Biomechanical analysis of gait adaptation in the nematode Caenorhabditis elegans

To navigate different environments, an animal must be able to adapt its locomotory gait to its physical surroundings. The nematode Caenorhabditis elegans, between swimming in water and crawling on surfaces, adapts its locomotory gait to surroundings that impose approximately 10,000-fold differences in mechanical resistance. Here we investigate this feat by studying the undulatory movements of C. elegans in Newtonian fluids spanning nearly five orders of magnitude in viscosity. In these fluids, the worm undulatory gait varies continuously with changes in external load: As load increases, both wavelength and frequency of undulation decrease. We also quantify the internal viscoelastic properties of the worm's body and their role in locomotory dynamics. We incorporate muscle activity, internal load, and external load into a biomechanical model of locomotion and show that (i) muscle power is nearly constant across changes in locomotory gait, and (ii) the onset of gait adaptation occurs as external load becomes comparable to internal load. During the swimming gait, which is evoked by small external loads, muscle power is primarily devoted to bending the worm's elastic body. During the crawling gait, evoked by large external loads, comparable muscle power is used to drive the external load and the elastic body. Our results suggest that C. elegans locomotory gait continuously adapts to external mechanical load in order to maintain propulsive thrust.     

Transposition of Tc1 in the nematode Caenorhabditis elegans.

We have identified a strain of Caenorhabditis elegans in which the transposable element Tc1 is genetically active. Most spontaneous mutations affecting the unc-54 myosin heavy chain gene of C. elegans variety Bergerac are due to insertions of Tc1 within unc-54. The Bergerac genome contains an unusually high number of Tc1 elements, but this is not responsible for transpositional activity. Another variety of C. elegans, strain DH424, contains an equally high number of Tc1 elements, but transpositions are not detected. Tc1 insertion mutations are genetically unstable. They revert to unc-54+ in both germ-line and somatic cells. Germ-line revertants are wild type and contain precise or nearly precise excisions of Tc1. Somatic revertants are genetic mosaics; they contain small patches of revertant muscle tissue in otherwise mutant animals. The pattern of mosaicism often allows us to know when and where during muscle development the excisions occur. Somatic reversion can be over 1000-fold more frequent than germ-line reversion.     

Nutritional alteration of life span in the nematode Caenorhabditis elegans

The longevity of the free-living nematode Caenorhabditis elegans was studied under two different nutritional regimes, one axenic and the other monoxenic. Axenic nematodes showed typical sigmoidal survival curves with exceptionally long tailing. Monoxenic worms died off much faster and the maximum life-span in bacterial culture was generally three to four times shorter than that obtained in axenic culture. When nematodes were transferred from axenic to monoxenic culture and vice versa at near adulthood the survival patterns observed were reminiscent of the final medium. These results are in agreement with the hypothesis that worms may die off prematurely in bacterial culture by toxins given off by the bacteria.     

Effects of vitamin E on the nematode Caenorhabditis elegans

Vitamin E at 200 μg/ml significantly extended the mean lifespan and extended maximum lifespan of the nematode Caenorhabditis elegans when supplied early in the prereproductive stage. At this concentration, vitamin E increased growth, but did not affect fecundity or the length of the reproductive period. The vitamin E effect was not passed from the parents to the progeny. Evaluations of the effects of vitamin E on lipofuscin accumulation were inconclusive. The results are compared to previous studies on C. briggsae and Turbatrix aceti.     

Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.

When grown at a temperature from 16 degrees to 25 degrees and placed on a thermal gradient, the nematode Caenorhabditis elegans migrates to its growth temperature and then moves isothermally. Behavioral adaptation to a new temperature takes several hours. Starved animals, in contrast, disperse from the growth temperature. Several mutants selected for chemotaxis defects have thermotaxis defects as well; these behaviors depend on some common gene products. New mutants selected directly for thermotaxis defects have unusual phenotypes which suggest mechanisms for thermotaxis.     

