How do you distinguish male and female Drosophila flies in a genetics experiment?

Males are slightly smaller with a darker, solid pigment spot on the end of their abdomen, while females are larger with striped patterns rather than solid spots. Accurate sex identification is critical before crosses, as any males in the female vial indicate the females have already mated and are unusable for the experiment.

Why must virgin females be used in fly genetics crosses?

Virgin females are unmated and free from prior sperm, ensuring that observed offspring traits result only from the intended cross. If males are present in the female vial, the females likely have sperm from males in their own strain, making them unsuitable for determining dominance and sex-linkage patterns.

What is the purpose of using Punnett squares before performing fly crosses?

Punnett squares allow you to formulate testable hypotheses by predicting offspring genotypes and phenotypes under different dominance scenarios. By comparing predicted outcomes with actual data, you can determine whether red or white eye color is dominant and whether the trait is sex-linked.

How does a reciprocal cross help determine if a trait is sex-linked?

A reciprocal cross reverses the sexes of parents in the original cross. If results differ between the two crosses, the trait is likely sex-linked. For example, if red-eyed females crossed with white-eyed males produce different offspring ratios than white-eyed females crossed with red-eyed males, sex-linkage is indicated.

What should you do with adult flies after larvae appear in the vial?

After about three days, when larvae are moving around the vial, remove the ten adult flies and dispose of them in a morgue tube containing 70% ethanol. Return the tube to the incubation rack and allow larvae to mature into adults over up to two weeks before analyzing phenotypes and sex ratios.

How do you prepare flies for phenotype and sex analysis after they reach adulthood?

Anesthetize the adult flies using a fly anesthesia wand dipped in anesthetic solution. Once immobilized, sort the flies by sex and phenotype on a white paper, then record the counts for each category to compare against your Punnett square predictions.

What does it mean if your experimental data does not match your Punnett square predictions?

Mismatched data may indicate the gene is sex-linked, meaning the trait is controlled by alleles on the X chromosome. Compare your results with other lab groups and analyze whether sex-specific inheritance patterns emerge. If data still does not fit standard hypotheses, consider alternative genetic mechanisms or experimental variables.

Genetics of Organisms

Concepts Instructor Prep Student Protocol

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An inheritance is something passed from one generation to another. In some contexts, this means stuff like houses and money. In the context of biology, however, the study of genes and how they are inherited, is called genetics. Gregor Mendel is credited with being the father of modern genetics. His work is responsible for our understanding of how noticeable physical traits, or phenotypes, are passed from one generation to the next. Famously, he studied such traits in pea plants.

The pieces of information controlling those phenotypes are called genes. As there are two copies of each gene, known as alleles, we can represent these as letters. Here, we'll use the letter P. This is the first step of Mendel's famous experiment on pea flower coloration, represented here, using a tool called the Punnett square. Mendel found that when he crossed purple flowers with white ones, all of the progeny, or the first-generation plants, had purple flowers. This is because the purple color is dominant, shown using uppercase P. And carrying even one dominant allele means the phenotype will be expressed. But interestingly, when these purple flowers were crossed again, 1/4 of them were white. Where were the white flowers in the first generation? All of the first generation of purple-flowered plants were heterozygous, meaning that they had one capital purple P and one lowercase, or white, P. When they passed on their alleles in the F2 generation cross, this meant that 1/4 of the offspring received two small P alleles, and so, expressed the white phenotype. Mendel didn't have the Punnett square tool to use, he had to figure all of this out by keeping track of thousands of plants, and then noticing patterns in their numbers.

From this evidence, and a lot more, we now know that genes are also present in two copies in other organisms, too, like humans, and flies. In following up on Mendel's work, several scientists found that not all inheritance patterns followed the simple, basic model that Mendel proposed. For example, in hemophilia, a genetic clotting disorder, unaffected mothers were capable of transmitting the disease to their male children. The reason behind this lies in chromosomes, which were studied by Thomas Hunt Morgan, using his famous fruit flies, Drosophila. Because of Morgan and others, we now know that chromosomes are long strands of DNA that typically exist in pairs. Here, we can see that Drosophila has four of them. These chromosomes have genes on them for different traits, much like how a cookbook contains lots of different recipes. Nowadays, using modern microscopy, we can actually see these chromosomes and even organize them. The product of this process is called a karyotype. Here, you can see a human one. In both humans and flies, there are autosomes, and sex chromosomes. Humans, like flies, have X and Y chromosomes controlling their sex. However, most of the genes on these chromosomes control things that have nothing to do with sex. In the rare form of hemophilia that we mentioned earlier, the reason that it occurs more frequently in males, is because the phenotype is controlled by a gene found on the X chromosome, in a section which has no partner on the Y chromosome. If a female has a bad copy of the gene, and her male child inherits this copy, he will have the disorder, he has no backup copy of the X chromosome. Since one copy of the gene is sufficient for a person to clot normally, a female must inherit two bad alleles of the gene, one from each parent, in order to exhibit the disease. Which, in this case, is not possible, because the father is unaffected. As a result, this type of hemophilia affects more males than females.
In this lab, we'll look at inheritance in Drosophila. Eye color in flies is controlled by a series of genes, some controlling what kinds of pigments are made, and one particularly important gene, called the ABC transporter, which controls transports of pigments into granules in the eye. If that gene is broken, even if the fly is making pigment, that pigment will be invisible, and the fly will have white eyes. The lab exercise is to recreate one of Thomas Hunt Morgan's most famous experiments, and explore the genetic inheritance pattern of the gene encoding the pigment transporter. Is it inherited like the purple color of Mendel's pea flowers, or is it sex-linked, like hemophilia?

Print Procedure Steps

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REF and WEF Fly Genetics
- NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
- Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
- Draw a line in the middle of the paper to make a male side and a female side.
- Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
- Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
- Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
- Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male. NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
- If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
- Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
- Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
- To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
- Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
- Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
- Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
- Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
- Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
- Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
- Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
- Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
- Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 – 18).
- Finally, once you have counted all of your flies, dispose of them in the morgue vial.
Results
- To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
- Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
- If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
- If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
- If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
- When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
- Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
- Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.