Caenorhabditis isolation guide (Source: www.wormbase.org)

The prize. As announced by Paul Sternberg at the 2007 International Worm Meeting, there will be a cash prize and species naming right awarded to the first person to isolate a sister species to C. elegans. Cash prize as of this writing is $4,000 US. Criteria and details are given below. The narrow purpose is to identify one or more close phylogenetic relatives to C. elegans for genome sequencing and comparative studies. The broader purposes are to sample the Caenorhabditis genus more densely for phylogenetic and evolutionary studies, and to sample other genera if possible.

Background. The combined experiences of Antoine Barrière, Marie-Anne Félix, Scott Baird, Michael Ailion, Takao Inoue, Walter Sudhaus, and James Thomas suggest the following rough picture of the natural life cycle of C. elegans, C. briggsae, and C. remanei. Dauer larvae inhabit soil, mature compost, leaf litter, and similar media, and may also be associated with snails, isopods (pill bugs) and other invertebrates. Invertebrate associations may be important for transport to new growth sites (C. drosophilae is transported by Drosophila species to rotting cacti and C. remanei is transported to rotting apples by isopods). When a dense bacterial culture arises, the dauers somehow locate the site, recover from the dauer, and initiate a burst of rapid growth. As the bacteria become depleted, dauers form and presumably disperse from the site to await a new food source. A prominent and easily-identified site of rich bacterial growth is rotting fruit and vegetables. Some Caenorhabditis nematodes may also grow in other bacteria-rich windfalls such as feces and carrion. C. perrieri was isolated from manure (but is not available in culture) and C. plicata was isolated from carrion. Worms recovered from rotting fruit are at all stages, including egg-laying adults, and they usually appear healthy and similar to those grown on OP50 on agar plates. Fruit fly larvae, mites, millipedes, and springtails are also common in rotting fruit, and presumably compete with or predate the nematodes. Though C. elegans, C. briggsae, and C. remanei have all been repeatedly isolated from fruit, many other nematodes from the rhabditid, panagrolaimid, and cephalob groups are also common. Recently, Michael Ailion isolated a new Caenorhabditis species (sp. 6) from a rotting apple in a yard in Portugal. Two other apples under the same tree contained C. elegans. This result suggests that additional species are likely to be found with more intensive sampling even in human-disturbed habitat from areas already sampled. Fruits from undersampled regions, including much of Asia and Africa, Central and South America, oceanic islands, and uncultivated habitats are also good bets to yield new species.

Sampling. Growing populations of C. elegans, C. briggsae, or C. remanei have thus far been isolated from rotting apples, apricots, plums, pears, figs, tomatoes, olives, unidentified fruits, and flowers. There is not yet any obvious correlation between species and fruit and there is every reason to believe that many other fruits are good candidates. Nematodes are most abundant on fruits in an advanced state of rot, presumably because the bacterial density is highest at that time. Look for collapsed brown mushy fruits. The fruit identity may only be apparent from fresher nearby fruits. Collect by donning latex lab gloves and putting the fruit into a zip-lock bag. You may want to put one fruit per bag since different fruits from the same location often contain different nematode species. For berries and other small fruits, it might be advisable to put a group from one spot into a bag. Seal a volume of air into the zip-lock bag to minimize anoxia, which can eventually kill the worms. Bags can be stored for some time as long as they remain aerated and don’t overheat, though presumably the population of nematodes will eventually dwindle.

