1. Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University Institute of Molecular & Cellular Biology College of Life Science Laboratory course: Model organism C. elegans Week 2: 1.Sensory neurons 2.Behavior 3.Worm mating (crossing)

2. Features of the nervous system Neurons can be categorized in: interneurons, sensory neurons and motorneurons interneuron sensory neuron motorneuron can be made visible with a special dye: DiI/DiO

3. Labial process bundles Amphid process bundles Ring ganglia Head neurons The nerve ring contains mostly sensory neurons and almost all interneurons UNC-104:: GFP

4. Front head Middle head Posterior head UNC-104:: GFP

5. 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 Identifying amphids

6. 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 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

7. 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 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 A hermaphrodite produces up to 1300 eggs during its lifetime

8. 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

9. Dauer larvae usually appear dark, thin, rigid and motionless Recovered dauer larvae retain their transparent appearance and begin feeding with increasing motion

10. 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 Imagine a human that is supposed to become 90 years old might become 900 years old Dauer worm L3 worm

11. 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

12. Behavior, learning and memory

13. 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

14. Gentle touch (eyelash) mechanosensory neurons Harsh touch mechanosensory neurons Nose touch and osmolarity sensors Texture sensing neurons Mechanosensory neurons

15. Chemotaxis Thermotaxis Traces of a worm which was allowed to freely move within one hour on a plate with an attractant (B = biotin) (right circle = buffer) 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 Simple behavior

16. 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) 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 short- interval habituation faster compared to long-interval habituation Habituation occurrence can be stored for more than 1 hour in the nervous system Habituation (non-associative learning)

17. 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 Na + = food Cl - = no food (short incubation) 5 hours deprivation worms go to Na +

18. Male mating behavior

19. WAmating.mov