Breeders SCW (1980) Worm Breeder's Gazette "a modified egg-plate method for obtaining large numbers of dauer larvae."

Breeders SCW

[Worm Breeder's Gazette, 1980]

In our laboratory, we routinely use dauer larvae to obtain large, synchronous cultures of L4's and adults for biochemical purposes. We have found that large numbers of dauer larvae can be obtained using eggwhite plates (a modification of the method of D. Baillie and R. Rosenbluth, WBG 2 (1)). Egg-white plates are prepared by stirring the white of one chicken egg with 50 ml of boiling distilled water for several seconds, homogenizing the mixture for one minute in a Waring blender, and layering 3-4 ml of the resultant liquid slurry onto a standard 100mm NGM plate containing a lawn of E . coli . After drying overnight, egg-white plates are seeded with about 1500 dauer larvae and incubated at 20 C . These dauer larvae develop into adults, but for some unknown reason a large proportion of their progeny develop into dauer larvae and become arrested at this stage . Approximately 1x10+E5 dauer larvae are usually recovered per plate after 5-7 days incubation time . Yields as high as 2x10+E5 per plate have been obtained in some instances. For purification of dauer larvae, animals washed from individual egg- white plates are incubated with 5-10 ml of 1% SDS for 30-60 minutes, collected by low speed centrifugation, resuspended in 0.5 -1 ml of buffer, and centrifuged through a 2 ml cusion of ice-cold 15% ficoll for 10 minutes at 300 xg. Intact dauer larvae pellet through the ficoll, while egg matter and worm carcasses remain at the interface . Because a small number of non-dauer animals sometimes escape this first SDS treatment, the entire procedure is usually repeated . Purified dauer larvae can be stored in Mg buffer at 16 C until use and retain good viability for 30-60 days . It has been our experience that dauer larvae obtained from egg-white plates recover much more synchronously than do dauer larvae isolated from starved E . coli plates, especially if they are used within 2 weeks of isolation. Also we have noted that the synchrony of recovering populations tends to decrease as dauer larvae are stored for longer periods of time.

Hodgkin JA (1982) Worm Breeder's Gazette "informational suppressors: a cautionary note."

Hodgkin JA

[Worm Breeder's Gazette, 1982]

The pleiotropic suppressors sup 5 III and sup 7 X (Waterston,1981) have been used by several worm breeders as a means of identifying null alleles ( = amorphs ), because they appear to act only on a subset of null alleles, and not on missense alleles. Also, the extent and efficiency of suppression ( i.e., whether suppression is dominant or recessive) have been used as an indication of whether a particular gene product is needed in catalytic or stoichiometric amounts. While these indications are probably fairly reliable in general, it is possible to find exceptions. I have identified eight sup-7 suppressible alleles of tra-1 III : one (e1781) was derived from a general screen for tra mutants and seven were obtained by reversion of the dominant feminizing allele e1575 ( 'her-2'). Six of these have similar properties: they appear to be null alleles causing XX animals to develop into phenotypic XX males instead of hermaphrodites ) and suppression is recessive. The other two alleles are anomalous. One, e1825, is a weaker allele (i.e., not a complete null) because e1825 XX animals often have abnormal male tails, and they are never fertile males, nor are e1825/e1781 XX heterozygotes. The other anomalous allele, e1835, appears to be a null allele but it is unusual in that suppression is dominant rather than recessive. Since six out of eight alleles exhibit a consistent pattern, it is reasonable to regard the other two as exceptional. Precedents for such exceptions can be found among microbial amber mutations. However, the existence of the exceptions shows that, as usual, genetic data can sometimes be misleading.

Nelson GA (1976) Worm Breeder's Gazette "further characterization of intersex mutants."

Nelson GA

[Worm Breeder's Gazette, 1976]

