-
[
International Worm Meeting,
2015]
The Caenorhabditis Intervention Testing Program (CITP) is a multi-institution effort, sponsored by the National Institute of Aging, designed to screen bioactive compounds for their ability to extend lifespan and enhance health using nematodes as a model system for the potential effects of background genetic variation. The Intervention Testing Program (ITP), began assaying pharmacological interventions for lifespan extension in mice in the early 2000s [1]. However, with a median lifespan over two years, this project has decreased capacity to test multiple compounds in a robust manner [2]. The CITP capitalizes on the short lifespan of hermaphroditic C. elegans, C. briggsae and C. tropicalis. Selected natural isolates represent a biologically and geographically diverse population, yielding a total of 22 test strains. A compound effective across a diverse worm panel increases the likelihood of potency across other species, including heterogeneous human populations.Reproducibility marks a key goal of the CITP; the collaborative group generated detailed standardized protocols to minimize among and within lab variability. A data center facilitates near-instantaneous and global access to all CITP data. Compound candidates are selected based on the scientific community's recommendations and advice from the project's steering committee.A subset of strains has already undergone initial testing of four compound interventions: (R) alpha-lipoic acid, Aspirin, Quercetin, and NP1. Resveratrol, propyl gallate, and Thioflavine T are to be tested shortly. In addition to manual lifespan experiments, each lab built over 20 automated lifespan machines which use flatbed scanner technology [3], allowing for high throughput and precise longevity measurements. Mid- and late-age health assays are also underway.Initial results indicate significant differences in among-isolate variation in longevity within species (e.g., C. elegans low variation, C. briggsae high variation), as well as strain- and species-specific differences in longevity responses to compound treatments.1. Warner et al (2000) Mech Ageing Dev 115:199-2072. Miller et al (2007) Aging Cell 6:565-5753. Stroustrup et al (2013) Nature Methods, 10:665-670.
-
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
The last twenty years has seen tremendous strides in the use of C. elegans and its relatives for evolutionary studies. One area in which nematodes have the potential to play a unique role is in studies of experimental evolution. Short generation times and large population sizes allow experiments on scale that is difficult to achieve in most animal systems, while the ability to focus on the biology and behavior of specific individuals allows a quality of phenotypic analysis that is difficult replicate in microbial systems. In this talk, I will provide an overview of some of the experimental evolution studies carried out in my lab and will discuss how these approaches can be used to address fundamental questions related to the evolution of sex and outcrossing, the evolution of sex determination, the evolution of aging, and the genetic basis of complex traits. In particular, I will focus on how these studies can interface with modern approaches to whole-genome functional analysis and innovative phenotypic manipulation systems, such as microfluidics.
-
[
Development & Evolution Meeting,
2008]
When individuals mate in order to reproduce, potential competition among mates can lead to both sexual selection and to sexual conflict if the reproductive goals of both sexes do not correspond. One of the defining features of the nematode genus Caenrohabditis is the evolutionary transition between mating systems, from separate sexes in C. remanei and most other members of the group to the independent origin of hermaphroditism within C. elegans and C. briggsae. Because C. remanei mates much more frequently than C. elegans, we might expect (1) that there would much more opportunity for sexual conflict and sexual selection within C. remanei, and (2) that if mating frequency within C. elegans were to be increased, this would result in the emergence of sexual selection and sexual conflict within this species as well. Examination of the reproductive and life history patterns of multiple strains of C. remanei reveals that this species is sperm limited and that both male and female components of reproductive fitness vary dramatically among strains. In particular, males differ in both the total number of offspring produced and in their effects on females after mating. One strain, EM464 from New York, is especially harmful to females, with only a single day of exposure to males leading to a 50% decline in female longevity compared to the lifespan of virgins. EM464 males also generate overall low female fecundity when sperm is not limiting. Further, sperm from EM464 males far out-completes sperm from other strains, indicating that male-male competition may have driven selection for sperm-specific characteristics that are harmful to females. To test the role that male-male competition plays in these phenotypes, we experimentally evolved
fog-2 lines of C. elegans in which hermaphrodites are effectively transformed into females, thereby mimicking the C. remanei mating system. After 60 generations of evolution, males evolved harmful effects on female longevity, increased sperm competitive ability and larger sperm. Together, these natural observations and experimental tests strongly support the hypothesis that male-male competition generated by multiple mating can drive the rapid evolution of male-specific traits, frequently to the detriment of female fitness.
