Eddy Egan

January 27, 2008

Mrs. Contreras

Determining Patterns of Inheritance with Wild, White Eyed, Yellow Bodied, Sable
Bodied, Eyeless, and Curved wing D. Melanogaster “Fruit Flies” using Chi- square
analysis

The Chi-square is a statistical test that makes a comparison between the data

collected in an experiment versus the data you expected to find. In genetics, the expected

result can be calculated using the Laws of Probability and even a Punnett square.

Variability is always present in the real world. If you toss a coin 10 times, you will often

get a result different than 5 heads and 5 tails although this is not guaranteed. The Chi-

square test is a way to evaluate this variability to get an idea if the difference between real

and expected results are due to normal random chance, or if there is some other factor

involved (like an unbalanced coin or any other things that can skew the data). The Chi-

square is used in genetics to evaluate data from experimental crosses to determine if the

assumed genetic explanation is supported by the data. The Chi-square test helps to decide

if the difference between your observed results and your expected results is probably due

to random chance alone, or if there is some other factor influencing the results. Our

experiment allows us to use Chi-square in order to test our experimental results against

our actual results. Although the Chi-square allows us see if random chance is involved in

our experiment it cannot prove or disprove that chance is the only thing affecting our

results. (Foglia)

The Chi-square is calculated based on the formula below:

X2 = ∑ (observed – expected)2
Expected
 Drosophila melanogaster exhibit complete metamorphism, meaning that their life

cycle includes an egg, larval (worm-like form), pupa and final emergence as a flying

adult. The same process can be witnessed when studying the butterfly go through its life

cycle. The larval has three main stages which can also be called instar or molts. A

simplified version of the life cycle as it happens day by day is broken down below.

Day 0: Female lays eggs

Day 1: Eggs hatch
Day 2: First instar (one day in length)
Day 3: Second instar (one day in length)
Day 5: Third and final instar (two days in length)
Day 7: Larvae begin roaming stage. Pupariation (pupae formation) occurs 120 hours after
egg lying
Day 11-12: Eclusion (adults emerge from the pupa case). Females become sexually
mature 8-10 hours after exclusion.
The eggs are small, oval shaped, and have two filaments at one end. They are

usually laid on the surface of a culture medium and can usually be seen with the naked

eye. After a day when the eggs hatch into the larval stage, the mouth can be seen moving

back and fourth as the larval eats almost continuously. This means that the larval then

make tunnels through the medium and can indicate good growth. The larva sheds its skin

twice during this stage since its size is increasing so rapidly. When the larvae mature

enough, they become Pupa. The pupae then usually climb up the side of the culture

bottles. The last larval covering then becomes harder and darker, forming the pupal case.

Through this case the later stages of metamorphosis to an adult fly can be observed.

Meaning, the legs, arms, eyes, and wings become more visible. (Handout)

In our experiment we predict that when crossing a Wild Male D. Melanogaster

with a Female one with Sable body color, the allele for Sable body color will not be sex

linked and will be recessive. When crossing a Female with White eyes and a Wild Male

D. Melanogaster White eyes will be sex linked on the X chromosome (female) and will
be dominant. When crossing a Yellow Male D. Melanogaster with and Wild Female, the

allele will not be sex-linked and yellow will be recessive.

Procedure*:
1. Acquire a vial of wild type flies from your teacher.
2. In order to distinguish the difference between male and female flies, use the
guidelines below:
a. Males are usually smaller than females
b. Males have dark, blunt abdomens, and females have lighter, pointed
abdomens.
c. Only the males have sex combs, which are groups of black bristles on the
uppermost joint of the forelegs.
d. Follow diagrams to further recognize differences.
3. In order to acquire virgin females for the experiment, you must breed two wild
fruit flies. After the two fruit flies are bred, one must wait until the eggs finally
hatch and eventually just reach the adult stage. No longer than about 8 hours
should one wait to collect the female virgins since they become sexually active
after this time. The reason that one must collect virgin females for the parental
generation is because when the flies mate, the female receives several thousand
sperm from the male and stores them internally. As the eggs are laid, so is the
sperm to fertilize them. Once a female has mated there is a chance that she will
always have some of the male’s sperm, meaning that the offspring will have the
genes from a male that our study is not tracking and therefore would skew our
results.
4. Practice immobilizing and determining the gender of these flies while not letting
any of the flies escape.
5. Examine and record data for the characteristics of the eyes, wings, bristles, and
antennae of the flies.
6. Immobilize the flies by using a chemical called ether, follow the steps below for
more detailed
a. Hold the vial containing the flies at an angle with only a breathable
stopper at the top of it. Open your jar of ether and place the rims of both
bottles together and shake for a few seconds to knock the flies out.
b. After the flies are immobilized, place them on a small Petri dish
containing #1 Whatman filter paper (used to contrast with the color of the
flies)
c. Use a light microscope (dissecting microscope) to view the flies. For more
detailed viewing, do not put the lid on the Petri dish. After a few moments
the cover should be put in place in order to stop flies from escaping
7. Obtain a vial containing pairs of experimental flies. Record the cross number of
the vial to recognize which cross you have observed. These are the parental
generation and have already mated. The eggs represent the F1 generation. It is not
necessary to isolate the virgin females for the F1 cross because all of the males
 have the same genotype as the females and we want to cross the F1 generation
females with the F1 generation males.
8. First Week (Today): Immobilize and remove the adult flies
9. Observe them under the dissecting microscope.
10. Separate the males from the females and look for any mutations
11. Identify the mutation(s) and give it/them a made-up name and symbol. Record the
phenotype and symbol in Table 7.1. Consult your teacher for any help or guidance
needed
12. Place the parents in a jar containing either alcohol or baby oil thus killing them.
We need to do this because if you leave the adult parental flies in the vial, once
the larvae pupate and hatch into the next generation of adults, these adults will
back-cross to their parents. Our study crosses F1 with F1 and not F1 with P. If the
adults were left in there, the experiment would be extremely skewed and would
need to be redone
13. Label the vial containing the eggs or larvae with symbols for the mating, your
name, and the date.
14. Place the vial in a warm location
15. Second week: Observe the F1 flies. Immobilize and examine all of the flies.
Record their sex and the presence or absence of the mutations observed in the
parental flies in table 7.1. Draw conclusions from this data.
16. Place 5 or 6 pairs of F1 flies in a new culture bottle and place the other flies in the
alcohol or baby oil. Label the new bottle F1 x F1 and also label the symbols that
signify what cross it is, the date, and your name.
17. Third Week: Remove the F1 flies from the vials and place them in the alcohol or
baby oil. The F2 generations are the eggs of larvae in the vial. Place the vial back
in the warm location
18. Fourth Week: Begin removing the F2 flies. Record their sex and the presence or
absence of the mutant phenotypes as we did before. Try to collect at least 200
flies.
19. Analyze your data using the chi-square test.

