At the end of this experiment, we are able to know
1. the basic procedures how to culture Drosophila melanogaster
2. to distinguish the sexes and types of mutant
3. to cross Drosophila in order to get F2 that illustrate
a. a. Mendel’s law
a. a. Mendel’s law
b. The pattern of transmissions from parent to offspring
INTRODUCTION
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately vinegar flies, wine flies, pomace flies, grape flies, and picked fruit-flies, a reference to the characteristic of many species to linger around overripe or rotting fruit. A second, related fly family, the Tephritidae, are also called fruit flies; these feed primarily on unripe or ripe fruit, with many species being regarded as destructive agricultural pests, especially the Mediterranean fruit fly. One species of Drosophila in particular, D. melanogaster, has been heavily used in research in genetics and is a common model organism in developmental biology. Indeed, the terms "fruit fly" and "Drosophila" are often used synonymously with D. melanogaster in modern biological literature. The entire genus, however, contains about 1,500 species and is very diverse in appearance, behavior, and breeding habitat. Drosophila are found all around the world, with more species in the tropical regions. They can be found in deserts, tropical rainforest, cities, swamps, and alpine zones. Some northern species hibernate. Most species breed in various kinds of decaying plant and fungal material, including fruit, bark, slime fluxes, flowers, and mushrooms. A few species have switched to being parasites or predators. Many species can be attracted to baits of fermented bananas or mushrooms, but others are not attracted to any kind of baits. Males may congregate at patches of suitable breeding substrate to compete for the females, or form leks, conducting courtship in an area separate from breeding sites. Males of this genus are known to have the longest sperm cells of any organism on Earth, including one species, Drosophila bifurca, that have sperm that are 5.8 centimetres long. The cells are mostly tail, and are delivered to the females in tangled coils. The other members of the genus Drosophila also make relatively few giant sperm cells, with D. bifurca's being the longest. D. melanogaster sperm cells are a more modest 1.8 millimetres long, although this is still about 300 times as long as a human sperm.
Drosophila vary widely in their reproductive capacity. Those such as D. melanogaster that breed in large, relatively rare resources have ovaries that mature 10–20 eggs at a time, so that they can be laid together on one site. Others that breed in more-abundant but less nutritious substrates, such as leaves, may only lay one egg per day. The eggs have one or more respiratory filaments near the anterior end; the tips of these extend above the surface and allow oxygen to reach the embryo. Larvae feed not on the vegetable matter itself but on the yeasts and microorganisms present on the decaying breeding substrate. Development time varies widely between species (between 7 and more than 60 days) and depends on the environmental factors such as temperature, breeding substrate, and crowding.
Drosophila melanogaster is a popular experimental animal because it is easily cultured in mass out of the wild, has a short generation time, and mutant animals are readily obtainable. In 1906 Thomas Hunt Morgan began his work on D. melanogaster and reported his first finding of a white (eyed) mutant in 1910 to the academic community. He was in search of a model organism to study genetic heredity and required a species that could randomly acquire genetic mutation that would visibly manifest as morphological changes in the adult animal. His work on Drosophila earned him the 1933 Nobel Prize in Medicine for identifying chromosomes as the vector of inheritance for genes.
MATERIALS:
1. Morgue containing 70% ethyl alcohol
2. Appreciate stock of Drosophila
3. Stereo dissecting microscope
4. Vial/bottles of culture medium (prepared)
5. Self-adhering labels
6. Etherizer
7. Re-etherizer ( petri dish with material or filter paper taped to the inside )
8. Ether in dropping bottles
9. Dropper
10. Fine camel’s hair brush
METHODS:
A. Lab session 1 (culture, distinguish sex, and recognize type of mutant)
1. Numerous media have been developed for the culture of D.melanogaster. in this experiment, we prepared the culture media first by using:
a. Stove, pod and ladle.
b. Chemical-propionic acid.
c. Apparatus-vial with rack
d. 875ml distillation water
e. 18g agar (SIGMA) EC 232-658-1
f. 70g soft brown sugar
g. 25g yeast
h. 35g corn meal
i. 250ml distillation water
j. Propionic acid (to avoid contaminated fungus)
2. Etherize and examine the adult flies:
a. A few drop of ether was place on the absorbent material of the etherizer.
b. The base of the vial was lightly strike on the palm of the hand so the flies will drop to the bottom.
c. The culture vial plug was remove, and quickly placed the mouth of etherizer, inverted the vial over the etherizer, and shaken flies into etherizer.
d. Waited for 30 second.
PRECAUTIONS: avoid overetherization. The flies will die if left in the etherizer too long
e. The flies was transfer and examine the etherized flies with a dissecting microscope at 10/25 magnifications. A soft brush was use to move the flies on the stage.
3. To distinguish the sexes of the flies, four basic characteristics should be considered;
a. Size – females are usually larger than males.
b. shape – the caudal extremity og the males is round blunt, whereas that of the females is sharp and protruding, the abdomen of the male is relatively narrow and cylindrical, whereas that of the female is distended and appears spherical or ovate. Adults newly emerged from the pupa case are relatively long and slanders, and the sexual differenced described here are not so readily noted.
c. Color – black pigment is more extensive on the caudal extremity of the male than on the female. On the male, the markings extend completely around the abdomen and meet on the ventral side. On the female, the pigment occurs only in the dorsal region.
d. Sex comb – only males have a small tuft or black bristles called a sex comb on the anterior margin at he basal tarsal joint of each leg. Magnification is necessary to see the sex comb.
