Fruit Fly Lab Alycia Fletcher Biology IB HL March 25th 2010 Fruit Fly Lab Introduction Genes can either be sex-linked or autosomal. If a gene appears mostly in one sex chances are the gene is sex-linked and if it appears frequently in both sexes it is most likely autosomal. Using Drosophila melanogaster, also known as the fruit fly, we will determine whether the gene is sex-linked or autosomal.

Drosophila melanogasters have a relatively short life span and are an excellent organism for genetic studies because it has simple food requirements, occupies little space, is hardy, completes its life cycle in about 12 days at room temperature, produces large numbers of offspring, can be immobilized readily for examination and sorting, and has many types of heredity variations that can be observed with low power magnification. Drosophila has a small number of chromosomes (four pairs). These chromosomes are easily located in the large salivary gland cells. Purpose

The idea of this experiment is to give a clearer understanding between autosomal, sex-linked, recessive and dominant genes. Furthermore, with this knowledge we can then understand how a gene is passed down through its generations and why it would appear in one individual and not in another. The experiment also helps give a better understanding of genotypes and phenotypes and what role they play in determining and predicting a genotype. Hypothesis Null: The wingless mutation is autosomal recessive Alternative: the wingless mutation is sex-linked The Life Cycle of Drosophila

The eggs: The eggs are small, oval shaped, and have two filaments at one end. They are usually laid on the surface of the culture medium and, with practice, can be seen with the naked eye. The eggs hatch into larvae after about a day. The larval stage: The worm like larvae eats most continuously, and its black mouth parts can be seen moving back and forth even when the larvae are less distinct. Larvae tunnel through the culture medium when eating; thus channels are a good indication of a successful growth culture. The larvae molt twice as it increases in size.

In the last of the three larval stages, the cells of the salivary glands contain giant chromosomes, which may be seen readily under low-power magnification after proper staining. The pupal stage: When a mature larvae in a laboratory culture is about to become a pupa, it usually climbs up the side of the container or on to a paper strip provided in the culture bottle. The last larval covering becomes harder and darker, forming a pupal case. The adult stage: When metamorphosis is complete, the adult flies emerge from the pupal case. They are fragile and light in color and their wings are not full expanded.

These flies darken in a few hours and take on the appearance of an adult fly. They live a month or more and then die. A female does not mate for 4-6 hours after emerging from the pupa. Once she has mated, she stores a considerable quantity of sperm in receptacles and fertilized her eggs as she lays them. To ensure a controlled mating, it is necessary to use females that have not been mated before (virgins). Design of the Exercise This genetics experiment will be carried on for several weeks. Drosophila with well-defined mutant traits will be assigned to you by your teacher. You re responsible for making observations and keeping records concerning what happens as mutant traits are passed from generation to the next. You will be assigned to study a certain mode of inheritance using particular genetic crosses of flies having one or two mutations. The modes of inheritance most commonly used are: •Monohybrid. In these experiments, the mode of inheritance is determined when a single contrasting pair of traits is involved. •Dihybrid. In these experiments, the mode of inheritance is determined when two pairs of contrasting traits are considered at the same time. Sex-linked. In these experiments, the mode of inheritance is determined when the mutant characteristic is associated with the X chromosome. Procedure: 1. Obtain a vial of wild- type flies. Practice immobilizing and sexing these flies. Examine these flies and note the characteristics of their eyes, wings, bristles, and antennae. 2. To make handling easier, immobilize the flies with fly-nap, or by twirling the vial in ice for several minutes. Place the immobilized flies on a piece of filter paper inside a petri dish. Place this under a dissecting microscope to view the flies. 3.

Distinguish male flies from female flies by looking for the following characteristics: a). Males are usually smaller than females. b). Males have dark, blunt abdomens, and females have lighter, pointed abdomens. c). Only males have sex combs. which are groups of black bristles on the upper most joint of the forelegs. 4. Obtain a vial containing pairs of experimental flies. Record the cross number of the vial. This number will serve as a record as to which cross you obtained. These flies are the parental generation (P1) and have already mated. The female should have already laid eggs on the surface of the culture medium.

The eggs represent the first filial, F1 generation and will be emerging from their pupal cases in about a week. 5. First week (today): Immobilize and remove the adult flies. Observe them carefully under the dissecting microscope. Separate the males from the females and look for the mutation(s). Note whether the mutation(s) is/are associated with the males or females. Identify the mutation(s) and give them a made up name and symbol. Record the phenotype and symbol in your journals. Note: I will be giving you all wild type & one mutant strain. 6. Place the parents in the morgue (fly & be free).

