 generations. We find that: O_LITotal progeny decreased by 65% after inbreeding C_LIO_LIThere were many nucleotides in the genome that remained heterozygous after inbreeding C_LIO_LIThere was an excess of inbreeding-resistant nucleotides on the X chromosome C_LIO_LIThe number of progeny remained low after 300 generations of recovery from inbreeding C_LIO_LI30 genes changed significant in allele frequency during recovery, including genes involved in the alimentary, muscular, nervous and reproductive systems C_LI Together, our results demonstrate that recovery from inbreeding is difficult, likely due to the fixation of numerous deleterious alleles throughout the genome.

evolutionary biology↗

bioRxiv2019

An open source microfluidic sorter for Caenorhabditis nematodes

Rapid and accurate sorting of biological samples is extremely useful in a wide variety of applications. The model nematode Caenorhabditis elegans lends itself to automated fluid-flow based sorting because of its ability to live in aqueous solutions. Here, we build upon previous developments to construct a microfluidic device capable of sorting individuals based on a variety of characteristics, with a specific application toward differentiating fluorescently marked individuals. We find that our new design generates highly repeatable pools of sorted individuals. In general, there tends to be a tradeoff between precision and speed that can be optimized based on several different factors. Importantly, sorting does not decrease offspring production, and individuals can be sorted multiple times for increased precision. We provide detailed parts lists, schematics and software to allow implementation of these methods in other laboratories. Our results demonstrate that custom-made sorter chips can be a flexible and versatile addition to the nematode experimental toolbox.

bioengineering↗

bioRxiv2019

Automated Lifespan Determination Across Caenorhabditis Strains and Species Reveals Assay-Specific Effects of Chemical Interventions

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach towards aging-intervention screening.

physiology↗

bioRxiv2019

Limits to Genomic Divergence Under Sexually Antagonistic Selection

Since the autosomal genome is shared between the sexes, sex-specific fitness optima present an evolutionary challenge. While sexually antagonistic selection might favor different alleles within females and males, segregation randomly reassorts alleles at autosomal loci between sexes each generation. This process of homogenization during transmission thus prevents between-sex allelic divergence generated by sexually antagonistic selection from accumulating across multiple generations. However, recent empirical studies have reported high male-female FST statistics. Here, we use a population genetic model to evaluate whether these observations could plausibly be produced by sexually antagonistic selection. To do this, we use both a single-locus model with nonrandom mate choice, and individual-based simulations to study the relationship between strength of selection, degree of between-sex divergence, and the associated genetic load. We show that selection must be exceptionally strong to create measurable divergence between the sexes and that the decrease in population fitness due to this process is correspondingly high. Individual-based simulations with selection genome-wide recapitulate these patterns and indicate that small sample sizes and sampling variance can easily generate substantial male-female divergence. We therefore conclude that caution should be taken when interpreting autosomal allelic differentiation between the sexes.

evolutionary biology↗

bioRxiv2019

Complex pleiotropic genetic architecture of evolved heat stress and oxidative stress resistance in the nematode Caenorhabditis remanei

The adaptation of complex organisms to changing environments has been a central question in evolutionary quantitative genetics since its inception. The structure of the genotype-phenotype maps is critical because pleiotropic effects can generate widespread correlated responses to selection and potentially restrict the extent of evolutionary change. In this study we use experimental evolution to dissect the genetic architecture of natural variation for acute heat stress and oxidative stress response in the nematode Caenorhabiditis remanei. Previous work in the classic model nematode C. elegans has found that abiotic stress response is controlled by a handful of genes of major effect and that mutations in any one of these genes can have widespread pleiotropic effects on multiple stress response traits. Here, we find that acute heat stress response and acute oxidative response in C. remanei are polygenic, complex traits, with hundreds of genomic regions responding to selection. In contrast to expectation from mutation studies, we find that evolved acute heat stress and acute oxidative stress response for the most part display independent genetic bases. This lack of correlation is reflected at the levels of phenotype, gene expression, and in the genomic response to selection. Thus, while these findings support the general view that rapid adaptation can be generated by changes at hundreds to thousands of sites in the genome, the architecture of segregating variation is likely to be strongly parcellated by the pleiotropic structure of the underlying genetic networks.

evolutionary biology↗

bioRxiv2018

Experimental evolution of independent genetic pathways for resistance to Pseudomonas aeruginosa pathogenicity within the nematode Caenorhabditis remanei