*Actual procedure was done using a Simulation Laboratory on the internet

Data
- See Attached Papers

Discussion
When looking at our P value in our Chi-square analysis we have concluded that

our hypothesis about Wild type males and Sable body females was proved to be not

correct. Our P value for that allele was 461.7107 and in order for the P value to be

accepted it must be below .05. Yellow body color still seems to be recessive since mostly

all wild D. Melanogaster have body colors that are closer related to a tan color. In
accordance with out hypothesis that this trait was not sex-linked, our data was not

supportive and therefore we can infer that the opposite is true.

Our hypothesis regarding females with white eyes crossed with wild males was

not supported since the P value had read to be a staggering 27,426.44. We have also

learned that the trait for white eyes is, in fact, recessive and that the trait is on the X

chromosome. By crossing a White eyed female and a wild male, we should have known

that the result would show all the males with white eyes and all of the females to be wild.

According to our results we can infer that white eyes are not sex linked. Our P value is

too high for any inkling that it would be and therefore white eyes is autonomic.

When crossing Wild Female D. Melanogasters with Males with Yellow bodies

our hypothesis was supported exceptionally well. The P value for this cross had been a

mere .0025, a marginal number compared to our other P values. This means that our data

supports that yellow bodies are not sex linked.

Our last cross between Wild females and Eyeless, Curved winged Males gave us

another unsupported hypothesis. Our number had been relatively close but our P value

had been 2.1104 which, of course, is larger than .05.

Of course, to fully prove that our analysis was correct many more experiments

must be made since repetition proves or disproves hypotheses.

A few sources of error could be found in our experiment. A large one that could

have occurred is in our sorting techniques, sorting the males from the females, and the

female virgins from the males. If our timing was wrong on when we choose to take out

the females from the growth and development jar, then the females could have risked the

possibility of becoming pregnant and skewing the data to not show the alleles that we had
wanted in our results. Instead, the flies could possibly show a third allele that we had not

even seen in our two original flies because the female was carrying sperm from a

previous encounter with another male. Another source of error could have been not

removing the parents from the first bottle soon enough. This ties into my previous source

of error and is just as likely but can cause vast damage to our results. One more source of

error could have been from knocking out the fruit flies. The ether we had used actually

from over-anesthetized the fruit flies and thus killed a large quantity in one of our

previous trials. In our experimental trials the same thing could have happened with some

of the flies being overexposed to ether and therefore skewing the final results of our fly

populations.

Further studies that seem to be relevant to this experiment include many different

traits being crossed at once. What I mean by this is using something like a male with ten

different traits and crossing it with a wild male. In turn would most likely result in an

extremely large analysis and lots of information to go through. Even so, the result seems

interesting to find out. Another study I would like to induce is one that I had actually

heard about through a friend. He supposedly found the gene that led to an organism being

homosexual. He did his experiment with a drosophila melanogaster “fruit fly”. With this

experiment we could determine if homosexuality is sex linked and passed from parent

generation to offspring or was it acquired. His results showed that homosexuality was sex

linked and did it by some form that could either turn on or off the gene of the parent fly

and would result in the change in the offspring. I actually find that this experiment could

lead to much greater and better things. Our experiment might have been not very hands

on since we were basically just pressing buttons and reading what the computer was
telling us. In college we actually get to do these experiments ourselves therefore learning,

seeing and touching all of these different crosses. Tracking diseases and pathogens

through families and determining who express the genes from their parents and

grandparents could be analyzed and research could be expanded limitless.

References

Campbell, Neil A., and Jane B. Reece. Biology: AP* Student Edition. 7th ed. San

Francisco, CA: Pearson Education, Inc., 2005.

Foglia, Kim, Biozone, www.bio.kimunity.com

Lab 7: Genetics of Organisms. Lab 7 Handout.