Laboratory course: Model organism C. elegans
Week 2:
1. Sensory neurons
2. Behavior
3. Worm mating (crossing)
王歐力 助理教授
Oliver I. Wagner, PhD
Assistant Professor
National Tsing Hua University
Institute of Molecular & Cellular Biology
College of Life Science
Features of the nervous system
Neurons can be categorized in: interneurons, sensory neurons and motorneurons
interneuron
sensory neuron
 can be made visible
with a special dye: DiI/DiO
motorneuron
Head neurons
The nerve ring contains mostly sensory neurons and almost all interneurons
Labial process bundles
Amphid process bundles
Ring ganglia
UNC-104:: GFP
Posterior head
Front head
Middle head
UNC-104:: GFP
Identifying amphids
• Amphids: are a pair of laterally located sensilla in the head which are open to
outside at the base of the lips
• These chemosensory organs can be stained with FITC or DiI
• Some mutants fail to be stained => dyf = dye filling (mutant)
• Each amphid is made up of 12 sensory neurons (ADF, ADL, ASH…) with
ciliated dendrites as well as one sheath and one socket cell
• The axon of the amphid is located in the nerve ring
Reproduction
• 99% of adult C. elegans are self-fertilizing hermaphrodites
• This feature enables scientists to easily generate homozygous mutant stocks
• Hermaphrodites are protandrous: the gonads produces germ cells which first
differentiate as sperm (about 250 sperm cells) and then produces eggs
• In C. elegans hermaphrodites reproducibility is determined by the sperm supply
• C. elegans produce males to about 0.05% which are fully functional but produce
sperm only; the sperm is transferred to the hermaphrodites during mating => this
feature enables scientists to transfer mutant alleles (to wildtypes or other mutants)
spermatheca
• Oocyte nuclei are produced by meiosis at the distal end
of the gonad and grow in a syncytium
• Just before fertilization the single nuclei are enclosed by
a separate plasma membrane
• Produced sperm is stored in the spermatheca
• After fertilization the egg-shell is added:
=> self-fertilization produces up to 300 eggs
General biology of C. elegans
• Fertilization takes place by squeezing mature oocytes through the spermatheca
• The eggs are laid through the vulva at a 40-cell stage
• Adult hermaphrodites have about 10 mature eggs inside; the older eggs are laid as
fast as new eggs are generated
A hermaphrodite
produces up to 1300 eggs
during its lifetime
• Males cannot produce eggs but they can cross-fertilize hermaphrodites making
them very suitable in genetics to create genetic combinations (male-fertilizing)
• The male sperm outcompetes the hermaphrodites sperm during cross-fertilizing
• XO combination in male is a spontaneous loss of X chromosome: XX => XO
The C. elegans lifecycle
• The 4 larval stages (“juveniles”) are common features of nematodes
• The lifecycle takes about 2.5 days at 25°C, 3.5 days at 20°C and 5.5 days at 15°C
• C. elegans also has an alternative L3 stage known as dauer (“enduring”) stage
• The dauer stage is a metabolic diapause to survive extreme conditions (mainly lack
of food); in the wild, the dauer stage might serve to geographically disperse
• The entry into the dauer stage is
determined by worm-crowding, high
temperature and lack of food
• As a dauer, C. elegans can survive
for up to 3 month highly extending
its lifespan
• On exposure to improved
conditions (availability of food…)
the L3 dauer exits and resumes
development
• Parasitic nematodes use the dauer
to infect hosts
Dauer larvae usually appear dark,
thin, rigid and motionless
Recovered dauer larvae retain their
transparent appearance and begin
feeding with increasing motion
During dauer-formation the mouth closes
• There is no aging at dauer state! Due to the dauer stage worms can live 10 times
longer than their normal lifespan!
• Due to the mouth closure the worms are restricted from eating
L3 worm
Dauer worm
Imagine a human that is supposed to become 90 years old might become 900 years old
• During post-embryonic development the number
of somatic cell nuclei increases to 959
• Total life span under suitable living
conditions: 2 weeks (300 eggs every
4 days) = 1300 eggs during a lifetime
• Wildtype-worms: Bristol (most commonly
used strain) has been isolated from
mushroom compost in Bristol (England) =
N2 strain
• Other strains isolated from soil and
moist environment are C. briggsae
and C. remanei
about 1.3 mm in length and 80 µm in diameter
Behavior, learning and memory
Mechanosensing: Avoidance reflex circuits
Mechanosensory neurons can sense different strength of touch:
eyelash touch
“Mec response”:
• touching the anterior
side of the body results
in a backward movement
• touching the posterior
side results in a forward
movement
Mechanosensory neurons
Gentle touch (eyelash) mechanosensory neurons
Harsh touch mechanosensory neurons
Nose touch and
osmolarity sensors
Texture sensing neurons
Simple behavior
Chemotaxis
Traces of a worm which was allowed
to freely move within one hour on a
plate with an attractant (B = biotin)
(right circle = buffer)
Thermotaxis
• Traces of a worm in a radial thermal
gradient
• Chooses an optimal temperature
(similar to its cultivation temperature)
• Circles in isotherms at that temp.
• Can detect thermal gradients < 0.1°C
Habituation (non-associative learning)
• Tapping at the side of the petri dish
stimulates backing response
• Backing movement decreases if
stimulus is continuously applied (every
10 seconds for total 30 minutes)
Habituation occurrence can
be stored for more than
1 hour in the nervous system
• If stimulus is applied less often (every
60 seconds) decrease of backing
response is less abrupt (indicating
habituation and not worm fatigue)
• Also habituation can be rapidly
abolished with an electroshock stimulus
(dishabituation)
• Further, worms recover from shortinterval habituation faster compared to
long-interval habituation
Classical conditioning (associative learning)
• Non-associative learning (habituation): an animal alters its behavior to a single
stimulus
• Associative learning: an animal learns to use a previously neutral stimulus to
predict a second more significant stimulus
• Example: after food deprivation, one ion is associated with food and the second
ion is associated with the absence of food => a conditioned animal will move to
the ion associated with food (even if no food is present)
• lrn-1 and lrn-2 (lrn = learn) are genes involved in learning
worms go to Na+
5 hours deprivation
Na+ = food
Cl- = no food
(short incubation)
Male mating behavior
Male mating behavior
WAmating.mov