Pathogenic host-microbe interactions can result from continuous evolution of a hosts ability to resist infection and a pathogens ability to survive and replicate. Pseudomonas aeruginosa is a versatile and opportunistic pathogen, ubiquitous in the environment, and capable of damaging plants, vertebrates, and invertebrates. Previous studies in nematodes suggest that the pathogenic effects of P. aeruginosa can result from multiple distinct pathways: a toxin-based effect that kills within a few hours and a generalized virulence that kills over the course of multiple days. Using experimental evolution in the highly polymorphic nematode Caenorhabditis remanei, we show that nematode resistance to the two modes of pathogenesis in P. aeruginosa evolves through genetically independent pathways. These results demonstrate that multiple virulence patterns in a pathogen can result in multiple responses in the host, and the genetic lines established here create resources for further exploration of the genetic basis for resistance to P. aeruginosa.

evolutionary biology↗

bioRxiv2018

The Stress-Chip: A microfluidic platform for stress analysis in Caenorhabditis elegans

An organisms ability to mount a physiological response to external stressors is fundamental to its interaction with the environment. Experimental exploration of these interactions benefits greatly from the ability to maintain tight control of the environment, even under conditions in which it would be normal for the subject to flee the stressor. ere we present a nematode research platform that pairs automated image acquisition and analysis with a custom microfluidic device. This platform enables tight environmental control in low-density, single-worm arenas, which preclude animal escape while still allowing a broad range of behavioral activities. The platform is easily scalable, with two 50 arena arrays per chip and an imaging capacity of 600 animals per scanning device. Validating the device using dietary, osmotic, and oxidative stress indicates that it should be of broad use as a research platform, including eventual adaptation for additional stressors, anthelmintic-drug screening, and toxicology studies.

physiology↗

bioRxiv2018

Proteomic and Evolutionary Analyses of Sperm Activation Identify Uncharacterized Genes in Caenorhabditis Nematodes

BackgroundNematode sperm have unique and highly diverged morphology and molecular biology. In particular, nematode sperm contain subcellular vesicles known as membranous organelles that are necessary for male fertility, yet play a still unknown role in overall sperm function. Here we take a novel proteomic approach to characterize the functional protein complement of membranous organelles in two Caenorhabditis species: C. elegans and C. remanei.\n\nResultsWe identify distinct protein compositions between membranous organelles and the activated sperm body. Two particularly interesting and undescribed gene families--the Nematode-Specific Peptide family, group D and the here designated Nematode-Specific Peptide family, group F--localize to the membranous organelle. Both multigene families are nematode-specific and exhibit patterns of conserved evolution specific to the Caenorhabditis clade. These data suggest gene family dynamics may be a more prevalent mode of evolution than sequence divergence within sperm. Using a CRISPR-based knock-out of the NSPF gene family, we find no evidence of a male fertility effect of these genes, despite their high protein abundance within the membranous organelles.\n\nConclusionsOur study identifies key components of this unique subcellular sperm component and establishes a path toward revealing their underlying role in reproduction.

evolutionary biology↗

bioRxiv2018

Auxin-Mediated Sterility Induction System for Longevity and Mating Studies in Caenorhabditis elegans

The ability to control both the means and timing of sexual reproduction provides a powerful tool to understand not only fertilization, but also life history trade-offs resulting from sexual reproduction. However, precisely controlling fertilization has proved a major challenge across model systems. An ideal sterility induction system should be external, non-toxic, and reversible. Using the auxin-inducible degradation system targeting the spe-44 gene within the nematode Caenorhabditis elegans, we designed a means of externally inducing spermatogenesis arrest. We show that exposure to auxin during larval development induces both hermaphrodite self-sterility and male sterility. Moreover, male sterility can be reversed upon cessation of auxin exposure. The sterility induction system developed here has multiple applications in the fields of spermatogenesis and mating systems evolution. Importantly, this system is also a highly applicable tool for aging studies. We show that auxin-induced self-sterility is comparable to the commonly used chemically-induced FUdR sterility, while offering multiple benefits, including being less labor intensive, being non-toxic, and avoiding compound interactions with other experimental treatments.

