The red death meets the abdominal bristle: Polygenic mutation for susceptibility to a bacterial pathogen in Caenorhabditis elegans

Understanding the genetic basis of susceptibility to pathogens is an important goal of medicine and of evolutionary biology. A key first step toward understanding the genetics and evolution of any phenotypic trait is characterizing the role of mutation. However, the rate at which mutation introduces genetic variance for pathogen susceptibility in any organism is essentially unknown. Here, we quantify the per‐generation input of genetic variance by mutation (VM) for susceptibility of Caenorhabditis elegans to the pathogenic bacterium Pseudomonas aeruginosa (defined as the median time of death, LT50). VM for LT50 is slightly less than VM for a variety of life‐history and morphological traits in this strain of C. elegans, but is well within the range of reported values in a variety of organisms. Mean LT50 did not change significantly over 250 generations of mutation accumulation. Comparison of VM to the standing genetic variance (VG) implies a strength of selection against new mutations of a few tenths of a percent. These results suggest that the substantial standing genetic variation for susceptibility of C. elegans to P. aeruginosa can be explained by polygenic mutation coupled with purifying selection.     

Short-term heritable variation overwhelms 200 generations of mutational variance for metabolic traits in Caenorhabditis elegans

Metabolic disorders have a large heritable component, and have increased markedly in human populations over the past few generations. Genome-wide association studies of metabolic traits typically find a substantial unexplained fraction of total heritability, suggesting an important role of spontaneous mutation. An alternative explanation is that epigenetic effects contribute significantly to the heritable variation. Here, we report a study designed to quantify the cumulative effects of spontaneous mutation on adenosine metabolism in the nematode Caenorhabditis elegans, including both the activity and concentration of two metabolic enzymes and the standing pools of their associated metabolites. The only prior studies on the effects of mutation on metabolic enzyme activity, in Drosophila melanogaster, found that total enzyme activity presents a mutational target similar to that of morphological and life-history traits. However, those studies were not designed to account for short-term heritable effects. We find that the short-term heritable variance for most traits is of similar magnitude as the variance among MA lines. This result suggests that the potential heritable effects of epigenetic variation in metabolic disease warrant additional scrutiny.     

Rapid fitness recovery in mutationally degraded lines of Caenorhabditis elegans

Abstract Deleterious mutation accumulation has been implicated in many biological phenomena and as a potentially significant threat to human health and the persistence of small populations. The vast majority of mutations with effects on fitness are known to be deleterious in a given environment, and their accumulation results in mean population fitness decline. However, whether populations are capable of recovering from negative effects of prolonged genetic bottlenecks via beneficial or compensatory mutation accumulation has not previously been tested. To address this question, long-term mutation-accumulation lines of the nematode Caenorhabditis elegans , previously propagated as single individuals each generation, were maintained in large population sizes under competitive conditions. Fitness assays of these lines and comparison to parallel mutation-accumulation lines and the ancestral control show that, while the process of fitness restoration was incomplete for some lines, full recovery of mean fitness was achieved in fewer than 80 generations. Several lines of evidence indicate that this fitness restoration was at least partially driven by compensatory mutation accumulation rather than a result of a generic form of laboratory adaptation. This surprising result has broad implications for the influence of the mutational process on many issues in evolutionary and conservation biology.     

A mutational analysis of Caenorhabditis elegans in space

The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.     

Variation in pleiotropy and the mutational underpinnings of the G-matrix

The pattern and extent of pleiotropic gene action can contribute substantially to the internal structure and shape of the additive genetic variance-covariance matrix (G)--a key determinant of evolutionary trajectories. We use data from our study (Estes et al. 2004) on the univariate effects of mutation in a mismatch-repair-defective strain, msh-2, of Caenorhabditis elegans to address the impact of increasing levels of selection on the magnitude and pattern of genetic covariance due to new mutations. Mutational covariances between three life-history traits are shown to exhibit a weak pattern of decline with increasing population size (increasing selection), while the orientation of mutational matrices remains reasonably constant. This suggests that mutations with smaller effects on fitness may tend to be slightly more confined in their influence than large-effect mutations (i.e., small-effect mutations reduce the magnitude of covariation between characters), but do not change the direction of this covariation.     

The fitness effects of spontaneous mutations in Caenorhabditis elegans

Abstract. Spontaneous mutation to mildly deleterious alleles has emerged as a potentially unifying component of a variety of observations in evolutionary genetics and molecular evolution. However, the biological significance of hypotheses based on mildly deleterious mutation depends critically on the rate at which new mutations arise and on their average effects. A long-term mutation-accumulation experiment with replicate lines of the nematode Caenorhabditis elegans maintained by single-progeny descent indicates that recurrent spontaneous mutation causes approximately 0.1% decline in fitness per generation, which is about an order of magnitude less than that suggested by previous studies with Drosophila . Two rather different approaches, Bateman-Mukai and maximum likelihood, suggest that this observation, along with the observed rate of increase in the variance of fitness among lines, is consistent with a genomic deleterious mutation rate for fitness of approximately 0.03 per generation and with an average homozygous effect of approximately 12%. The distribution of mutational effects for fitness appears to have a relatively low coefficient of variation, being no more extreme than expected for a negative exponential, and for one composite fitness measure (total progeny production) approaches constancy of effects. These results are derived from assays in a benign environment. At stressful temperatures, estimates of the genomic deleterious mutation rate (for genes expressed at such temperatures) is sixfold lower, whereas those for the average homozygous effect is approximately eightfold higher. Our results are reasonably compatible with existing estimates for flies, when one considers the differences between these species in the number of germ-line cell divisions per generation and the magnitude of transposable element activity.     

