Download C. elegans Background Information

C. elegans Background Information
The purpose of this document is to provide teachers with sufficient background information on C. elegans
anatomy, physiology and behavior to be able to instruct students in the accompanying lab activities and to
be able to answer basic student questions. For further reading, teachers are encouraged to investigate
the following resources:
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WormBook at http://www.wormbook.org/
WormAtlas at http://www.wormatlas.org/index.htm
WormClassroom at http://www.wormclassroom.org/
WormBase at http://elegans.swmed.edu/.
General Background (Altun and Hall, 2005; Worm Classroom)
C. elegans is a free-living (non-parasitic) soil nematode (roundworm). It is an important model organism
that is used to study such topics as aging, genetics, neuroscience, innate immunity, development,
apoptosis (programmed cell death), and drug response. It is an ideal model organism because it is small,
easy to propagate, inexpensive, and very well studied. The entire genome has been sequenced (The C.
elegans Sequencing Consortium, 1998), and the origin and fate of each of its 959 cells is known.
In 1998, C. elegans became the first multicellular organism with a completely sequenced genome (The C.
elegans Sequencing Consortium, 1998). The genome consists of approximately 100 million base pairs,
roughly twenty times the size of the E. coli genome but only 3% of the size of the human genome. C.
elegans shares many genes and gene products with humans (about 40% homology) (Worm Classroom,
http://www.wormclassroom.org/ac/celegansModel.html#experiment).
Nematodes are cylindrical, unsegmented members of the phylum Nematoda (in contrast to earthworms
and other segmented worms in the phylum Annelida). Adults are approximately 1 mm long. The C.
elegans body consists of a clear outer tube, called the cuticle. This allows visualization of internal organs
with only the aid of a light microscope. The cuticle serves as an external skeleton, helping to maintain
body shape and protecting the worm from its environment. There are also two internal tubes—a large
tube containing the reproductive organs, and a smaller tube containing the pharynx, gut, and nerve ring (a
primitive “brain”). A ventral nerve cord runs the length of the body, as does a smaller dorsal nerve cord.
Four longitudinal bands of muscles run the length of the body. The alternating flexing and relaxing of
these muscles causes the worm to move in an “elegant” sine-curve motion. This is why the worm was
named C. elegans (Altun and Hall, 2005).
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Digestion and Defecation (Hart, 2006)
In the wild, C. elegans live in soil and compost, eating bacteria that grow on decaying plant matter. In the
laboratory, the worms are fed a strain of E. coli, called OP50, which is harmless to humans. Bacteria is
ingested through the worm’s pharynx and digested in the gut. Wastes are released from the anus every
45-60 seconds.
The C. elegans alimentary system contains 127 cells and consists of a foregut (buccal cavity and
pharynx), midgut (intestine), and hindgut (rectum and anus in hermaphrodites and cloaca in males). The
intestine consists of 10 pairs of cells with each pair forming a ring. A pharyngeal valve and a rectal valve
cap the intestine at the anterior and posterior ends, respectively.
Worms defecate using the Defecation Motor Program (DMP). Defecation begins with peristalsis of the
posterior body wall muscle, followed by anal contractions several seconds later. Feces appear as a cloud
emanating from the anus. Defecation can be observed at 25x or higher magnification on a high quality
dissecting microscope. Worms will not eat or defecate if disturbed, so allow five minutes of recovery time
before attempting to observe either function if worms have been recently picked or washed. A movie of
the DMP is available at: ftp://calliope.gs.washington.edu/movies/simpleDMP.avi
Gustation, Olfaction and Chemotaxis (Bargmann, 2006; Pierce-Shimomura, et al 2005; PierceShimomura, et al, 1999)
C. elegans can detect a variety of volatile chemicals through olfaction (smell) and soluble chemicals
through gustation (taste). A worm uses these senses to detect food, danger, and other animals. Olfaction
and gustation also play a role in development and mating. Additionally, chemical cues prompt chemotaxis
(moving toward a stimulus), avoidance (moving away from a stimulus), entry into or exit from the dauer
larval state, and changes in motility. As much as 5% of the C. elegans genome is dedicated to sensing
environmental chemicals (Bargmann, 2006).
Worms detect chemical signals through ciliated neurons in the head (the amphid and inner labial sensory
organs) and near the tail (phasmid sensory organ). Receptor proteins that bind chemical ligands (soluble
or volatile chemical stimuli) are found primarily in these cilia, not in the axons or dendrites. Mutants that
lack chemosensory cilia (e.g., che-2, che-3, daf-6, daf-10) are unable to detect chemicals in their
environment and will not move preferentially toward a stimulus that would normally attract a wild-type
(N2) worm. Other mutants are able to chemotax normally in virtually all situations except one. The mutant
odr-10, for example, has a substantially reduced ability to detect the chemical attractant diacetyl but has
normal sensitivity to other volatile attractants. ODR-10 is a receptor protein for diacetyl (Bargmann, 2006).