genetics↗

bioRxiv2018

Two levels of host-specificity in a fig-associated Caenorhabditis

BackgroundBiotic interactions are ubiquitous and require information from ecology, evolutionary biology, and functional genetics in order to be completely understood. However, study systems that are amenable to investigations across such disparate fields are rare. Figs and fig wasps are a classic system for ecology and evolutionary biology with poor functional genetics; C. elegans is a classic system for functional genetics with poor ecology. In order to help bridge these disciplines, here we describe the natural history of a close relative of C. elegans, C. sp. 34, that is associated with the fig Ficus septica and its pollinating Ceratosolen wasps.\n\nResultsTo understand the natural context of fig-associated Caenorhabditis, fresh F. septica figs from four Okinawan islands were sampled, dissected, and observed under microscopy. C. sp. 34 was found in all islands where F. septica figs were found. C. sp. 34 was routinely found in the fig interior and almost never observed on the outside surface. Caenorhabditis was only found in pollinated figs, and C. sp. 34 was more likely to be observed in figs with more foundress pollinating wasps. Actively reproducing C. sp. 34 dominated younger figs, whereas older figs with emerging wasp progeny harbored C. sp. 34 dispersal larvae. Additionally, C. sp. 34 was observed dismounting from plated Ceratosolen pollinating wasps. C. sp. 34 was never found on non-pollinating, parasitic Philotrypesis wasps. Finally, C. sp. 34 was only observed in F. septica figs among five Okinawan Ficus species sampled.\n\nConclusionThese observations suggest a natural history where C. sp. 34 proliferates in young F. septica figs and disperses from old figs on Ceratosolen pollinating fig wasps. The fig and wasp host specificity of this Caenorhabditis is highly divergent from its close relatives and frames hypotheses for future investigations. This natural co-occurrence of the fig/fig wasp and Caenorhabditis study systems sets the stage for an integrated research program that can help to explain the evolution of interspecific interactions.

ecology↗

bioRxiv2018

Environmental and evolutionary drivers of the modular gene regulatory network underlying phenotypic plasticity for stress resistance in the nematode Caenorhabditis remanei

In response to changing environmental conditions, organisms can acclimate through phenotypic plasticity or adapt by evolving mechanisms to cope with novel stressors. Changes in gene expression, whether dynamic or evolved, are an important way in which environmental responses are mediated; however, much is still unknown about how the molecular networks underlying plastic phenotypes evolve. Here, we compare transcriptional responses to acute heat stress among four populations of the nematode Caenorhabditis remanei--one selected to withstand heat stress, one selected under oxidative stress, an unselected control, and the ancestral population. We used a weighted gene coexpression network analysis within these lines to identify transcriptional modules, which are sets of genes that respond similarly to stress via plastic responses, evolutionary responses, or both. The transcriptional response to acute heat stress is dominated by a plastic response that is shared in the ancestor and all evolved populations. However, we also identified several modules that respond to artificial selection by (1) changing the baseline level of expression, (2) altering the magnitude of the plastic response, or (3) a combination of the two. Our findings reveal that while it is possible to perturb the nature of the transcriptional response network with short bouts of intense selection, the overall structure of transcriptional plasticity is dominated by inherent, ancestral regulatory systems.

evolutionary biology↗

bioRxiv2017

Quantifying male and female pheromone-based mate choice in Caenorhabditis nematodes using a novel microfluidic technique

Pheromone cues are an important component of intersexual communication, particularly in regards to mate choice. Caenorhabditis nematodes predominant rely on pheromone production for mate finding and mate choice. Here we describe a new microfluidic paradigm for studying mate choice in nematodes. Specifically, the Pheromone Arena allows for a constant flow of small molecule signals to be passed in real time from signaling worms to those making a choice without any physical contact. We validated this microfluidic paradigm by corroborating previous studies in showing that virgin C. remanei and C. elegans males have a strong preference for virgin females over mated ones. Moreover, our results suggest that the strength of attraction is an additive effect of male receptivity and female signal production. We go on to explicitly examine female choice and find that females are more attracted to virgin males. However, a females mate choice is strongly dependent on her mating status.

animal behavior and cognition↗

bioRxiv2017

Dramatic evolution of body length due to post-embryonic changes in cell size in a newly discovered close relative of C. elegans

Understanding morphological diversity--and morphological constrainto--has been a central question in evolutionary biology since its inception. Nematodes of the genus Caenorhabditis, which contains the well-studied model system C. elegans, display remarkable morphological consistency in the face of extensive genetic divergence. Here, we provide a description of the broad developmental patterns of a recently discovered species, C. sp. 34, which was isolated from fresh figs in Okinawa and which is among the closest known relatives of C. elegans. C. sp. 34 displays an extremely large body size and can grow to be nearly twice as long as C. elegans and all other known members of the genus. Observations of the timing of developmental milestones reveal that C. sp. 34 develops about twice as slowly as C. elegans. Measurements of embryo and larval size show that the size difference between C. sp. 34 and C. elegans is largely due to post-embryonic events, particularly during the transition from larval to adult stages. This difference in size is not attributable to differences in germ line chromosome number or the number of somatic cells. The overall difference in body size is therefore largely attributable to changes in cell size via increased cytoplasmic volume. Because of its close relationship to C. elegans, the distinctness of C. sp. 34 provides an ideal system for the detailed analysis of evolutionary diversification. The context of over forty years of C. elegans developmental genetics also reveals clues into how natural selection and developmental constraint act jointly to promote patterns of morphological stasis and divergence in this group.

evolutionary biology↗