The genetics of caloric restriction in Caenorhabditis elegans

Low caloric intake (caloric restriction) can lengthen the life span of a wide range of animals and possibly even of humans. To understand better how caloric restriction lengthens life span, we used genetic methods and criteria to investigate its mechanism of action in the nematode Caenorhabditis elegans. Mutations in many genes (eat genes) result in partial starvation of the worm by disrupting the function of the pharynx, the feeding organ. We found that most eat mutations significantly lengthen life span (by up to 50%). In C. elegans, mutations in a number of other genes that can extend life span have been found. Two genetically distinct mechanisms of life span extension are known: a mechanism involving genes that regulate dauer formation (age-1, daf-2, daf-16, and daf-28) and a mechanism involving genes that affect the rate of development and behavior (clk-1, clk-2, clk-3, and gro-1). We find that the long life of eat-2 mutants does not require the activity of DAF-16 and that eat-2; daf-2 double mutants live even longer than extremely long-lived daf-2 mutants. These findings demonstrate that food restriction lengthens life span by a mechanism distinct from that of dauer-formation mutants. In contrast, we find that food restriction does not further increase the life span of long-lived clk-1 mutants, suggesting that clk-1 and caloric restriction affect similar processes.     

Evidence of a mate-finding cue in the hermaphrodite nematode Caenorhabditis elegans

When males of the roundworm Caenorhabditis elegans come into association with their hermaphroditic counterparts they cease foraging behavior and begin to mate. Here we detail several assays used to demonstrate that a diffusible cue is correlated with this process. This cue is sexually dimorphic, given off only by the hermaphrodite and eliciting a response only in the male. Males are attracted to, reverse direction of movement frequently, and remain in regions of agar conditioned with hermaphrodites. From our studies we suggest a form of kinesis that works by attracting males to their mating partners from a distance and functions, once males arrive, in holding attracted males in close proximity. The hermaphrodite vulva is not required for the cue. Males from general sensory mutants osm-5 and osm-6 fail to respond to the cue, whereas male-specific mutants lov-1 and pkd-2 respond. Finally, that males from multiple isolates of C. elegans also respond similarly to this cue indicates that this cue is robust and has been maintained during recent evolution.     

The longevity effect of dietary restriction in Caenorhabditis elegans

The nematode Caenorhabditis elegans has been subjected to DR by food (Escherichia coli) dilution, growth in axenic medium and using animals having defects in feeding behavior or in specific nutrient transporter proteins. There is evidence that DR causes increased resistance against environmental stressors but no decrease of metabolic rate. The insulin/IGF-1 signaling pathway does not mediate the longevity effect of DR in this species, but TOR signaling may be involved. The metabolic stability-longevity theory offers a plausible explanation of the longevity effect of DR but needs experimental validation.     

Cell lineages of the embryo of the nematode Caenorhabditis elegans.

Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.     

A second trans-spliced RNA leader sequence in the nematode Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the 22-nucleotide RNA sequence called the spliced leader (SL) is trans-spliced from the 100-nucleotide-long SL RNA to some mRNAs. We have identified a trans-spliced leader (SL2) whose sequence differs from that of the original spliced leader (SL1), although both are 22 nucleotides long. By primer-extension sequencing, SL2 but not SL1 was shown to be present at the 5' end of the mRNA encoded by one of the four glyceraldehyde-3-phosphate dehydrogenase genes. The other three glyceraldehyde-3-phosphate dehydrogenase genes encode mRNAs that have the SL1 but not the SL2 sequence at their 5' ends. Therefore, the trans-splicing process can discriminate the transfer of SL1 from that of SL2 in a gene-specific manner.     

Extrachromosomal copies of transposon Tc1 in the nematode Caenorhabditis elegans.