In Volume 1 #1 of the newsletter an autosomal temperature-sensitive sterile mutation (TS17) was reported to have sexually dimorphic phenotype: spermatogenesis defect in hemaphrodites and intersexuality in 'males'. Further mapping of this mutation by Ken Lew and Johji Miwa has located TS17 in linkage group IV. The second allele of this gene is also linked to IV. Therefore, the intersex gene is distinct from either of the two known transformer genes. Observation of Feulgen stained mutant worms shows that TS17 hermaphrodites grown at 25 C are totally devoid of spermatocytes and mature sperm. At the time of normal spermatogenesis gonadial cells in the position of normal spermatocytes begin to mature in the pattern characteristic of oocytes. These oocytes can be fertilized by outcross sperm, but in the absence of mating they pass through the empty spermatheca and undergo endoduplication of their DNA becoming highly polyploid. These polyploid oocytes are laid as 'squashy eggs'. TS17 intersexes are variable in morphology. They always have a normal-looking bursa and nearly always contain mature oocytes. However, the vulva is not always present and when present is often abnormal so that matings of intersexes to males have not yet been successful. Consequently we do not know whether the intersex oocytes are functional. The intersex gonads vary from normal hermaphrodite shape to male configuration. Occasionally they contain sperm. Often the gonad is broken up into lumps packed solid with nuclei, or it loops around in odd patterns and may contain highly polyploid cells distinct from polyploid oocytes. For analyzing morphology I recommend the use of a camera lucida with stained preparations. Feulgen is already in wide use amongst worm breeders and Hematoxylin/eosin or toluidine blue complements Feulgen by providing information about cytoplasm and nucleoli.

Russo T et al. (1993) Worm Breeder's Gazette "Using C. elegans in High School"

Russo T, Way T

[Worm Breeder's Gazette, 1993]

We have been trying to develop a set of classroom experiments that would use C. elegans to teach basic genetic concepts to high school students. For this purpose, C. elegans has obvious advantages over Drosophila, the current standard organism. Our goal is to have students doing the following experiments: isolating soil nematodes from the wild, and performing simple genetic crosses that would demonstrate dominant, recessive, and X-linked mutations. The immediate problem is teaching a high school student how to pick up and transfer a worm. First, in a class of 20-30 students, each student would ideally learn how to do this in a few minutes. Most worm-breeders will recall that it took us much longer to become proficient at worm-picking. However, a high school student will only use this skill for one or two experiments before moving on to another lab exercise. Also, most high schools only have a few low-quality dissecting microscopes that students must share, so microscope time becomes limiting. Second, the purchase of platinum wire and making of picks are inconvenient. We have had some recent success (ourselves, not with students) using bamboo skewers available in local supermarkets. It is possible to dab the skewer with sticky bacteria, and then dab up large worms to set up a mating. In this context, niceties like sterility or scratching the surface of the agar are not so important. So far we have not even tried to use C. elegans with a whole class. However, a few high school students have used C. elegans for longer-term after-school projects, and one recently won first prize in a county science fair. Please contact us if you have any suggestions or experience from teaching labs, etc.

Wallenfang MR et al. (1997) Worm Breeder's Gazette ""IT'S A BOY!" - USING GFP TO SEX EMBRYOS AND YOUNG WORMS"

Wallenfang MR, Seydoux GC, Dunn MA

[Worm Breeder's Gazette, 1997]

Chromosomally-integrated transgenes that express GFP throughout embryogenesis could serve as useful chromosomal markers, enabling worm breeders to genotype embryos, when the use of larval/adult markers is not possible. We have generated such a GFP transgene (pJH1.16) using the pes-10 promoter. Embryonic expression of pJH1.16 is detected as early as the 28-cell stage in all somatic blastomeres; by the 2-fold stage, expression becomes restricted to the gut lineage, and persists there for the remainder of development. We have generated an integrated array containing this transgene using dpy-20 DNA as a selectable marker. By chance, this array integrated on the X chromosome; the resulting strain was outcrossed six times (JH103) and has a mild Dpy phenotype. We realized that this X-linked transgene could be used to determine the chromosomal sex of embryos and young worms before the appearance of any obvious sexual dimorphism. To test this prediction, we mated JH103 males to tra-2(q122) (spermless) hermaphrodites and scored resulting embryos for GFP expression. Green and non-green embryos were transferred to separate plates and allowed to grow to adulthood to score their sex. Since hermaphrodites receive their X chromosomes from both parents, whereas males receive their single X chromosome from their mothers, we expected all green embryos to be hermaphrodites, and all non-green embryos to be males. Results were as follows: Stage at which GFP Green Non-green expression was scored 28-cell to comma 7/7 hermaphrodites 9/9 males comma to 2 fold 3/3 hermaphrodites 3/3 males 3-fold 3/3 hermaphrodites 1/3 males L1 10/10 hermaphrodites 7/7 males TOTAL 23/23 hermaphrodites 20/22 males In summary, 43 out of 45 embryos/L1s were sexed correctly. The 2 embryos which were sexed incorrectly were both 3-fold, a stage at which gut GFP expression is sometimes harder to detect. We conclude that this strain can be used reliably to sex embryos, with the possible exception of 3-fold embryos. The availability of a transgene that expresses reliably throughout embryogenesis should prove to be a useful tool for tracking the segregation of specific chromosomes, and has many conceivable uses besides the specific one described here. Transgene pJH1.16 and strain JH103 are available to anyone interested. (Contact Matt at mwallenf@welchlink.welch.jhu.edu). We are grateful to Andy Fire for creating the GFP used in this study.