Label the vial containing the eggs or larvae with the symbols for the mating. Also label the vial with your name and date. Place the vial in a warm location (on the shelf). 7. Second week: Begin by observing the F1 flies. Immobilize and examine all the flies. Record their sex and phenotype. Place as many F1 flies in a fresh culture bottle. For this cross the females need not be virgins. Label the vial with the symbols, name, and date. 8. Third week: Remove the F1 flies from the vials and place them into the morgue. The F2 generation are the eggs and /or larvae in the vial. Place the vial in a warm place. 9.

Fourth week: Begin removing the F2 flies. Record their sex and the presence or absence of mutation(s). The more F2 flies collected, the more reliable the data will be. You may have to collect flies over a three-or four day period (or more). Try to collect at least 200 flies (probably quite a bit lofty). 10. To analyze your data, you will need to learn how to use the Chi-Square Test. The Chi-Square Test (pronounced kahy) will be a part of your lab exam. NB: All suggested times are approximate; your schedule does not have to exactly matching the procedure timeline Procedural Flowchart for Drosophila melanogaster F1 Predictions

A. If the wingless mutation is autosomal Dominant on either the female or male then we can expect all of the offspring in our F1’s to have a wingless phenotype and the genotype to be heterogeneous. n n NNnNn NNnNn B. If the wingless mutation is recessive and the wild type is dominant we can expect all of the offspring to appear wild and the genotype to be heterogeneous. n n NnNnN NnNnN C. If the wingless mutation is sex-linked and on the female we can expected the phenotype and genotype of the F1’s to be 2:1 ratio all of the females will appear wild but be carries and all of the males will be wingless X¬n Xn XNXNXnXNXn YXnYXnY

B. If the wingless mutation is sex-linked and on the male then we can expected the phenotype of all the F1’s appear wild although the females will be carriers of the wingless mutation, the genotype being 2:1. X¬N XN XnXNXnXNXn YXNYXNY F1 results Vial one had AP female > wingless And wild male > winged > F1’s both male and females are winged Vial Two had AP male > wingless And wild female > winged > F1’s, both sexes are all winged Based on the results of the F1’s, both males and females appeared winged, we can assume that the wingless trait is recessive, and therefore either prediction A or C is correct.

However, whether or not the trait is sex-linked or autosomal cannot be determined until the f2’s. F2 Predictions Taking the F1’s from prediction A, autosomal recessive, and crossing them we can expect the offspring to have a 3:1 phenotype ratio and a 2:1:1 genotype ratio N n NNNNn nNnnn Taking the F1’s from prediction C, sex-linked on the females, and crossing them we can expect the offspring to have a 1:1:1:1 phenotype as well as a 1:1:1:1 genotype X¬N Xn XnXNXnXnXn YXNYXnY F2 results: Vial one had AP female > wingless And wild male > winged > F2’s from this group are Female winged: 18 Female wingless: 4 Male winged: 5

Male wingless: 0 Vial Two had AP male > wingless And wild female > winged > F2’s from this group are Female winged: 15 Female wingless: 0 Male winged: 6 Male wingless: 2 Percentage calculations Female total + Male Total > 33 winged 11 winged 4 wingless 2 wingless 37 total 13 total 33/37 = 89% winged Female 11/13 = 85% winged male 4/ 37 = 11% wingless Female 2/ 13 = 15% wingless male

Total winged and wingless percentages 44 winged 6 wingless 50 flies 44/50 = 88% winged (both sexes) 6/50 = 12% wingless (both sexes) Chi – Squared calculation N n NNNNn nNnnn Phenotype: 3:1 Degrees of Freedom (phenotype): 3-1 = 2 Observed Values WingedWingless Sum Males11213 Female33437 Sum44650 Expected Values WingedWingless Males13x44 = 11. 44 5013×6 = 1. 56 50 Female37x44 = 32. 56 5037×6 = 4. 44 50 fofefo – fe(fo – fe)2 (fo – fe)2 fe 1111. 44 -0. 440. 19360. 0176 3332. 56 0. 440. 19360. 0059 21. 56 0. 440. 19360. 0986 44. 44 -0. 440. 19360. 0484 Total : 0. 1705 Critical value at 0. 5 for 2 degrees of freedom = 5. 91 Because 0. 1705 < 5. 991 we can say that the phenotype of the fly is independent of its sex and therefore not sex- linked and based on these results we can devise that the wingless mutation is autosomal recessive Percentage Error = measured value – accepted value x 100% Accepted value PhenotypeObserved Expected(Observed- Expected)(O-E)/EX 100% Error Winged Male 11 11. 44 -0. 440. 03853. 853. 85% Winged Female 33 32. 56 0. 440. 01351. 351. 35% Wingless male 2 1. 56 0. 440. 282128. 228. 2% Wingless female 4 4. 4 -0. 440. 09919. 919. 91% Conclusion In all the experiment turned out well as the Null hypothesis was not rejected and the wingless mutation was autosomal recessive. Our percentages of F2 phenotypes were very close to the expected 3:1 ratio. The chi-squared supported the hypothesis and all the percentage error values were relatively small except for one. This could be due to the lack of specimen collected which would be one suggestion for the next time this experiment is done, which is to collect and produce a greater yield. As more specimens would have made for better and more accurate results.