Therefore, a defect in this gene only prevents worms from detecting diacetyl. Diacetyl is the chemical that
gives synthetic butter its characteristic aroma. In contrast, odr-3 mutants exhibit weak olfaction to several
different odorants due to defective responses from several different classes of neurons.
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Most chemoreceptor neurons occur in left-right pairs. One pair of neurons can express up to nine different
genes, producing nine different odorant receptors. Humans have only one receptor per neuron. Thus, in a
sense, worms are able to do more with less. However, with up to nine receptors per neuron, worms can
have difficulty differentiating between specific odorants. For example, if receptors on the same neuron
simultaneously detect two or three different odors, the worm cannot tell the difference. Since each
receptor activates the same signal transduction cascade, each odorant will “smell” the same to the worm.
With each odor receptor responsible for detecting only one odorant, humans do not experience this
problem.
C. elegans feed on bacteria and locate their food sources through chemotaxis. As a result, worms are
attracted to a variety of alcohols, ketones, amines, esters, and other volatile organic molecules that are
produced as a by-product of bacterial metabolism (Bargmann, et al, 1993). When feeding, worms spend
about 80% of their time “dwelling” in a localized area of worms. When dwelling, worms move very slowly
and do not stray far from their starting point. Occasionally, they will “roam,” which involves rapid
movement across the bacterial lawn. Chemosensory neurons are involved in this switch (Fujiwara et al.,
2002; Bargmann, 2006).
Dwelling on a bacterial lawn
Pivoting, in search of food
Roaming across lawn
(Bargmann, 2006, adapted from Gray et al., 2005)
Worms have a rapid withdrawal (repulsion) response to odorants they do not like, such as copper and
copper compounds, detergents, guanine, and low pH (Colbert et al., 1997; Culotti and Russell, 1978;
Dusenbery, 1974; Hilliard et al., 2002; Hilliard et al., 2004; Ward, 1973). When a worm forages, it moves
its nose in a rhythmic dorsal/ventral pattern. When it detects food, or other attractive odors, it will
chemotax toward the odorant. The overall movement toward an attractant is made in response to
gradients that form as the chemical diffuses. The worm accomplishes chemotaxis in a somewhat
inefficient and indirect way by making long movements toward an attractant with occasional reversals in
the wrong direction. These reversals are known as pirouettes. Over time, the worm makes more long
movements toward its goal than short pirouettes away from it, thus eventually reaching the peak of the
chemical gradient. The “pirouette model” of movement is not the optimal method of chemotaxis in a
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perfect linear gradient, but it is well-suited for a more complex, natural environment (Pierce-Shimomura et
al., 1999).
The Pirouette Model: The worm starts a point X and the attractant is at point Y.
(From Bargmann, 2006)
Mechanosensation (Hart, 2006; Goodman, 2006)
In the wild, C. elegans rely on mechanoreception to avoid collisions with rocks, dirt clods, and other
animals as well as sense their own movements. Hermaphrodites have 22 mechanoreception neurons
(MRNs); males have 46 (the extra 24 neurons are required for mating behaviors). Touch is important to
several behaviors in worms, including mating, defecation, locomotion, feeding, and egg-laying. Touch
receptor neurons are located along the body and are coupled with the skin or cuticle.
Worms respond to touch in several different ways. A gentle tapping of the plate will cause worms to move
in response to the tapping with adults usually moving backwards. Worms also generally recoil or move
their head when gently poked in the nose. This can be done with a toothpick, a nichrome nematode pick,
or even a tightly twisted end of tissue. There are also mutant strains that do not respond to touch. Rapid
repetition of plate or nose tapping will cause habituation (i.e., the worms learn that their response to the
stimulus does not stop the stimulus and they cease to respond). Recovery of the response to either
stimulus will return with time.
The nose touch response is mediated by ciliated, sensory neurons located at the tip of the nose, where
they can detect light touch to the nose, high osmolarity, and volatile chemicals. glr-1and eat-4 mutants
are defective in the nose touch response.