-
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
Although C. elegans has served as a wonderful model for genetic and functional analysis, it is clear that most of recent and current evolutionary history is dominated by its selfing mode of reproduction, especially from the standpoint of population genetics. A growing number of studies have shown that the closely related gonochoristic, fully outcrossing species C. remanei displays orders of magnitude more molecular and quantitative genetic variation. Indeed, levels of polymorphism within C. remanei exceed those of most animals, providing unique opportunities, as well as serious difficulties (such as profound inbreeding depression). Here, I summarize what is known about the evolutionary genetics of this species and present an update on the efforts that my lab has made in creating a set of canonical recombinant inbred lines, using next generation sequencing to produce a high density genetic map, in fully assembling the genome, and in creating a drug-based transformation system.
-
[
International Worm Meeting,
2011]
Numerous studies have implicated the insulin/IGF-1 signaling (IIS) cascade as playing a major role in both stress response and aging. In C.elegans, this cascade has been shown to negatively regulate various transcription factors including DAf-16, SKN-1 and HSF-1 in response to environmental conditions such as nutrients, heat and oxidative stress. To date, little is known regarding which, if any of these transcription factors, play a significant role in the observed phenotypic variation in both stress response and lifespan variation observed in natural populations of nematodes. To address these questions we have begun dissecting natural populations of C.remanei to identify the genetic networks that contribute to stress resistance and lifespan variation. First, we are determining the extent of genetic variation downstream of DAF-16 in C.remanei populations. We are utilizing commercially available antibodies against DAF-16 to perform ChIP-Seq experiments under environmental stress conditions and will correlate DAF-16 binding site patterns with total RNA changes as measured by RNA-Seq. In addition, we will are taking a traditional quantitative genetic approach by mapping lifespan variation and stress resistance in a set of recombinant inbreed lines (RIL's) that we are generating. To date, we have 55 lines which exhibit significant phenotypic variation in both lifespan and stress resistance. Progress on both fronts will be presented.
-
[
International Worm Meeting,
2017]
Selection can drive sub-populations to become differentiated, both in phenotype and genotype, but gene flow between them can affect the evolutionary trajectories of the phenotypes in question, potentially by mitigating the effects of selection and slowing the rate of adaptation to novel environments. Migration-selection dynamics are one of the fundamental aspects of speciation and population divergence, but they have not been rigorously investigated in an experimental context. Stress resistance is a heritable complex trait involving interactions between numerous genes and pathways. Previous studies in Caenorhabditis elegans have identified correlations between single genes and stress responses, but few have investigated genome-wide, causative relationships. Multiple questions remain in understanding how complex, polygenic traits evolve on the whole-genome scale under the opposing influences of strong selection and gene flow. The goals of this project are to dissect the genetic basis of chronic heat stress, a model complex trait, in C. remanei; to elucidate how gene flow affects rates of adaptation to a novel environment; and to investigate migration-selection dynamics. We utilized an evolve-and-resequence framework, which allowed for selection on standing genetic variation in the ancestral strain of C. remanei that was derived from a wild isolate and lab-adapted for 75 generations. Populations of C. remanei derived from this ancestral population were evolved in pairs to either a control (20 deg C) or heat stress (31 deg C) environment for forty generations. We tested the effect of migration between sub-populations with three migration rates: 0 (no migration), 5, and 20 percent. Female fecundity was measured to estimate the strength of selection in the heat stress environment. We observe a significant effect of selection and a significant interaction between selection and migration. The no-migration 31 deg C-evolved, 5-percent migration 31 deg C-evolved, and ancestor populations were sequenced via whole genome pooled population sequencing. The ancestral and descendant populations were compared on a locus-by-locus basis, allowing us to identify the number and location of putative loci under selection in the heat stress environment. We find that the 5-percent 31 deg C-evolved populations show fewer divergent regions than the no migration 31 deg C-evolved populations, as expected due to the homogenizing effects of migration. Preliminary analysis shows that there are more sites with significant divergence in the no migration populations.