One other thing to keep in mind for future experiments is to create a detailed journal. This is another possibility for slight error in the type and numbers of specimen as the journal was difficult to understand and draw information from at times. http://www. mun. ca/biology/dinnes/B2250/DrosophilaGenetics. PDF Fruit Fly Lab Journal November 2, 8:55am -Flies were released (they were not separated in time) -The next person must separate the flies (as long as they are there in time) Corey and Matt November 4, 12:36pm -Flies were frozen and separated -2 wild males -2 wingless females -AP male + wild female in one vial AP female + wild male in other vial Matt November 5, 8:51am -No larvae in either new vial -vial #1 – 1 AP female, 2 (+) male -vial #2 – 2 AP male, 1 (+) female -Check back later to see if flies have reproduced Corey and Matt November 5, 3:40pm Checked flies, there was no change. Please release the flies tomorrow morning (Friday) and then freeze and sex them tomorrow at lunch, adding them into the 2nd generation vials until you see many larvae. Matt THESE ENTRIES WERE NOT DATED SO I HAD TO GUESS… November 6, 9:00am The flies were released (just the vials containing (+) and AP) aka the one with all the larvae is left.

The larvae are quite brown, they will probably hatch soon, please check back at lunch, sex and separate them into vials. Corey November 6, 12:20pm The few flies that were sexed from the (+), and the two that were sexed from the AP were added to the flies attempting to reproduce and left for the weekend. Check for larvae on Monday. Vial #Female Male 12 AP3 (+) 24 (+)2 AP Added: Female (+) 3 Male (+) 1 Female AP 1 Corey and Kim November 7, 8:05am The flies in F1 vial #1 and #2 were released. Apparently there ARE eggs in the vials, you just cannot see them, so I guess we have to wait. Corey November 7, 1:00pm

The wrong vials were released this morning. The flies in the P1 vials #1 and #2 were released now. Do NOT release cross-breeds!!! Kim November 7, 3:30pm -2 female (+) -1 male (+) Added to the F1 vials, there were no new wingless virgins. Kim November 8, 8:30am P1 Vial #1 and #2 were released. Kim November 8, 12:50pm There are no flies in the (+) male vial. There is one fly in the (+) female vial. The flies were separated into four vials: -Wingless Female -Wingless Male -Wild Female -Wild Male ** All are virgin and with the same sex and species. Vial #2 – mixed 3 wild female with 0 wingless males

Vial #1 – mixed 1 wild male with 3 wingless female Kim and Alycia November 9, 8:30am We released the flies in the two original vials. Kim and Corey November 9, 12:30pm We sexed the flies from the original vials. One fly was lost, the gender was unknown. Vial #FemaleMale 17 AP2 (+) 23 (+)0 AP Kim, Corey, and Alycia November 10, 8:45am The flies were released; they are already starting to hatch. Please sex and separate fruit flies at lunch. Corey November 10, 12:30pm Sexed the flies: 3 female wingless and 3 male wild were put into vial #1. One fly was lost from the vial. Alycia November 12, 8:10am

Released the flies from the original vials. Corey THERE IS A BIG GAP BETWEEN WHEN WE MADE OUR LAST “UNDATED” ENTRY AND OUR FIRST DATED ENTRY November 17, 8:30am I talked to Mr. Allen, he said we need to release our F¬1`s. The next step is to put the adults from the F1`s into vials. He also said that we need to start dating our entires. Kim November 18, 8:30am The P1`s were released. Some larvae in the F1 vials look like they are close to hatching. Check frequently to monitor them. Matt and Kim November 18, 12:30pm F1s from vial #1 hatched and were sexed and put in F2 vials Vial #1 > F2¬