Lab isolation. Use gloves and forceps to pull chunks of rotten material out and place them on the edge of seeded NG agar plates. On a prime sample, hundreds to thousands of animals will crawl out onto the plate within a few hours. It also seems to be effective to pipette some water into the bag, agitate, and pipette the fluid onto plates. Pick or micropipette several animals to a fresh plate to establish stocks. If there is more than one morphotype apparent, obviously you should pick them to separate plates. The sample is likely to contain all kinds of other nasty things such as mites, fungi, and slime molds, so it is advisable to pick or chunk the worms onto fresh plates and to seal, discard, and eventually autoclave the remainder. Many fruits contain large numbers of fruit fly larvae, which crawl around rapidly, spreading contaminants and making it harder to interpret what you are seeing. They are easy to get rid of by picking and flaming them (death to fruit flies!) or picking the worms off to fresh plates. If you used the water isolation method, nearly all the other animals can be removed by passing the fluid through a nylon stocking (L’eggs Knee-Highs work well). Keep new isolates well away from standard lab stocks to minimize the risk of mite infection. New species may be hermaphroditic or gonochoristic (male-female), so be sure to pick gravid females or pick males along with the females if found. Male-female species usually suffer from inbreeding depression, so it is especially important in these cases to pick multiple animals or establish multiple stocks. Letting the newly isolated animals move for a while on a fresh plate and then repicking animals that have moved well away from the original pick spot will help reduce bacterial contamination. If done several times over a period of hours, this often suffices to get rid of all contaminants. Cultures can also be bleached by the protocol used for C. elegans. Some species (though probably not Caenorhabditis) will not thrive on OP50 on NG agar plates and may require special culture conditions (ask Karin Kiontke for advice). Many nematodes and all close relatives of C. elegans freeze reasonably well using the standard C. elegans method. Isabelle Nuez and Marie-Anne Félix have frozen recalcitrant isolates after heat shock (1 hour 37º) and addition of 2mM CaCl2 to the standard freezing solution.

Records to keep. Note the precise location (GPS coordinates would be great), date, and content of the collected sample. For male-female species note how many animals were used to establish the stock. Include any descriptive information that you have acquired.

What to send. Fruit cannot be mailed to the United States, but can be mailed to France. Marie-Anne Felix is interested in obtaining such fruit, especially samples from new locations. Isolated nematodes growing on plates can be sent to and within the United States. For mailings to U.S. labs, the following should be attached to outside of the package, with a copy inside as well: "Contents include: non-hazardous, non-pathogenic, non-parasitic biological sample of a free-living rhabditid nematode. I hereby declare that this organism is NOT considered to be pathogenic for livestock, poultry, or humans. This shipment follows USDA APHIS Guideline #1116 and does NOT require a permit. DO NOT REFRIGERATE, FREEZE OR EXPOSE TO HEAT! If undeliverable, please immediately return to sender, contact the sender, and contact the intended recipient [include contact info]. Purpose: material for US federally sponsored research." If possible, check by email beforehand with one of the individuals willing to process and help identify the nematodes: kk52@nyu.edu (Karin Kiontke), felix@ijm.jussieu.fr (Marie-Anne Félix), ailion@biology.utah.edu (Michael Ailion), or jht@u.washington.edu (James Thomas).

Dissecting scope identification. Many different groups of nematodes are found on fruits. If you keep everything you will quickly be overwhelmed. One plan is to immediately discard anything that is obviously not a Caenorhabditis or another interest group. Here are dissecting microscope characters for various other groups that we have commonly encountered in fruit. Comparative remarks are relative to C. elegans.

Panagrellus sp.: usually larger, often have a posterior vulva, and often are live-bearing. The stoma of Panagrellus and Panagrolaimus species is diagnostic with Nomarski (see below).

Panagrolaimus sp.: usually long and thin with elongated eggs.

Cephalobus sp.: usually slightly smaller, gut dark, eggs often nearly spherical, less active, most are hermaphroditic or parthenogenetic (no males), often slower growing. The stoma is diagnostic with Nomarski (see below).

Pristionchus sp.: usually slightly dumpy looking, all have a longitudinally striated cuticle (seen best off of the bacterial lawn) and no grinder in the pharynx. These dissecting scope characters are readily seen and are diagnostic of Pristionchus.

Oscheius sp.: often smaller and usually darker, the rectum is long and the defecation cycle is distinctive - the anal depressor contraction and expulsion occur during the pBoc relaxation (rather than well after as in all other tested nematodes) and the intestinal muscle contraction is a full second or two later. Most are hermaphroditic. The long rectum is diagnostic with Nomarski (see below).