-
[
International Worm Meeting,
2015]
Little is known about the relationship between the evolution of microRNAs and the mRNAs they target during gene regulation. To gain insight we have taken a genomic, bioinformatic and molecular approach to identify both unique and conserved microRNAs and their targets in the hyperdiverse nematodes, C.remanei and C.latens. These obligate outcrossing worms have genomes ~20% larger then C.elegans with close to 4000 more genes and significantly greater natural genetic variation at the population scale. To identify novel microRNAs we sequenced small RNA libraries using the Illumina platform. Between both species, we have generated over 400 million reads covering multiple developmental stages including biological replicates and both sexes as young adults. Using a combination of miRDeep2 and miRdentify we have stringently identified over 100 new microRNAs between both species for which we also have developmental profiles MicroRNAs display a wide variety of developmental patterns, with specific microRNAs displaying peak expression at each stage analyzed. Overall, expression appears to group within a small number of clustered patterns (5-6) within each species. In addition to characterizing the developmental profiles of microRNAs in these species, we are endeavoring to identify the suites of mRNAs that are targeted by individual microRNAs. To this end, we are using a in-vitro pull down assay similar to the LAMP assay developed in the Tsai lab (NAR, 2009). Using biotinylated, synthetic mature microRNAs and cell extracts we have been able to sequence RNA libraries from streptavidin bead pull downs. Utilizing both wild type and Argonaute mutant extracts from C.elegans in addition to microRNAs with normal and mutated seed sequence we hope to identify mRNA targets for any microRNA. Additionally, we have made peptide derived polyclonal antiobodies against the C.remanei ALG-1 protein and will present data on the efficacy of these antibodies for pulling down the RISC complex for a more comprehensive analysis of microRNA function.
-
[
International Worm Meeting,
2015]
C. elegans is a bacteriovore capable of consuming a variety of bacterial species. When presented a choice between bacterial foods C. elegans exhibits dietary preferences that generally correlate with the ability to support fast developmental rates. These preferred foods have therefore been described as "good quality" foods. However, it is not clear how well these foods mimic the worms' natural diet, nor whether these models are relevant to human diets for health research. Common lab practices skew experimental conditions even further to extremes by exposing worms to a vast excess of food which can have unaddressed consequences on their metabolism. Our goal is to use different bacterial strains of variable quality to link C. elegans food preference with observed physiological outcomes. This dietary profile allows a better understanding on the internal calculus of resource acquisition behaviors and dietary choice as well as how resources become internally allocated. In order to create this profile we use an array of novel and traditional physiological assays that allow for fine resolution of life-history traits such as longevity, health, and reproduction.Through the integration of the Lifespan machine system developed by Stroustrup et al. and microfluidic technology we investigate the internal calculus that mediates the preferences shown by C. elegans. With Lifespan machines we automate the measurement of longevity from thousands of worms grown on foods of differing quality. By also integrating the Lifespan system with a microfluidic device that allows worms to experience and choose between different bacterial strains in a controlled environment, we have achieved a high-throughput method of food preference determination that eliminates the majority of extraneous signals. In addition, we use a series of physiological and behavioral recordings to look at how internal resources are allocated when individuals are provided different foods as adults. Physiological health is determined from the maximal pump rate of the pharynx as recorded by a microfluidic chip that measures the electrical activity given off by pharyngeal muscles. The nutritional effects on reproduction are determined via rates of egg-laying. In summation, we present the physiological profile of C. elegans grown on different bacterial diets as well as the technology used to collect such data. .
-
[
International Worm Meeting,
2019]
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 response, ranging from <72 hours to ~120 hours. We performed genome-wide mapping and epistatic analysis of the responses of 7,855 individuals spread across 72 mapping lines, identifying a number of different chromosomal regions responsible for this variation. Genetic differences for a subset of lines were confirmed using a multi-generational introgression analysis using backcrossing with selection. Overall, there is a clear genetic basis for natural variation in the response to food availability within this species.
-
[
International Worm Meeting,
2021]
Body size is a fundamental trait that drives multiple evolutionary and ecological patterns. Caenorhabditis inopinata is a fig-associated nematode that is exceptionally large relative to other members of the genus, including C. elegans. We previously showed that C. inopinata is large primarily due to postembryonic cell size expansion that occurs during the larval-to-adult transition. Here, we describe gene expression patterns in C. elegans and C. inopinata throughout this developmental period to understand the transcriptional basis of body size change. We performed RNAseq in both species across the L3, L4, and adult stages. Most genes are differentially expressed across all developmental stages, consistent with C. inopinata's divergent ecology and morphology. We also used a model comparison approach to identify orthologs with divergent dynamics across this developmental period between the two species. Notably, among such genes were two transcription factors previously shown in C. elegans to be important for body size that are regulated by the TGF-beta signaling pathway. Multiple hypodermal collagens were also observed to harbor divergent developmental dynamics across this period. C. elegans-specific ontology enrichment reveals genes with divergent developmental dynamics tend to be expressed in neurons and regulate behavior; they also include genes important for molting and body morphology. A comparison of such genes with previous C. elegans experiments reveals overlap with stress response, developmental timing, and small RNA/chromatin regulation. These results have identified candidate genes that will be further investigated to test their roles in cell size divergence and broaden our understanding of the genetic bases of body size evolution.