Diapositiva 1 by S3D5Ng

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									Model Organisms in Teaching Life Sciences:
         Caenorhabditis elegans
          Angela Crimi Anna Pascucci




                      In collaboration with Paolo Bazzicalupo
                               IIGB CNR Napoli Italy
                  Why it is              Equipment
                a good choice               and
                 for school              Materials
                science labs?
                                                         Transport
What are                                                    and
                                                        preservation
C.elgans?




   Activity 1                                        Activity 3

                                Activity 2
                      C.elegans




                  C. elegans is a nematode
             It is small, growing to about 1 mm
                        It lives in the soil
It survives by feeding on bacteria such as Escherichia coli
There are two sexes: a self-fertilizing hermaphrodite and
                               a male
   C.elegans




Philum: Nematoda
Class: Secernetea
Order: Rhabdita
Genus: Caenorhabditis
Species: elegans
           Why we use C. elegans
                at school?
  It is easy to grow on Petri plates seeded with bacteria
It’s very small and on just one Petri plate thousands can
                          be grown;
             It has a short life span ( 3-4 days)
 The body is transparent and visible with a microscope
         Organisation and process of development
                      Numerous spawn
                        Mutant supply
              Why we use C. elegans at
                      school?
   Embryogenesis                        Hermaphroditism


   Sex determination                           lifecycle
                                   Different behaviours following
                                          genetic mutations




 Slow down of metabolism                 Different behaviours
process (dauer larval stage)            following gustative and
                                            olfactory stimuli
 self-fertilising hermaphrodites
Morphology and Anatomy,
      Physiology                       Hereditary characters
                                           transmission
        Equipment and Materials
Microscope
Stereomicroscope
Petri dishes with grow agar + E.coli + C.elegans;
Petri dishes with grow agar + E.coli;
Petri dishes with grow agar + E.coli + mutant of C.elegans
(dumpy, unc, blister, him, lin );
Petri dishes for crossing;
Eyelash
Platinum wire;
1 ml pipettes
Sterile water
Microscope slide, cover slip
Agarose or agar
Scalpel
Nail varnish
    Transport and preservation

The Petri plate with the worms can be conserved in
the fridge at 4° C for a long time (many months)
It’s important to open the plate less it is possible to
avoid the contamination of the moulds
                 1°level activities
Observing:
•Lab 1 anatomy,
•Lab 2 developmental stage;
•Lab 3 sexual dimorphism;
•Lab 4 embryos;
•Lab 5 dauer larval stage;
•Lab 6 mutants;
•Lab 7 behaviour

           Technique: Looking at worms with a
            stereomicroscope and microscope

  This teaching method allows graded experiments and laboratory techniques
                       based on complexity of theme
         1°level activities
     Observing Anatomy and Morphology



Head
Pharynx
Gut
Spermatheca
Ovary
Tail
            1°level activities
      Observing Anatomy and Morphology




The adult essentially comprises a tube, the exterior
cuticle, containing two smaller tubes, the pharynx and
gut, and the reproductive system.
Most of the volume of the animal is taken up by the
reproductive system.
C. elegans has no eyes, it might respond slightly to light
                      1°level activities
                   Observing developmental stages

The short life span ( 3-4 days) permit:

• Observation with
  stereomicroscope and
  microscope of the
  different larval stages of
  lifecycle
• Experiment at
  different temperatures
  of the duration of the
  larval phases
    1°level activities
   Observing developmental stages




C. elegans develops through four larval
 stages which are separated by moults
                    1°level activities
                  Observing developmental stages
      L3
     moult
     9 hours
                                                             L1
                                                            16 hours




L4 moult                                                     L2        moult
                                                                9 hours
   11 hours
spermatogenesis


               In L4 the hermaphrodite produces about 320
                      spermatozoa (only in this phase)
   1°level activities
Observing developmental stages




Oogenesis happens in the adult
The adult produces only oocyte
          1°level activities
     Observing the embryogenesis phases