Spontaneous mutational variation for body size in Caenorhabditis elegans

We measured the impact of new mutations on genetic variation for body size in two independent sets of C. elegans spontaneous mutation-accumulation (MA) lines, derived from the N2 strain, that had been maintained by selfing for 60 or 152 generations. The two sets of lines gave broadly consistent results. The change of among-line genetic variation between cryopreserved controls and the MA lines implied that broad sense heritability increased by 0.4% per generation. Overall, MA reduced mean body size by approximately 0.1% per generation. The genome-wide rate for mutations with detectable effects on size was estimated to be approximately 0.0025 per haploid genome per generation, and their mean effects were approximately 20%. The proportion of mutations that increase body size was estimated by maximum likelihood to be no more than 20%, suggesting that the amount of mutational variation available for selection for increased size could be quite small. This hypothesis was supported by an artificial selection experiment on adult body size, started from a single highly inbred N2 individual. We observed a strongly asymmetrical response to selection of a magnitude consistent with the input of mutational variance observed in the MA experiment.     

The fine structure of Caenorhabditis elegans N-glycans

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.     

Selection against males in Caenorhabditis elegans under two mutational treatments

Within populations with mixed mating systems, selfing is expected to be favoured over outcrossing unless a countervailing process such as severe inbreeding depression is present. In this study, we consider the relationship between the expression of deleterious alleles and the maintenance of outcrossing in the nematode species, Caenorhabditis elegans. This species is characterized by an androdioecious breeding system composed of males at low frequency and self-fertilizing hermaphrodites that can only outcross via males. Here, we find that experimentally increasing the mutational load in four different isogenic wild isolates using 10 generations of Ethylmethane sulphonate (EMS) and UV irradiation mutagenesis significantly diminishes the cost of males. Males are maintained at higher frequencies in mutagenized versus non-mutagenized populations. Nevertheless, males still tend to be driven to low frequencies within isolates that are known to be prone to lose males. Further, we determine the viability effects of a single round of mutagen exposure and find that, for EMS, outcrossing overcomes the almost completely recessive and nearly lethal effects generated. We briefly interpret our results in light of current evolutionary theory of outcrossing rates.     

Developmental regulation of energy metabolism in Caenorhabditis elegans

Changes in energy metabolism during larval development in Caenorhabditis elegans have been investigated using phosphorus nuclear magnetic resonance (31P NMR). The relative concentrations of ATP, ADP, AMP, sugar phosphates, and other metabolites were observed to change during larval development, producing stage-specific spectra. These spectra are consistent with enzyme assays for isocitrate dehydrogenase and isocitrate lyase, indicating that high activity of the glyoxylate pathway during embryonic development decreases during the first larval (L1) stage, and respiration during the L2, L3, and L4 stages occurs preferentially through the TCA cycle. Metabolic strategies were further studied using mutants that are predisposed to enter the dauer stage, a developmentally arrested third-stage larva formed under conditions of overcrowding and limited food. After the L1 molt, energy metabolism in animals destined to become dauer larvae diverges from that of animals committed to growth. Relative to the L1, the L2 larvae committed to growth exhibit increased isocitrate dehydrogenase activity as well as increases in ATP and other high-energy phosphates, but predauer (L2d) larvae exhibit declining enzyme activities and declining levels of high-energy phosphates. The predominant phosphorus NMR signal in dauer larva extracts corresponds to inorganic phosphate. We conclude that metabolism is regulated during C. elegans larval development, with a major transition apparent after the L1 stage. This transition does not occur in larvae destined to form dauer larvae.     

Transposable elements, mutational correlations, and population divergence in Caenorhabditis elegans

Transposable element activity is thought to be responsible for a large portion of all mutations, but its influence on the evolution of populations has not been well studied. Using mutation accumulation experiments with the nematode Caenorhabditis elegans, we investigated the impact of transposable element activity on the production of mutational variances and covariances. The experiments involved the use of two mutator strains (RNAi-deficient mutants) that are characterized by high levels of germline transposition, as well as the Bristol N2 strain, which lacks germline transposition. We found that transposition led to an increase in mutational heritabilities, as well as to the intensification of correlation patterns observed in the absence of transposition. No mutational trade-offs were detected and mutations generally had a deleterious effect on components of fitness. We also tested whether the pattern of mutational covariation could be used to predict observed patterns of population divergence in this species. Using 15 natural populations, we found that population divergence of C. elegans in multivariate phenotypic space occurred in directions only partially concordant with mutation, and thus other evolutionary factors, such as natural selection and genetic drift, must be acting to produce divergence within this species. Our results suggest that mutations induced by mobile elements in C. elegans are similar to other spontaneous mutations with respect to their contribution to the microevolution of quantitative traits.     

