Rand JB et al. (1982) Worm Breeder's Gazette "wild and wooly strains."

Russell RL, Rand JB, Johnson CD

[Worm Breeder's Gazette, 1982]

During the last 10 years, our lab has been accumulating wild isolates of nematodes from soil samples. We have sent some of them to a number of labs, and we are now sending all of them to the CGC for distribution to one and all. We expect that these will be of use to those people interested in enzyme polymorphisms, DNA polymorphisms, developmental and behavioral variants, and dysgenesis, to say nothing of population genetics and nematode taxonomy. Knowledge about many of these strains is sketchy, but what we do know is summarized below. [See Figure 1] The following strains are known to be interfertile with C. elegans ( Bristol): CL2a, all the GA strains, HA8, all the PA strains, and PaC1. The following strains have been tested for interfertility with N2 males without apparent success: FU2, FU4, HA1-7, PG1-3. All of the strains are monoecious. None of the strains has (to our knowledge) been tested for interfertility with C. briggsae. All strains from any one location (i.e., bearing the same letter designation) cannot be guaranteed to be independent of each other, although some of them probably are. It is also remotely possible that some of the GA strains may, in fact, be laboratory strains which Carl inadvertently brought home to his garden. (However, Scott Emmons has data showing independence of at least several of the GA strains.) We request that anyone obtaining data (protein, DNA, behavioral, etc.) bearing on the independence or lack of same for any strains send their conclusions to the CGC, so that other users of these strains can design their experiments accordingly.

(1976) Worm Breeder's Gazette "levamisole-resistant mutants with a wild type visible phenotype."

[Worm Breeder's Gazette, 1976]

As described by Brenner, a large fraction of levamisole-resistant mutants are worms with a wild type visible phenotype when grown on NG plates. We find that most of these mutants grown in the presence of levamisole acquire the peculiar uncoordinated phenotype characteristic of the levamisole-resistant uncs. In our cut worm assay, several of these 'wild type' mutant strains showed near wild type susceptibility to all drugs tested (including levamisole) when grown in the absence of levamisole. Grown and tested in the presence of levamisole, the worms showed extreme resistance to cholinergic agonists but sensitivity to the muscle contracting agent ouabain. The latter pharmacological phenotype is characteristic of levamisole-resistant uncs and N2 wild type worms treated with cholinergic blocking agents. As we mentioned previously, levamisole also acts as an agonist on naive worms and is blocked by cholinergic antagonists. From these observations, we infer that levamisole is a cholinergic depolarizing blocking agent. We believe that mutants with a normal visible phenotype have less of and/or less functional acetylcholine receptors than the wild type-but still workable receptors. Possibly, levamisole depolarizes in binding to the Ac receptor but once bound, has a blocking effect. N2 wild type worms grown on levamisole would always have a significant amount of muscle-depolarization due to constant dissociation and reassociation of levamisole with the receptor, whereas the wild type mutants would have an insignificant absolute amount of receptor undergoing this flux-hence only the blocking effect is seen after recovery from initial exposure to the drug. By far the largest fraction and the most resistant 'wild type' mutants fall into a gene probably corresponding to tmr-1 described by Brenner. Very rare mutants in this gene are levamisole-resistant uncs when homozygous and semi-dominant in the growth test on levamisole! Most of the other wild type mutants appear to be leaky alleles of the levamisole-resistant unc loci. A couple of rare birds are sex-linked and apparently sexually dimorphic. Four rather different mutants have been isolated with close to wild type visible phenotype and extreme resistance to levamisole even when grown in the absence of the drug. In the cut worm assay they show extreme resistance to levamisole and varying susceptibility to other cholinergic agonists (unfortunately not a wild type susceptibility to acetylcholine). These might be mutants having the minimal amount of functional Ac receptor necessary for a wild type visible phenotype; we prefer to think that they are acetylcholine binding protein mutants. Three of the four mutants are in unc-29, LG I.

Anderson P (1984) Worm Breeder's Gazette "collecting wild isolates of C elegans."