The embrional phase takes about 800 minutes
  (13h 20m). Eggs: 0.1 mm
      1°level activities
 Observing the embryogenesis phases




The eggs are deposited by the adult four
hours after fertilization, you can see
inside the abdomen of the big adult, the
zygote and the first mitosis of the eggs
      1°level activities
     Observing sexual dimorphism




                                              The male




With some practice it is possible to recognize:
       the hermaphrodite and the male
             1°level activities
            Observing sexual dimorphism




The hermaphrodite has a longer tail, the male has a
tail cut with a small fan-tail which is needed to keep
the hermaphrodite during coupling
                    1°level activities
                   Observing sexual dimorphism




                                                 The male




On rare occasions, nondisjunction occurs during meiosis with the
loss of one X chromosome. Animals with a single X are males and
are able to fertilize the eggs of the hermaphrodites (with more
success than they have themselves). Most of the time they fertilize
themselves, so that some recessive alleles quickly become
homozygous and affect the phenotype
                         1°level activities
                         Observing sexual dimorphism




A XO combination in the 6th chromosome pair will produce    a male. Occasionally in a
hermaphrodite population a male appears (2 / 1000) because of an error (nondisjunction)
that happens during the meiotic process; in this way the gametes have been formed that
haven’t the sexual chromosome. With the fecundation we’ll have the male zygote.
                           1°level activities
                              Observing dauer stage
                                      Embryo



                   Adult                                   L1
   oogenesis




                           moult
                                        Dauer

                     L4                                         moult
                               (until some months)        L2
 spermatogenesis



                                        L3              moult

During bad environmental condition for the survival , like the absence of food, the larva
L2, after a moult, they become a dauer. During the phase the metabolic process they
slow down
1°level activities
  Observing dauer stage
              1°level activities
                   Observing mutants




                                          Dumpy 8




The mutants dpy-8 body appears shorter and wider. The
  mutation is on the chromosome 3 (a recessive gene)
         1°level activities
              Observing mutants




                                      Lin 18




The mutants lin 18 appears with multi vulva.
            1°level activities
                  Observing mutants




                                          Him 5




The mutation of him-5 promote the birth of males, so in
the plate is possible to observe a high number of males.
         The mutation is on the chromosome 5
                    (a recessive gene)
            1°level activities
                Observing mutants




                                         Blister 6




The phenotype appears very changed, in fact one or
 more excrescence like wart deform the body. The
        mutation is on the chromosome 4
               (a recessive gene)
             1°level activities
                 Observing mutants




                                      Mutants unc-33




The worms appears limp paralysed and lots of them
looks like a C. The mutation is on the chromosome
                 4 (a recessive gene)
                        1°level activities
                              Observing behaviour




     • In competition for the food the worms crowd and kink.
     • The worms reunite in bunches (dauer phase).

A diversity of behaviours, it can taste, smell, and sense light and temperature. These
characteristics make C. elegans a good experimental organism for students to study
behaviour. They can learn how worms react to different chemical gradients and
realize how mutations can influence behaviours.
                  2°level activities

•Lab 1 Verifying experiences of self-fertilising hermaphrodites
•Lab 2 Experience: from dauer to L4




               Technique: Picking worms
                       2°level activities
          Verifying experiences of self-fertilising hermaphrodites




Take with a pipette some drops of sterile water and put a drop on Petri plate
 containing E.coli without worms. Under the stereomicroscope and using
 the platinum wire, take one worm and place it on the drop of water on Petri
 plate. Check the worm is well and close quickly the Petri plate to avoid
 mould contamination. Observe at regular intervals the Petri plate, you will
 be able to notice in a short time a rapid multiplication of the number of
 worms.
           2°level activities
Verifying experiences of self-fertilising hermaphrodites




    C. elegans has five pairs of autosomes and
    one pair of sex chromosome. If the 6th
    chromosome pair is XX, then C. elegans
    will be a hermaphrodite.
              2°level activities
              Experience: from dauer to L4