Papp A et al. (1991) Worm Breeder's Gazette "Improved Injection Setup!"

Papp A, Mancillas JR

[Worm Breeder's Gazette, 1991]

We have devised a very convenient, semi-automated injection setup closely based on the system of Mello and Ambros (wbg 11(4):7). It provides many of the advantages of the digital automated systems available, at a minute fraction of the cost. Our system is very similar to that described by Mello and Ambros, and anyone with a system like theirs can upgrade. The main difference is that the 3-way ball valve, used to control the duration of injection flow, has been replaced with an electronic solenoid-operated valve. This permits foot pedal (or any other way you'd prefer to operate an electric switch) control, and leaves both hands free to deal with micro- manipulation and focusing. In addition, the solenoid operated valve can open and close in a fraction of a second, permitting very precise control of flow. If the needle happens to be in the wrong place, a quick tap will show this without appreciably damaging the worm. at high pressure, quick on/off pulses also appear to aid in unclogging clogged needles, while at low pressure the system is ideal for pulse- filling oocyte nuclei or gonads. By varying the pulse time, it is possible to compensate for the flow characteristics of individual needles. By selecting the right match of components, it is possible to construct a system as drawn below which is extremely compact, attaching directly to the pressure source, requiring no cut hypodermic needles or tubing (except the piece connected to your needle) and occupying no bench space at all. Shown not to scale below, the entire system that attaches to a pressure regulator measures only 3' x 5' x 3/4', and minimizes dead gas space. Fittings such as those described by mello and ambros can be used; however, the key is to replace the 3- way ball valve (b41x52) with a solenoid valve such as the nupro 110vac model. All metal-to-metal connections should be wrapped in teflon-based tape. Controlled pressure can be supplied to this system with a nitrogen tank via a high-pressure regulator or with a pump or in-house compressed air via a low-pressure regulator. For a complete parts list and any additional information, please contact us. In addition, a pre-assembled and tested complete system including foot pedal controller and the tubing that attaches directly to your needles can be obtained from tritech research (see above address and phone number). As an introductory offer to worm breeders, the system is available for $210, or $360 with high-quality pressure regulator and tank fitting (approximately the retail cost of the unassembled components). [See Figure 1]

Carta LR et al. (1992) Worm Breeder's Gazette "Call for Worms"

Edgley ML, Thomas WK, Carta LR, Fitch DHA

[Worm Breeder's Gazette, 1992]

In response to the growing interest in worms related to C. elegans for comparative studies, we are coordinating an effort to collect a comprehensive array of cryopreservable species belonging to the order Rhabditida. This collection would be maintained at the Caenorhabditis Genetics Center, would be freely available to all interested scientists, and would provide an excellent resource for worm breeders who are interested in applying a broader phylogenetic viewpoint to comparative biological investigations. Of course, an important advantage of a universally accepted canonical set of living type species is that species identifications can be tested biologically through cross-mating experiments. The utility of such a collection has already been demonstrated in the Drosophila system a remarkable collection of species from around the world is maintained, for example, at the Bowling Green Stock Center in Ohio. We request that interested parties please send their wild isolates to David Fitch, Lynn Carta or Kelley Thomas, along with the following data: the date, source and method of isolation, any ecological information concerning the isolate, pertinent literature references, the names and addresses of the collector, the depositor and the taxonomist(1), and any specifics about stock maintenance. Other data about the species should also be included, such as measurements(2) and male tail characteristics. Scale illustrations and any anatomical, developmental, cytogenetic or molecular data are greatly appreciated. If the isolate is hermaphroditic, males should also be provided, since most of the morphological characters used in species identifications are associated with males. Males may occur spontaneously or can be induced by heat-shocking L4 or young adult hermaphrodites (usually, but not always, at 30 C for 6 hours). Males obtained in this way can be mated to hermaphrodites to maintain a stock containing males. Kelley will provide a molecular "identification tag". David and Lynn will determine if the species has been previously identified in the literature and serve as liaisons to nematologists with taxonomic expertise to help verify the species identification. We will then deposit the species with the CGC. Eventually, we hope to make all of the information associated with each species in the collection available in a database. (See the abstract by Fitch et al. in this issue for the latest information on current CGC species depositions). So hesitate not to share your pet species with us! We think that the effort in building a phylogenetically broad and comprehensive live collection of Rhabditida will be more than compensated by the valuable opportunities it will provide for developing novel approaches to many areas of nematode research.