Mechanisms of aging and energy metabolism in Caenorhabditis elegans

Aging studies on diverse species ranging from yeast to man have culminated in the delineation of several signaling pathways that influence the process of senescent decline and aging. While understanding these interlinked signal-transduction cascades is becoming even more detailed and comprehensive, the cellular and biochemical processes they impinge upon to modulate the rate of senescent decline and aging have lagged considerably behind. This fundamental question is one of the most important challenges of modern aging research and has been the focus of recent research efforts. Emerging findings provide insight into the facets of cellular metabolism which can be fine-tuned by upstream signaling events to ultimately promote longevity. Here, we survey the mechanisms regulating aging in the simple nematode worm Caenorhabditis elegans, aiming to highlight recent discoveries that shed light into the interface between aging signaling pathways and cellular energy metabolism. Our objective is to review the current understanding of the processes involved and discuss mechanisms that are likely conserved in higher organisms.     

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii

Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best‐fitting mutational effects models were highly dependent on the genetic background of the ancestral strain.     

Spontaneous mutational correlations for life-history, morphological and behavioral characters in Caenorhabditis elegans

The pattern of mutational covariance among traits plays a central, but largely untested, role in many theories in evolutionary genetics. Here we estimate the pattern of phenotypic, environmental, and mutational correlations for a set of life-history, behavioral, and morphological traits using 67 self-fertilizing lines of Caenorhabditis elegans, each having independently experienced an average of 370 generations of spontaneous mutation accumulation. Bivariate relationships of mutational effects indicate the existence of extensive pleiotropy. We find that mutations may tend to produce manifold effects on suites of functionally related traits; however, our data do not support the idea of completely parcelated pleiotropy, in which functional units are separately affected by mutations. Positive net phenotypic and mutational correlations are common for life-history traits, with environmental correlations being comparatively smaller and of the same sign for most pairs of traits. Observed mutational correlations are shown to be higher than those produced by the chance accumulation of nonpleiotropic mutations in the same lines.     

Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditis elegans

Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol,defects in which may lead to hyperargininemia,a devastating developmental disorder.It is largely unknown if defective arginine catabolism has any effects on mitochondria.Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditis elegans.Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate(ATP) production in C elegans hypodermal cells.ARGN-1 localizes to mitochondria and its loss causes arginine accumulation,which disrupts mitochondrial dynamics.Heterologous expression of human ARGl or ARG2 rescued the mitochondrial defects of argn-1 mutants.Importantly,genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations.These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.     

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.     

SPONTANEOUS MUTATION ACCUMULATION IN MULTIPLE STRAINS OF THE GREEN ALGA,CHLAMYDOMONAS REINHARDTII

Estimates of mutational parameters, such as the average fitness effect of a new mutation and the rate at which new genetic variation for fitness is created by mutation, are important for the understanding of many biological processes. However, the causes of interspecific variation in mutational parameters and the extent to which they vary within species remain largely unknown. We maintained multiple strains of the unicellular eukaryote Chlamydomonas reinhardtii, for approximately 1000 generations under relaxed selection by transferring a single cell every ～10 generations. Mean fitness of the lines tended to decline with generations of mutation accumulation whereas mutational variance increased. We did not find any evidence for differences among strains in any of the mutational parameters estimated. The overall change in mean fitness per cell division and rate of input of mutational variance per cell division were more similar to values observed in multicellular organisms than to those in other single‐celled microbes. However, after taking into account differences in genome size among species, estimates from multicellular organisms and microbes, including our new estimates from C. reinhardtii, become substantially more similar. Thus, we suggest that variation in genome size is an important determinant of interspecific variation in mutational parameters.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Lysosome Inhibition Reduces Basal and Nutrient-Induced Fat Accumulation in Caenorhabditis elegans

A long-term energy nutritional imbalance fundamentally causes the development of obesity and associated fat accumulation. Lysosomes, as nutrient-sensing and lipophagy centers, critically control cellular lipid catabolism in response to nutrient deprivation. However, whether lysosome activity is directly involved in nutrient-induced fat accumulation remains unclear. In this study, worm fat accumulation was induced by 1 mM glucose or 0.02 mM palmitic acid supplementation. Along with the elevation of fat accumulation, lysosomal number and acidification were also increased, suggesting that lysosome activity might be correlated with nutrient-induced fat deposition in Caenorhabditis elegans. Furthermore, treatments with the lysosomal inhibitors chloroquine and leupeptin significantly reduced basal and nutrient-induced fat accumulation in C. elegans. The knockdown of hlh-30, which is a critical gene in lysosomal biogenesis, also resulted in worm fat loss. Finally, the mutation of aak-2, daf-15, and rsks-1 showed that mTORC1 (mechanistic target of rapamycin complex-1) signaling mediated the effects of lysosomes on basal and nutrient-induced fat accumulation in C. elegans. Overall, this study reveals the previously undescribed role of lysosomes in overnutrition sensing, suggesting a new strategy for controlling body fat accumulation.