Anderson P

[Worm Breeder's Gazette, 1984]

We have discovered a simple and convenient procedure for obtaining wild isolates of C. elegans. Many attempts to isolate wild-type strains by spreading soil samples on petri dishes were unsuccessful, but the following procedure yielded several isolates quickly. This procedure samples relatively large volumes of soil. It is essentially the same as the 'Baermann tray method' described in Methods for Evaluating Plant Fungicides, Nematicides and Bactericides, published by the American Phytopathological Society. The materials include 2 pie pans and a gauze milk filter (that's right -- a milk filter!). One pie pan is approximately 18 cm in diameter and 3.5 cm deep. The second is slightly smaller (approximately 17 cm diameter and 3 cm deep) and fits into the first; furthermore, it has a coarse wire mesh bottom (openings about 3.5 x 3.5 mm). The milk filter is approximately 19 cm in diameter and 2 mm thick. Cheesecloth might also work. Soil samples are sifted through the wire mesh pie pan and then through a 30-mesh wire sieve. This sifting removes debris and loosens the soil. The milk filter is placed over the mesh bottom of the smaller pie pan and covered with 2 facial tissues. A 50-100 ml sample of soil is spread evenly over the tissues and the smaller pan inserted into the larger one. The combination is filled with approximately 250 ml of water. This amount of liquid is sufficient to keep the soil saturated. The pan is covered with Saran Wrap and stored at room temperature for 3-7 days. Nematodes crawl through the filter and collect in the water. The nematodes can then be concentrated either by settling for several hours or by centrifugation. The worms are plated out on NGM agar and screened for those resembling C. elegans. ( Be prepared for a real zoo.) We collected soil from around the campus and isolated C. elegans from five different samples. Habitats ranged from a barnyard stock pen to a forest floor along the shores of Lake Mendoda. Southern blots show that four of these strains have a low copy number of Tc1 (similar to Bristol), while the fifth has a high copy number (similar to Bergerac).

Hodgkin JA (1988) Worm Breeder's Gazette "wild isolates and mating plug formation."

Hodgkin JA

[Worm Breeder's Gazette, 1988]

Many natural isolates of C. elegans differ from the standard laboratory strain, N2, in copulatory plug formation: the males from these strains deposit a gelatinous blob over the vulva of a mated hermaphrodite, but N2 males do not. Previously (Hodgkin, Doniach & Kenyon, WBG 8-3:36) we showed that the plugging trait, as discovered in the wild isolate Sta-5 (formerly named Cal-5) behaved as a Mendelian dominant, mapping to the locus plg-1 on LGIII. We suggested that N2 had lost the plugging trait at some point during its scientific career. A survey of natural isolates indicates that this is not the case; instead, it appears that many natural races are nonpluggers. Male stocks were established from the 24 strains listed in the table, and males were tested for plug formation. All non- plugging strains were crossed with N2, and F1 heterozygous males tested: all were negative. Thus, if non-plugging represents loss-of- function, then all these strains carry recessive mutations of the same gene, plg-1. Also, the plugging trait in two of the positive strains, AB3 and RC301, was examined genetically, by crossing with N2-derived mapping strains. In both cases the trait behaved as a dominant mapping to approximately the same location as plg-1.In almost all of these races (or at least the stocks of them presently held in Cambridge) males are present at less than 1% of the individuals in a growing population, but these males are always potent and male stocks are easily established. The only exceptions are the two Bergerac strains, RW7000 and N62, which are well known to produce males that mate very poorly. It is not obvious what advantage or disadvantage is associated with plugging. The trait is stable in culture: both plugging and non- plugging strains maintain their character over hundreds of generations.

Wild M et al. (1983) Worm Breeder's Gazette "the actin genes of Caenorhabditis elegans."

Hirsh DI, Krause MW, Wild M

[Worm Breeder's Gazette, 1983]

We have continued our study of the four nematode actin genes. The complete nucleotide sequences of the clustered genes I, II and III are now available, and most of gene IV has been sequenced. The coding regions of genes I and III are identical at the nucleotide level. When compared to genes I and III, genes II and IV have many silent substitutions. Gene II has three conservative amino acid changes; and gene IV has at least one amino acid change similar to gene II. Genes I, II and III each have two introns in identical positions; gene IV has three introns, only one of which interrupts at a position similar to the introns in the clustered genes. We are now studying actin transcription during development in wild- type worms and in revertant worms in which reversion is accompanied by DNA rearrangements of the actin cluster. When Northern blots are probed with actin DNA, a triplet of bands is seen at all developmental stages in wild-type worms. The amount of actin message increases markedly between the L2 and L4 stages. A gene I specific probe shows hybridization exclusively to the middle band of the triplet. In revertants, the number and size of the actin transcripts varies. We have used the gene I specific probe to demonstrate the absence of a stable gene I transcript in a revertant containing an insertion in gene I. The exact nature of the variations in this and other revertants is now being studied using other gene-specific probes.