Hodgkin JA et al. (1984) Worm Breeder's Gazette "the blob from California: a gene for mating plug formation."

Hodgkin JA, Kenyon CJ, Doniach T

[Worm Breeder's Gazette, 1984]

Males from several wild isolates of C. elegans exhibit a detectable difference from males of the Bristol N2 strain: when mated with an hermaphrodite of any genotype the males lay down (or conceivably stimulate the hermaphrodite to secrete) a gelatinous acellular blob or plug over the vulva. The plug increases in size with repeated matings, up to about 40 microns in diameter. It remains in place for several days after formation, not being dislodged by egg-laying. Hermaphrodites from a plugging strain of C. elegans from Sara Doniach's compost heap in Palo Alto) were crossed with him-5( e1490) males, and a him-5 strain carrying the plugging trait was recovered as a F2 segregant. The trait behaves as a single autosomal dominant mutation, and was mapped to a locus tightly linked to unc-69 III, designated plg-1 (for 'mating PLuG formation'). The dominant plugging allele has been designated e2001. It is probable that N2 is in fact a mutant, because the plugging trait is seen in other wild isolates and in other species such as C. remanei, and also because e2001 is dominant. The e2001 allele may therefore represent the true wildtype, but since all C. elegans genetics is based on N2, the plugging trait is regarded as a dominant mutation. N2 probably carries at least one other mutation, affecting burrowing behavior, because most wild isolates of C. elegans exhibit a strong burrowing tendency not seen in N2. The genetic basis of the burrowing trait is not known. The fact that N2 may be mutant in some properties is not surprising in view of its long sojourn in the unnatural environment provided by the worm community. N2 may also have lost other abilities; for example, it could be that some N2 sensory receptors are genuinely nonfunctional, rather than functioning as detectors for an unknown stimulus. We call the blob a mating plug by analogy with the plugs formed during copulation in many animal species: insects, rodents and so on. The plugs in these animals act to prevent sperm loss and to block further insemination, but as yet we have no evidence that C. elegans plugs have these functions. The mating efficiency of plg-1;him-5 males appears to be higher than that of him-5 males, but not dramatically so. Also, an N2 male can successfully fertilize a hermaphrodite after it has been plugged. So, the function of the blob is not clear. It is potentially useful to worm breeders as a means of showing that copulation has occurred, even in the absence of successful fertilization. For example, C. remanei males will plug C. elegans even though no progeny are produced.

Mello CC et al. (1990) Worm Breeder's Gazette "Worm Injection Setup and Parts List"

Mello CC, Ambros VR

[Worm Breeder's Gazette, 1990]