4. Wild type flies was first examine that are considered normal to all traits. To recognize the mutant flies, three basic aspect should be considered; eye (white or bar eye), wing ( vestigial/curly or lobe eye), body color (black or yellow).
B. Lab session 2 ( making crosses )
1. A cross between two varieties of flies begin when a virgin female must be secured. Once inseminated, females retain viable sperm for several days. Thus, the only where to ensure a controlled mating between different genetic stocks is to use virgin females. To ensure the females, this procedure was followed:
The appropriate vial of adult flies make sure empty. The time was record on the vial.
a. Within 8 hours of removing the adult flies, etherize any newly emerged adults. Such newly emerged flies were distinguished by their pale body color and characteristic dark spot on the ventral side of the abdomen, slightly to the left of the midline.
b. Females among those newly emerged adults will be virgins that can be used in appreciate cross.
2. The flies was placed into new vial as follows;
a. Vial 1: 10 females wild type (++) x 10 male white eye (ww)
b. Vial 2: 10 females white eye (ww) x 10 male wild type (++)
Be sure to label vial and keep the vial on its side until the flies have recovered from etherization.
3. After a week, the parent flies (P1) was removed to prevent their being confused with or mated with their offspring. The first filial generation (F1) will soon begin to emerge.
4. After several F1 flies have appeared, them were etherized and examined, especially with regards to characters which the P1 flies differed. In table 3.1 the phenotypes of the F1 flies of each sex was record. These F1 was placed in a fresh vial of medium (the vial must label) as follows:
a. Vial 1: 10 female wild type (++) x 10 male wild type (++)
b. Vial 2: 10 female wild type (++) x 10 male white eye (ww)
This mating will allow for the production od a second filial generation (F2). It is not necessary that the F1 female flies be virgins for this mating. However, remember to remove the parent flies after a week.
5. After several F2 flies have appeared, them were etherized, examine and calculated. All the data was record as below (result).
Result
RESULT
Experiment number 3 Name culture and crosses of drosophila
1. Cross male wild-type X female mutant
2. Dated P1 mated : 22nd July 2008
3. Date P1 is removed : 29th July 2008
4. Date F1 is first appeared : 7th August 2008
5. Phenotype of F1 males : Male mutant
6. Phenotype of F1 females : Female wild-type
7. Date F1 male and female placed in fresh vial : 7th August 2008
8. Date of F1 flies removed : 12th August 2008
9. Date F2 progeny appeared : 21st August 2008
10. Record data in the following table :
Table 3.1 : result of crosses 1
Phenotype | Genotype | Observed (O) | Expected (E) | |
female wild-type | X+XW | 6 | 6 | 0 |
Male mutant | XWY | 6 | 6 | O |
Total | ∑O | X2= 0 |
Degree of freedom: = n – 1 (n=2)
df=1
P = 0.05
X2 table = 3.84
X2 calculated = 0
Phenotype | Genotype | Observed (O) | Expected (E) | |
female wild-type | X+XW | 7 | ¼ X 49 = 12.25 | |
Female mutant | XWXW | 11 | ¼ X 49 = 12.25 | |
Male wild-type | X+Y | 3 | ¼ X 49 = 12.25 | |
Male mutant | XWY | 28 | ¼ X 49 = 12.25 | |
total | ∑O = 49 | X2 = 29.6123 |
n = 4
Degree of freedom: = 4 – 1
P = 0.05
X2 table = 7.81
X2 calculated = 29.6123
Discussion
In Drosophila the sex is determined by the number of copies of the X chromosome. An individual that has two X chromosomes is female and an individual with one X chromosome, which then joins with the Y chromosome, is male. During fertilization, if the egg joins with an X sperm, the zygote is XX, which becomes female. If the Y sperm is involved in fertilization, there is a XY zygote, which develops into a male. The reasoning for Morgan's results is due to the fact that the gene for while eyes in Drosophila is located on the X chromosome and not the Y chromosome. Genes on the X chromosome that determine a trait are called sex linked. After one understands how the white-eye trait is recessive to the red-eye trait, one can easily notice that Morgan's results follow Mendel's assortment of chromosomes. Morgan's experiment has been called one of the most important events in genetics. His work with Drosophila proved Sutton's theory that Mendel's "traits" are found on chromosomes.
Conclusion:
As a conclusion, we are able to know the basics procedure how to culture Drosophila and to distinguish the sexes and types of mutant. Besides that, we are able to cross drosophila in order to get illustrate the Mendel’s law and the pattern of transmissions from parent to offspring.
REFERENCES:
Brooker, R. J. (2009). Genetics Analysis & Principle. Third edition: Mc Graw Hill international edition.
Campbell O'N. (2005). Biology. Seventh Edition: Benjamin Cumming.
Eldra P. Solomon, Linda R. Berg, and Diana W.Martin. (2005). Biology. Third Edition: Thomson Brooks/Cole.
Kenneth R. Miller,and Joseph Levine. (2004). Prentice Hall Biology. Teacher's Edition: Pearson Publishing.
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