Xie GF et al. (1992) Worm Breeder's Gazette "A Simple Procedure for Capturing Wild Nematodes"

Ravi L, Xie GF, Aamodt EJ

[Worm Breeder's Gazette, 1992]

Croll NA et al. (1977) Worm Breeder's Gazette "behaviour of Caenorhabditis elegans (wild type)."

Rutherford TA, Sukul NC, Smith JM, Croll NA

[Worm Breeder's Gazette, 1977]

It remains the feeling of this laboratory that very little is known of the behavior of wild type C. elegans and that detailed analysis of the behavior of individuals still provides the best method of study. We are currently analyzing the behavior of adults, 4 1/2 days + 1/2 day at 20 C as our recent study (Croll, Smith & Zuckerman, 1977, Experimental Aging Research) has shown that age is critical in determining the rates of different behavioral actions. The wave patterns of individuals is significantly altered by L-tryptophan and D- tryptophan (5mM), an approach derived from Dusenbery (J. Exp. Biol. 1975, 193; 413). The patterns are further significantly affected by small temperature changes from the eccritic response. As well as any directional component attributed to chemical or thermal gradients, we are now considering them to be important 'behavior modifiers' in non- directional environments. A full description of defecation has been completed which supports our earlier contention that defecation is a feature of the 'feeding phase' and that its rate is dependent upon an endogenous signal(s) and not upon the rate of ingestion. When not ingesting rapidly there is a spontaneous 'pseudocrap' in which faeces are not voided! Further details (J. Zool. Lon.) can be provided of this study now in press upon request. C. elegans adults respond very strongly to electric currents and potentials between .01-.06 mA and 1.0 to 3.3 V/cm. This is now being investigated with thresholds, habituation phenomena and feeding factors being included. This response could provide a new set of mutants for those in the business of making them. Worms can go either towards the cathode or anode depending upon the current strength. We would like to develop this investigation to see if certain amphid-defective, chemoreceptive-defective mutants had also lost their galvanotaxes - any offers?

Fodor A et al. (1982) Worm Breeder's Gazette "discovery of a wild-type Caenorhabditis briggsae (var Gujarat)."

Fodor A, Riddle DL, Nelson K, Golden JW

[Worm Breeder's Gazette, 1982]