Wild Male + Wingless Females -winged male -winged male-winged female -winged female -winged female -winged female -winged female -winged female – winged female Two flies were lost…. The total is: 2 winged male 7 winged female They have been put into the F2 vial. Alycia and Corey November 19, 8:30am F1 from vial #1 were put in an F2 vial. AP /(+) males = 0, (+)/ AP females = 11. Kim November 19, 12:15 The new F2 vial from vial #2: -There is one fly in the jar -One winged female was added -Added to F2 vial -3 winged females -Total in F2¬ vial #1, 10 flies. Matt, Kim and Corey November 20, 8:30am

There are plenty of new flies from the F1¬ vial one. Accidentally killed one while switching vials (it was not sexed yet). Only one fly from vial #2 for F1’s. Kim November 21, 8:30am Vial #2 > F2 #2 1 winged female Total in F2 #2 is 2 winged females Vial #1 > F2 #1 3 winged males 4 winged females Total in F2 #1 is 17 flies. (We assumed that on the 18th two females were lost as the flies in the vial were breeding today. ) Corey and Kim November 22, 8:30am The flies from vial #2 were sexed and transferred into vial #2. #2: -5 winged females -3 winged females -1 winged male -2 winged males -2 winged females 2 winged males -2 winged females -3 winged males The total was 12 females and 8 males and so the total for the F2 #2 is 14 females and 8 males. #1 -1 winged male -1 winged female -2 winged females -2 winged males -4 winged males -1 winged female One male died. The total in F2¬ #1 is 6 males and 4 females. Kim and Corey November 22, 12:30pm We checked the vials, no new F1’s in either vial. Kim and Alycia November 24, 8:30am We gave our wild P1’s to Nadine Fitcher’s group. We have one new V2 F¬¬1, one female. We have two new V1 F¬¬¬1¬¬’s one male and one female. Kim November 24, 12:30pm F1 VIAL SPONGES WERE OFF!!!!!

I am not sure how this happened but I definitely put the sponges back on when I left the lab this morning. Luckily, nothing of importance was lost. Kim November 25, 8:30am No new flies in either vial. Kim November 26, 8:25am There are no new flies in either vial. The flies look fat, so I am pretty sure we will see some results soon. Corey November 26, 8:35am We must have just missed each other, strangely enough there are 8 flies in vial one now: 3 males and 5 females. Did not add females to the F2 vial. Kim November 26, 3:45pm Checked in after school, no new flies to sex. Potential larvae in one of the F2 vials. Matt

November 27, 8:45am MACINTYRE SAID TO ADD ALL FLIES!! Flies were sexed and moved from vial #1 to F2 #1: 2 males and 16 females. I lost a couple. I don’t see any larvae in the F2¬ vials yet. Corey November 27, 12:20pm More flies were sexed from vial #1 and transferred to F2 #1. There are 4 males and 10 females. I lost two flies. Corey December 2, 8:45am I moved the parents of vial F2 into new vials. So the old vial F2 just have the larvae and will be able to be sexed when they hatched. Parents can keep mating in new vials. I lost eight from vial F2¬ (+) male + AP female Alycia December 2, 12:30pm

Released the F1 vial, and grown-up, already sexed, F2. Plenty of larvae in the vials, we just need to wait for them to hatch. Kim December 2, 3:45pm Checked after school, plenty of larvae but none have hatched as of yet. A couple appear to be close though. Matt December 3, 8:30am Checked the vials marked ‘larvae’, none have hatched. Please check back at lunch for flies. Corey ps. There are white things crawling in the jar. I am thinking this is a good sign, haha. December 7, 9:10am There are plenty of flies to be sexed. I did not have time to start. I will be in at lunch to sex them, help would be appreciated.

Corey December 7, 11:50am Transferred F2¬ flies into vials. They are currently in the freezer, dying. End results F2 Vial #1 SexDescription -female -female -male -female -female -male -male -female -female -female -female -female -male -female -female -female -female -female -male -female -female -female -female -female-normal, winged -normal, winged -normal, winged -normal, winged -wingless -normal, winged -normal, winged -wingless -wingless -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -wingless -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged normal, winged -normal, winged -normal, winged -normal, winged F2 Vial #2 SexDescription -female -female -male -male -female -male -male -female -female -female -female -male -female -male -female -female -female -female -female -female -male -male -female-normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -wingless -normal, winged -wingless -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged -normal, winged