The accompanying diagram shows the system which we use to pressurize our microinjection setup. The key feature of this system is the three way ball valve through which pressure at the needle is applied and then rapidly relieved during each injection. Pressure must be off between injections because the high flow associated with optimal injections will float animals off the pad before the needle can be inserted. It may be possible to adapt your current system to include such a valve, or to construct a similar system with other parts. [See Figure 1] 1) Leitz microinstrument collar (part no. 520145). Holds the injection needle. 2) 3/32' outside diameter (1/32' I.D.) Tygon tubing. This tubing, once stretched, will fit snugly down the opening in the back of the microinstrument collar. This junction is taped with electrical tape. The tape keeps this joint from getting worked loose during the frequent handling of the microtool collar. The tape should not be necessary to make the pressure seal. 3) 21g metal tube (cut off of a syringe needle). Fitted halfway within the 3/32' tubing, the protruding portion is inserted into the 1/16' tubing and serves as a connecter. This connection is also wrapped with electrical tape. 4) 1/16' polyethylene tubing (rigid plastic or nylon tubing works best with the Swagelok adapters). Note; if you plan to HF treat your needles (Worm Breeders Gazette 11-1:18), then this tubing should be long enough to reach a nearby dissecting scope. S) Swagelok reducer (part no. B-100-R-2). Connects the 1/16' tubing to the Swagelok 1/8 connector which comes with the three way valve. 6) Swagelok three way ball valve (part no. B-41XS2). Should be mounted or clamped in place. This valve is used during each injection to apply pressure at the needle. It should be placed within easy reach but should not be on the vibration free table. 7) Swagelok male connector (part no. B-100-1-4). This connector threads into the 1/4 NPT female socket of the STREET TEE. 8) CAJON STREET TEE connector (part no. B-4-ST). Male end threads into the pressure regulator. Female ends accept the tubing connector and the pressure release valve. 9) Swagelok toggle operated shut off valve (B-1GM4-S4). This valve opens the system to allow a quick release of pressure. This is necessary when using the pressure regulator to make downward adjustments in pressure, for example if there is too much flow at the needle. 10) Pressure regulator. Must have a 1/4 NPT Female thread in order to accept the STREET TEE described above. Allows the pressure to be increased or decreased. Pressures required for adequate flow range from 10- to 100-lbs/sq.in. depending on the needle; higher pressures tend to blow connections or to propel the needle out of the holder at high velocity. 11) Nitrogen tank. Should be positioned so that the regulator handle can be reached easily without getting up during injection. Happy injecting!!

Horvitz HR et al. (1976) Worm Breeder's Gazette "egg-laying deficient mutants."

Sulston JE, Horvitz HR

[Worm Breeder's Gazette, 1976]

Mutants of C. elegans which do not lay eggs end up as 'bags of worms'. Progeny hatch internally and devour their parent; the residual adult cuticle encloses about 50 active young larvae. Eventually, these larvae crawl free of the cuticle. Because of its striking phenotype, a bag of worms is easy to detect on a plate containing F2 progeny of mutagenized nematodes. In addition, bags of worms are of lower density than living nematodes and can be isolated by centrifugation in a Ficoll step gradient. (At 4 C, bags of worms float and living worms pellet in 16 per cent w:w Ficoll,) Using these techniques, 57 mutants have been isolated from among approximately 15,000 independent F2 progeny produced by EMS- mutagenized worms. All mutants which have been examined appear to produce bags of worms because of deficiencies in egg-laying. We have begun to characterize both these mutants and similar egg- laying deficient mutants isolated by others. Many mutants have proved to have abnormal post-embryonic cell lineages (see Worm Breeders' Gazette, Vol. 1, No. 1). Virtually all of these cell lineage mutants show variable penetrance. Three mutants (E1309, dev-2 X; E1417, dev-3 IV; E1413, II) appear to be specifically blocked in the divisions of the ventral hypodermal cells which produce the vulva. No eggs can be laid because no vulva is present. Males of these strains appear essentially normal and are capable of mating. E912, rep-2 II, isolated by Babu and previously examined by Jonathan Hodgkin, is also defective in the ventral hypodermal cell lineages. Other lineages (such as those which produce the posterior lateral ganglia) are affected as well. Males are grossly abnormal and incapable of mating; most, if not all, male-specific cell lineages are defective. some cells in the male undergo extensive proliferation, leading to hundreds of extra cells in the tail region and, generally, to the death of the animal. Mutations in two genes -- unc-83 V (2 alleles) and unc-84 X (4 alleles) -- prevent the migrations of the ventral cord precursor nuclei into the ventral cord. Because these precursors normally generate the ventral hypodermal cells involved in vulva formation, these mutants fail to form vulvae. In addition, they are behaviorally uncoordinated because of their ventral nerve cord defects. The dorsal hypodermal ridge of a nuclear migration mutant contains nuclei of the hypodermal syncytium (In N2, this ridge is normally devoid of nuclei.). This defect is visible as early as the 'comma-stage' of embryogenesis; conceivably, the resulting hypodermal abnormality may be the reason for the subsequent deficiency in the migrations of the nuclei of the precursor cells located in the subventral hypodermis. Males of these strains fail to mate. All six alleles of these two genes are temperature sensitive. E1414, unc-85 II, shows blocks in late divisions in the ventral nerve cord cell lineage and is behaviorally uncoordinated (see White et al., this newsletter). Other lineages, such as those which produce the posterior lateral ganglia, are also affected (see Dew and Sulston, this newsletter). It is not known why this mutant fails to lay eggs. Males show gross deficiencies in their rays and are incapable of mating.