Carolina Labsheets™

Drosophila is widely used model organism for the study of genetics; however, one hindrance to using Drosophila is the need to select virgin female flies for crosses. The use of our Drosophila F₁ Crosses removes this obstacle. Our technicians select virgin female flies and set up the crosses to produce the F₁ cultures that we ship. The F₁ flies that emerge in these cultures can mate only among themselves, so your students need only to collect F₁ flies and set them up in fresh vials to produce an F₂. Each F₁ culture can be used to set up about 8 vials to produce F₂ flies.

172020 F₁ white × wild

A vial of F₁ flies from a cross between white-eyed (172220 white) females and red-eyed (172100 wild type) males. White (w) is recessive and is located on chromosome 1. Chromosome pair 1 consists of the sex chromosomes. The locus for white is on a position on the X chromosome that has no homolog on the Y; thus, white is inherited only through the X chromosome, making white a sex-linked (or X-linked) gene.

Since the crosses must be set up before shipment, please give 2-week notice for delivery of the cultures; otherwise, the crosses may not be available when you need them.

Needed Materials

F₁ Drosophila Cultures

empty culture vials and plugs (173076)

vial labels or wax pencils

Formula 4-24^® Instant Drosophila Medium (173200)

FlyNap^® Kit (173010)

alcohol

Drosophila sorting brushes (173094)

index cards to use as sorting trays

Carolina™ Drosophila Manual (452620)

stereomicroscopes

Optional Materials

Carolina Drosophila Stand (173030)

Our 173050 Drosophila Culture Kit includes 36 vials and plugs, labels, FlyNap^® Kit, Formula 4-24^®, sorting brushes, sorting cards, and a Carolina™ Drosophila Manual.

Drosophila Care Guide

Safety

Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Use personal protective equipment such as safety glasses or goggles, gloves, and aprons when appropriate. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.

Procedures

Students can work individually or in groups of 2–4.

When your cultures arrive, open the package immediately and inspect the cultures to verify that they have arrived in good condition. Examine the label on each vial. The label is divided into four parts. The upper left quadrant gives information on the female flies used for the cross, and the upper right quadrant gives information on the male flies. The lower left quadrant is stamped with the date that the parent flies were placed in the vial. F₁ flies should begin emerging 12–14 days after the date on the label.

Each culture will produce approximately 100 flies over a 10-day period and will serve 6–8 groups.

Either prepare a vial with media for each student group or provide materials and instructions to the students so they can prepare their own vials. Materials needed are culture vials with plugs and Formula 4-24^® Instant Drosophila Medium. Open the bag of medium and locate the small measuring cup and packet of yeast. Using the small cup, add a level cup of medium to each vial. When all the vials have dry medium, follow with a level cup of cool water. Open the yeast packet and sprinkle 6–8 grains of yeast onto the surface of the medium in each vial. Do not add too much yeast. The yeast produces CO2, which in large amounts can cause sterility or even death of the flies.

Either prepare the fly morgues with alcohol (one morgue is included in each FlyNap^® Kit) or provide materials and instructions so the students can do this themselves.

Observing F₁ Flies and Setting Up F₂ Cultures

Anesthetize flies before the lab and dump them onto sheets of paper. When the FlyNap^® wand has been inserted into a vial, either place the vial upside down on a Carolina Drosophila Stand or lay the vial on its side to prevent the flies from dropping onto the media and possibly becoming stuck. At the beginning of the lab students pick up flies on index cards.

Students will also need the following materials for doing initial observations and setting up cultures for F₂ flies:

vial with medium and plug
vial Label
FlyNap^® Kit (if flies are to be anesthetized by students during lab)
sorting brush
index card
stereomicroscope

Clearing F₁ Parents from the F₂ Vials

Seven to 10 days after the F₂ vials are set up, the F₁ flies need to be removed from the F₂ cultures. This is done to avoid including the F₁ flies in the F₂ counts.

The following materials are needed:

FlyNap^® Kit (for every 6–8 groups of students)
sorting brush
index card
stereomicroscope

Scoring Phenotypes

F₂ flies will begin emerging about 12 to 14 days after students have set up their vials. Begin scoring phenotypes of the F₂ on the day after they first begin emerging. More females than males emerge on the first day, but the ratio evens out on successive days. Scored flies should not be returned to the vial but must be disposed of in a morgue to prevent them from being counted twice. Phenotypes can be scored every other day for up to 10 days. Counts carried beyond 10 days risk including flies of the next generation.