Extrachromosomal copies of the 1.6-kilobase transposable element Tc1 are present at the level of between 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Bergerac. Extrachromosomal elements were detected and studied using Southern hybridizations employing a Tc1-specific probe. The amount of extrachromosomal Tc1 DNA was roughly constant during development in Bergerac, which has approximately 300 integrated chromosomal copies of Tc1 in its haploid genome. Extrachromosomal Tc1 DNA was not detected in strain Bristol, which has 30 chromosomal copies of Tc1. Three forms of extrachromosomal DNA were detected. The predominant form was a 1.6-kilobase linear molecule with ends corresponding to the ends of an integrated Tc1 element. The other two forms were, respectively, relaxed and supercoiled circular copies of the element. Structural assignments were based on electrophoretic mobility, the results of sedimentation velocity and equilibrium density gradient experiments, and on the sizes of the products produced by treatment of purified extrachromosomal DNA with restriction endonucleases. The suggestion is made that these extrachromosomal transposable elements are the products of excision events known to be occurring at high frequency in somatic cells in Bergerac.     

Toward a physical map of the genome of the nematode Caenorhabditis elegans

A technique for digital characterization and comparison of DNA fragments, using restriction enzymes, is described. The technique is being applied to fragments from the nematode Caenorhabditis elegans (i) to facilitate cross-indexing of clones emanating from different laboratories and (ii) to construct a physical map of the genome. Eight hundred sixty clusters of clones, from 35 to 350 kilobases long and totaling about 60% of the genome, have been characterized.     

A selection for myosin heavy chain mutants in the nematode Caenorhabditis elegans.

The unc-54 gene of Caenorhabditis elegans encodes an abundant myosin heavy chain protein expressed in body-wall muscle cells. We have designed genetic techniques that select directly for unc-54 mutants. This selection is based upon properties of the unc-54 dominant allele e1152. Mutations that eliminate dominance of e1152 are null alleles of unc-54. Deletions have been identified by their genetic properties. We have defined mutationally a number of essential genes near unc-54, and we have described the genetic fine structure of this region of linkage group I. As much as 27% of the unc-54 mutations induced by the bifunctional alkylating agent 1,2,7,8-diepoxyoctane are multisite deletions. Extrachromosomal free duplications that include unc-54 are also described.     

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions

The nematode Caenorhabditis elegans was cultured monoxenically with E. coli as a food source and the influence of the bacterial growth conditions on the life span was studied. When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematode's life span tended to be prolonged without a marked effect on postembryonic development. The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C. elegans) rather than during the larval to young adult stage. Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.     

Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes

In Caenorhabditis elegans, several manipulations that affect nutrition slow development, reduce fecundity, and increase life span. These are viewed as dietary restriction (DR) and include culture in semidefined, nutrient-rich liquid medium that is axenic (i.e., there is no microbial food source). Here we describe convenient ways to exert DR by culture on agar plates containing axenic medium. We used these to explore whether effects of axenic culture really reflect DR. Our results imply that major nutrient components of axenic medium, and overall caloric content, are not limiting for life span. However, adding growth-arrested Escherichia coli as an additional food source rescued the effects of axenic culture. We then sought to identify the component of E. coli that is critical for normal C. elegans nutrition using add-back experiments. Our results suggest that C. elegans has a nutritional requirement for live, metabolically active microbes or, possibly, an unidentified, heat-labile, nonsoluble component present in live microbes.     

Body size,insulin/IGF signaling and aging in the nematode Caenorhabditis elegans

A number of recent studies of aging in Drosophila, mice and dogs have shown an association between reduced body size and increased lifespan. It is unclear (a) whether such an association is a general feature of animal species; and (b) whether the association reflects an effect of body size on aging, or pleiotropic effects of common determinants of growth and aging. To address these issues, we have studied the relationship between size and lifespan in the nematode Caenorhabditis elegans, and surveyed related findings in Drosophila. In C. elegans, we compared 12 wild isolates with varying body size and lifespan, but saw no correspondence between these traits. We also examined aging in giant and dwarf mutants, but observed only reduced lifespan in all cases. In a comparison of 15 long-lived daf-2 insulin/IGF receptor mutants, we saw a positive correlation between body length and lifespan, and up to a 28% increase in daf-2 mutant body volume. Thus, in C. elegans, insulin/IGF signaling may limit growth rather than promote it. Studies of Drosophila show no consistent correlation between body size and lifespan. These results indicate that the negative correlation between body size and lifespan seen in some mammals is not typical of invertebrates, but support the view that co-variation of size and longevity may occur via effects on insulin/IGF signaling.