A hermaphroditic nematode strain isolated from the soil (by A. F.) in the State of Gujarat, India, has been identified as Caenorhabditis briggsae by genetic and morphological criteria. The Dougherty strain of C. briggsae, which has been cultured axenically in various laboratories since 1954, was obtained by the CGC from Dr. Bert Zuckerman. No other sub-lines of C. briggsae are known to exist (see Friedman, Platzer and Eby, 1977, J. Nematol. 9:198). A male stock of the Gujarat strain was established and used for mating tests, genetic analysis and comparisons of male copulatory bursa morphology together with N2 males and heat-shock-generated Zuckerman males. ( Historically, the number and arrangement of bursal rays has been the basis for species classification in this genus.) The Gujarat males mate very well with the Zuckerman strain and not at all with C. elegans N2. Conversely, N2 males fail to mate with either Gujarat or Zuckerman hermaphrodites. Examination of bursal ray patterns confirms the conclusion that the Gujarat strain is C. briggsae. In general, the behavior, appearance, and growth properties of the Gujarat strain are very similar to C. elegans strain N2. However, both of the C. briggsae strains grow and reproduce well at 27.5 C while N2 does not. The Gujarat generation time from egg to egg is about 37 hrs. at 27.5 C, and about 56 hrs. at 20 C. The Zuckerman C. briggsae strain differs markedly from both Gujarat and N2 in many respects. The Zuckerman strain is variably uncoordinated, it exhibits chemotaxis defects, it is dauer-defective the hermaphrodites produce relatively small broods (140 + 18 is the best at 22.5 C in comparison with 238 + 25 of Gujaratat the same temperature), spontaneous males will not mate but instead crawl off the plate, and the male bursa is abnormally small with stunted rays. Bursa size and ray length of Gujarat males are the same as N2, but 25% greater than Zuckerman males. Gujarat/Zuckerman hybrid males possess a bursa indistinguishable from Gujarat itself, so we conclude that this Zuckerman trait is autosomal and recessive. However, the hybrid males are uncoordinated, non-chemotactic, and dauer-defective. The dauer-defective phenotype can be scored as a failure to form dauer larvae in response to the Caenorhabditis 'crowding' pheromone (Golden and Riddle, 1981, C. elegans Meeting Abstracts). These traits are all X-linked, recessive, and may represent pleiotropic effects of a single mutation. An attempt was made to induce revertants of the Zuckerman dauer-defective trait with EMS, but no revertants were found in a screen of almost 10+E5 mutagenized chromosomes. This leaves open the possibility that the X-linked defect may be a non-revertible, multi-site mutation of some sort. What does all this mean? Perhaps not much, but we believe this may be a particularly grandiose example of evolution (or de-evolution) in the laboratory. The Dougherty strain of C. briggsae was reported in 1969 to form dauer larvae (Yarwood and Hansen, 1969, J. Nematol. 1:184), but the strain we have today forms dauers at very low frequency. We are tempted to speculate that prolonged liquid axenic cultivation may have actually favored the type of genetic variation in sensory behavior, movement and dauer formation that we have observed in the Zuckerman strain. Interestingly, the Zuckerman strain does not exhibit uncoordinated movement in liquid, but it seems to thrash about more slowly than N2 or Gujarat. Slower movement also might be an advantage in liquid suspension culture. Does cultivation on agar plates spread with E. coli also select particular types of variation from 'true wild-type'? Well, our N2 stock is not noticeably different in growth or behavior from the recently-isolated Gujarat C. briggsae strain, with one interesting exception. The exception involves sensitivity to the dauer larva pheromone. All three strains produce the Caenorhabditis specific 'crowding' pheromone. While the Zuckerman strain does not respond to the pheromone (it is dauer-defective), the Gujarat strain responds to even lower concentrations of pheromone than does N2. Presumably as a consequence of its pheromone sensitivity, the Gujarat strain actually forms some dauers prior to starvation on NGM plates. If N2 had such a characteristic when it was first cultured on E. coli, it almost certainly would have been at a selective disadvantage to spontaneous genetic variants less sensitive to the pheromone.

Xie GF et al. (1992) Worm Breeder's Gazette "Esterase Expression Patterns in Wild Nematode Species"

Ravi L, Xie GF, Aamodt EJ

[Worm Breeder's Gazette, 1992]

Beitel GJ et al. (1990) Worm Breeder's Gazette "lin-34/let-60: dominant Muv mutations increase wild-type gene activity, dominant Vul mutations antagonize wild-type gene activity."

Beitel GJ, Clark SG, Horvitz HR

[Worm Breeder's Gazette, 1990]

lin-34 mutations can cause a dominant Multivulva (Muv) phenotype, while let-60 mutations can cause dominant Vulvaless (Vul), recessive lethal or recessive Vul/lethal phenotypes. lin-34 and let-60 are likely to be the same gene because they map very close together and the tightly linked lin-34(n1046) suppressor n1981 is a recessive lethal mutation that fails to complement let-60 mutations. Preliminary molecular data are consistent with this interpretation ( Min Han and Paul Sternberg, personal communication; also our unpublished observations). We investigated the nature of the dominant Muv and dominant Vul alleles using a new duplication of chromosome IV, nDp5. Isolated by Saechin Kim, nDp5 appears to be a free duplication that spans lin-34/let-60 and extends from at least unc-44 to unc-26, covered. Using this duplication and a deficiency that spans the lin-34/let-60 region, we constructed strains containing varying doses of the wild-type and mutant alleles. As seen in the table below, the dominant Muv mutations n1046 and n1700 show an increasingly severe phenotype with increasing copies of the wild-type gene. This result suggests that these mutations cause an increase in a wild-type-like gene activity and are therefore hypermorphic mutations. The dominant Vul mutations n1631 and n2031 seem to antagonize the wild-type gene activity and thus are antimorphic mutations. However, the dominant Vul mutations are not able to antagonize the activity of the dominant Muv mutations, since dominant Muv/dominant Vul heterozygotes have the same phenotype as dominant Muv/+ heterozygotes. Since nDp5 appears to be a free duplication that is lost at a fairly high rate during meiosis, we will attempt to do mosaic analysis of the lin-34/let-60 locus using nDp5.[See Figure 1]