For scoring the phenotypes of the F₂ flies, students will need their F₂ vials and the following materials:

FlyNap^® Kit (one for every 6–8 groups of students)
sorting brush
index card
stereomicroscope

Optional

As an option, you can provide cultures of the parent flies of the cross. Students can compare the phenotypes of the parental flies to the F₁ culture to see which is dominant.

For a demonstration of the use of FlyNap^®, students can watch our Observing Phenotypes with Drosophila melanogaster video.

Chi-square analysis is not included as part of the activity, but it can easily be added. There are several Internet sites that will perform the calculation from input data.

Answer Key to Questions asked on the Student LabSheet

	Red Eyes	White Eyes
Female	6	0
Male	0	6

Numbers will vary, but students should find that all the females have red eyes and all of the males have white eyes.

1. Morgan’s original white-eyed fly was a male. When he crossed this male with red-eyed females, only the male offspring expressed the white-eyed phenotype. Morgan’s original assumption was that the allele for white eyes was lethal in females. Was Morgan correct? Give evidence for your answer.
   Morgan was not correct. The female parent flies we used to make this cross had white eyes. If white were lethal to female pupae, there would be no adult females with white eyes.
2. Why is it necessary to remove the F₁ flies?
   So that they will not be counted with the F₂ flies that emerge in the vial. Some students may think that it is because the F₁ flies might mate with the F₂; however, since the activity will end before the F3 flies emerge, this is not of much concern.

Table 2: F₂ Phenotypes and Numbers of Flies

Date Counted	Phenotype 1	Phenotype 2	Phenotype 3	Phenotype 4
Total	56	47	59	58

Numbers will vary but the totals should suggest a 1:1:1:1 ratio.

3. Assume that this is an autosomal monohybrid cross with the allele for red being dominant to the allele for white. What would be the expected phenotype ratio for the F₂? Does your data support this explanation of the inheritance of white eye color?
   The expected ratio would be 3:1. The observed ratio as about 1 red-eyed for every white-eyed fly. This does not support the assumption that this is an autosomal monohybrid cross.
4. Assume that this is an autosomal dihybrid cross with a gene locus for sex determination and a second unlinked gene locus for eye color. What would be the expected phenotype ratio for the F₂? Do your data support this explanation of the inheritance of sex and white eye color?
   The expected ratio would be 9:3:3:1. The observed ratio is about 1:1:1:1. This does not support the assumption that this is an autosomal dihybrid cross. Some students may point out that these results look like the results of a testcross. In a sense this is correct, but the results are not produced by backcrossing with the recessive parent stock.
5. Assume that the locus for white eye color is located on the X chromosome and that there is no corresponding locus for white eye color on the Y chromosome. Use this assumption to diagram the crosses you have been studying. Show your work below.
   X^w+    X chromosome with allele for wild-type eyes
   X^w     X chromosome with allele for white eyes
   Y       Y chromosome without locus for eye color

Parental Cross	Xw / Xw	x	Xw+ / Y
	Female Parent		Male Parent

F1 (give genotype and phenotype)

Xw / Xw+ (female, red eyes)      ,

Xw F / Y (male, white eyes)

Cross of F1 to produce F2 (give genotype)	Xw / Xw+	x	Xw / Y
	Female Parent		Male Parent

Draw Punnett square Here

6. On the basis of your Punnett square, give the expected results for the F₂.
   1 white-eyed female: 1 red-eyed female: 1 white-eyed male: to 1 red-eyed male.
7. Do your data support the assumption that the allele for white eyes is inherited on the X chromosome?
   Yes, the pattern of inheritance can be explained by assuming that the allele for white eye is on the X chromosome and that it is recessive to the wild-type (or normal) allele.
8. When Thomas Hunt Morgan began his work on white eye color, he was skeptical of sex determination by X and Y chromosomes. As he continued his work with Drosophila, he became a supporter of the XY hypothesis. What does this tell you about Morgan?
   That he was able to abandon his preconceived ideas in the face of evidence to the contrary