Mesorhabditis sp.: smaller and darker with a very posterior vulva, low activity level, males usually much smaller than females. A crinkled pharyngeal lumen is diagnostic with Nomarski (see below).

Miscellaneous: characters sometimes seen that are not found in any known Caenorhabditis include a blunt adult tail (female or hermaphrodite), very dark intestine, male tail less obvious and without a prominent fan, uncoordinated or very slow locomotion, failure to thrive and grow rapidly on NG agar plates. It is difficult to be certain, but none of these characters are likely to be associated with new Caenorhabditis species.

Caenorhabditis sp.: Basically, if it looks a lot like C. elegans it is probably a Caenorhabditis species. The female (or hermaphrodite) tail is gently and evenly tapered, the gut is relatively pale and may have a brownish tinge, intestinal nuclei are usually large and especially prominent in the L3 and L4. The vulva is in the mid-body. Movement is active and has an elegant wave form. The stoma (buccal cavity) is long and has marked longitudinal parallel lines visible with a good dissecting scope. The pharynx has a grinder and with a really good dissecting scope you can see a distinct anterior bulb. Eggs are distinctly oval but not long. If there are males, the tail has a prominent fan. It is likely that sister species to C. elegans will look nearly identical to C. elegans, though they could be hermaphroditic or gonochoristic. Mating tests between C. elegans and a sister species should be useful - look for successful mating but postzygotic lethality among cross progeny. Much more detailed characters are described in papers listed below.

Criteria for a C. elegans sister species. This is defined as a species that separated from C. elegans more recently than any currently known species. Among known species, C. briggsae, C. remanei, C. brenneri, and C. sp. 5 form a clade that separated from C. elegans most recently. DNA sequence will be used to establish the phylogenetic position of new species. Ideally, we would identify multiple C. elegans sisters with various levels of divergence, but for now the prize will go to the first sister species isolated. The complete genome sequence for a sister species will undoubtedly be a high priority because of the great utility for comparative studies of gene structure, etc.

Further reading.

Barrière and Félix, Guide to isolation of Caenorhabditis and related nematodes.

Kiontke and Fitch, WormBook chapter. Phylogeny of Caenorhabditis and related rhabditids.

Kiontke and Sudhaus, WormBook chapter. Ecology of Caenorhabditis species.

Sudhaus and Kiontke 1996. Morphological character-based phylogeny of Caenorhabditis.

Sudhaus and Fitch 2001. English translation of the classic Rhabditidae systematics work by Walter Sudhaus.

Barrière and Félix 2007. Temporal study of population genetics of C. elegans, including isolation of C. elegans from rotting fruit.

Barrière and Félix, 2005. Genetic diversity and population genetics of C. elegans, including evidence that dirt and old compost harbor only dauer larvae.

Sivasundar and Hey 2005. Isolation of wild C. elegans, including an RNAi method for species identification.

Sudhaus and Kiontke 2007. Formal description of the Caenorhabditis brenneri species.

Kiontke, Hironaka, and Sudhaus 2002. Formal description of the Caenorhabditis japonica species.

 

Easy Nomarski characters for some of the nematodes

C. elegans and C. briggsae. Stroma is longer than it is wide and that the edges form parallel straight lines. This is also found in other genera closely related to Caenorhabditis.

Panagrolaimid (unknown species). Panagrellus and related genera have a stoma that form parallel straight lines. The anterior bulb in the pharynx is absent.

Cephalob (unknown species). Cephalobs have a stoma that has two segments.

Oscheius (unknown species). All Oscheius species have an elongated rectum. The stoma is similar to C. elegans.

Mesorhabditis (unknown species). This looks subtle, but note waviness of the cuticular lining of the anterior pharyngeal lumen.

 

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Pharynx and rectum in C. elegans and O. tipulae
(Barrière and Félix 2006, WormBook)

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C. briggsae and O. tipulae rectum

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Pharyngial morphology in some families
(Chiang et al. 2006, J Exp Biol. 209, 1859-1873)

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