 To interrupt the dauer phase bring the worms and put
     them on the Petri plates with Escherichia coli
 In a few hours you can observe their transformation,
they will turn into L4, they will be lively and looking for
      food and they start again a normal life-cycle
                 3°level activities
•Lab 1 Use of microscope slide to observation the anatomy
•Lab 2 Embryo preparation and transfer to agarose pad; follow
 early all division
•Lab 3 Crossing wild type hermaphrodites x male
•Lab 4 Crossing mutant hermaphrodites x male
•Lab 5 C.elegans responds to bitter compounds
                                3°level activities
                Technique : To observe live worms under a microscope

A drop of agarose                           microscope slides             2

                                  1                                                         1

     Place a drop of 2% molten agarose
     on a microscope slide (1)
                                                          Squash the drop with another glass slide (2)
                                                                 Throw excess agarose away
                             Cover slip +          and allow agarose to solidify (wait for about 10 minutes)
                             drop + worm




On a cover slip put a PBS (M9 buffer)
drop, then choose a worm to observe and                                                    1
lay it down with care. On the four corner of
the cover slip, put some nail varnish.                Take off the slide2 from the
                                                      slide1 and overturn the cover slip
                                                      making a slight pressure
                               3°level activities
            Technique : Embryo preparation and transfer to agarose pad;
                                    follow early all division
                                              Gravid hermaphrodite
scalpel



                                              Cover slip
                                eggs


 On a cover slip put a PBS (M9 buffer)                                  Then go on on the
 drop, then transfer a gravid hermaphrodite                             example to observe
 and dissect it off the vulva (cut adult                                live worms under a
 transversely through his middle one-third)
                                                                        microscope
 with a scalpel (you will see the just
 feconded eggs, coming out). On the four
 corner of the cover slip, put some nail
 varnish.
                                                                                        1
                                                     Take off the slide2 from the
                                                     slide1 and overturn the cover
                                                     slip making a slight pressure
                    3°level activities
    Technique: Crossing wild type hermaphrodites x male




Take with a pipette some drops of sterile water and put a drop on
Petri plate for crossing, put two worms hermaphrodites WT and
ten males mutants him-5 (this mutation favour the birth of the
male) of the same size. Observe at regular intervals the Petri plate
                       3°level activities
         Technique: Crossing wild type hermaphrodites x male




According to Mendel’s law, crossing a hermaphrodite and a male the first
generation will grow 50% male and 50% hermaphrodites,
The male guarantees the genetic variability
                   3°level activities
      Technique: Crossing mutant hermaphrodites x male




Take with a pipette some drops of sterile water and put a drop on
  Petri plate for crossing, transfer two worms hermaphrodites
mutants (dumpy-8, unc, blister) and ten males mutants him-5 (this
mutation favour the birth of the male) of the same size. Observe at
                  regular intervals the Petri plate
                     3°level activities
        Technique: Crossing mutant hermaphrodites x male




If the crossing happens, after a few days, you will be able to notice
three different phenotypes: hermaphrodites mutants Blister males
and hermaphrodites wild type. This last type has reappeared
because the mutation Blister on account of a recessive gene located
on the chromosome 3
Repeat the same operations using hermaphrodites mutants unc-33
and hermaphrodites mutants blister-6.
                          3°level activities
               C.elegans responds to bitter compounds




                                  Drop test

C. elegans reacts to chemical, mechanical and thermal stimuli in the environment.
C.elegans responds to bitter compounds (e.g. quinine and other alkaloids ) showing
escape reaction.
                            3°level activities
            C.elegans responds to bitter compounds




                Wild type                              Mutant

Such chemiosensorial mechanisms seem to be part of a common strategy animals
adopt to protect from potentially toxic chemical compounds in the environment.
Suggestive classroom activities investigating c.elegans’ behaviour could lead to
discussion about the evolution of the sensory system in both animals and man

								
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