Alcohol (McIntire, In Press)
Ethanol causes a host of reversible effects on C. elegans. Body flattening is visible at environmental
ethanol concentrations as low as 100-200 mM (in agar). 400-500 mM ethanol causes nearly complete
inhibition of egg-laying and locomotion in C. elegans (Davies, et al, 2003). The worms can still move, but
they are extremely slow and uncoordinated. 400-500 mM ethanol in the agar corresponds to a 20-30 mM
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internal ethanol concentration. For humans, a 22 mM concentration of ethanol in the blood is comparable
to a 0.1% blood alcohol level, the legal limit for driving in some states (McIntire, In Press).
Researchers have identified a genetic component to these phenotypes. slo-1 mutants are resistant to the
effects of ethanol on locomotion and egg-laying. The slo-1 gene encodes a homolog of a type of
potassium channel that is also found in humans (the BK channel) (Wang et al., 2001). Researchers have
measured the current associated with SLO-1 channels in C. elegans and found that ethanol (at doses that
cause inebriation) causes an increase in SLO-1-dependent current. This effect was not seen in slo-1
mutants. Under the influence of intoxicating levels of ethanol, a similar increase in BK channel opening
has also been observed in mammals (McIntire, In Press).
A genetic link to alcohol tolerance has been discovered in C. elegans. Wild-type worms discovered in
Hawaii (strain CB4856) develop ethanol tolerance more quickly than N2 wild-type worms (McIntire, In
Press). This is believed to be due to genetic variation between the strains. Some studies have indicated
that a similar tolerance gene may play a role in the ethanol tolerance of rodents as well.
Reproduction and Life Cycle (Altun and Hall, 2005; Emmons, 2005)
C. elegans have 6 pairs of homologous chromosomes (5 autosomal and 1 sex). The 2 sexes are defined
by the number of sex chromosomes. An XX embryo will develop into a self-fertilizing hermaphrodite, while
an embryo with one X chomosome (XO genotype, where “O” denotes a missing X chromosome)
becomes a male. Males are uncommon, occurring approximately 0.1% of the time under normal
circumstances as a result of chromosome nondisjunction. Males can be produced at a higher rate through
breeding, particularly through the use of mutants defective in chromosome segregation such as him-5, or
they can be ordered from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota.
Males also occur at higher rates under certain types of stress, such as heat or food scarcity.
Healthy hermaphrodites tend to resist mating with males. They do this by moving away quickly when
touched by males and by ejecting their sperm after copulation. Males search for hermaphrodites through
chemotaxis.
In the diagrams below, note that a hermaphrodite possesses a tapered, pointed tail, whereas a male tail
is spade-shaped.
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Worms become gravid (full of ripe eggs) approximately 40 hours after reaching the late L4 stage of
development. L4 is the final juvenile stage prior to adulthood. L4 larvae can be distinguished by the
presence of a white crescent where the vulva is normally located. You will probably need 40x or higher
magnification to observe this L4-specific characteristic. As these worms mature to the late L4 stage, they
develop a black spot in the middle of the white crescent.
Self-fertilized worms lay approximately 300 eggs; those inseminated by males can lay as many as 1000
eggs. The newly hatched progeny pass through four developmental stages before becoming mature
adults. The entire life cycle takes approximately three days under ideal conditions, with a single worm
able to live up to three weeks. Each larval stage (L1-L4) is followed by a molt and the production of a new
cuticle.
At the end of the L2 stage, if conditions are unfavorable (e.g., overcrowding, insufficient food, or heat
stress), worms may enter the dauer stage, a form of arrested development. A dauer worm is thinner than
other larvae and does not eat as a result of its mouth being chemically plugged. It also moves more
slowly. When conditions become favorable, dauer worms will continue development and eventually
mature into adult worms. Nematodes that have gone through the dauer stage show an increased
resistance to stress and can live four to eight times longer than unstressed worms.
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References
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Bargmann CI. 2006. Chemosensation. in WormBook. (ed. The C. elegans Research Community),
WormBook, doi/10.1895/wormbook.1.123.1,
http://www.wormbook.org/chapters/www_chemosensation/chemosensation.html
Bargmann CI, Hartwieg E, and Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction
in C. elegans. Cell 74: 515–527.
C. elegans Sequencing Consortium, The. 1998. Genome sequence of the nematode C. elegans: a
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Colbert HA, Smith TL, and Bargmann CI. 1997. OSM-9, a novel protein with structural similarity to
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Culotti, JG and Russell RL. 1978. Osmotic avoidance defective mutants of the nematode Caenorhabditis
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McIntire SL. 2003. A central role of the BK potassium channel in behavioral responses to ethanol
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Worm Classroom, http://www.wormclassroom.org/ac/celegansModel.html#experiment
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