Lab 3. Inheritance in *Drosophila* • biol200

Notes:

For the pre-lab exercise, first you will learn a little about the life cycle of Drosophila melanogaster and apply your understanding of life cycles to the fruit fly life cycle. Second, you will work through some problems on basic inheritance. Both of these activities will help you prepare to work with the fruit flies in lab and for the inheritance problems we’ll tackle in Lab 4!
This lab is based on Bixler, A. and F. Schnee (2005). Proceedings of the 26th Workshop/Conference of the Association for Biology Laboratory Education
Link to PDF version of this handout.

Objectives:

Practice working with populations of flies (Drosophila melanogaster)
Demonstrate your understanding of mitosis and meiosis.
Be able to construct a Punnett square and predict offspring genotypes and genotype frequencies.

KEY WORDS: model organism; Drosophila; instar; mitosis and meiosis (and all their stages); cytokinesis; chromatid; sister chromatid; centromere; homologous chromosomes; synapsis; crossing over; chiasma; haploid; diploid; allele; genotype; phenotype; parental generation; F₁ generation

1 Background on Drosophila and Life Cycles

1.A The model organism Drosophila melanogaster

Next week in lab, we will apply the Hardy-Weinberg Model to populations of Drosophila melanogaster (pictured below), more commonly known as fruit flies. This week in lab, we will become better acquainted with Drosophila by practicing distinguishing male flies from female flies, and distinguishing flies with Bar eyes from flies with wild-type eyes.

Species of Drosophila occur in the wild all over the world, and they have been adopted for lab work in a variety of fields because they are easy to raise in large numbers in the lab, have a short generation time (about 2 weeks), and have only 4 pairs of chromosomes (2n = 8). Their wide use in laboratory work has made D. melanogaster a model organism: a species that is well-suited for lab work and has been studied extensively in order to provide insight into the biology of related organisms, including humans. For example, in 1933, Thomas Hunt Morgan won the Nobel Prize in Medicine for his work using Drosophila to identify chromosomes as the unit through which genes are inherited. Closer to home, in 2012-13, biology honors student Darrin Shultz used D. melanogaster to study neuronal longevity—work which has implications for Alzheimer’s research.

1.B Life Cycle

A new Drosophila zygote is created when sperm fertilize eggs inside the female parent’s oviduct. The sperm and eggs are haploid, the products of meiosis, while the new zygote is diploid. The female parent then lays the fertilized egg (zygote) on a suitable food source. Eggs are white, ovoid, and about 0.5 mm long. About 21 hours after they are laid, eggs hatch and the larvae emerge.

The larvae develop in stages known as instars, which are common to many insect species. The newly emerged larvae, known as the first instar, are voracious eaters. They are tiny and difficult to see with the naked eye; however, the tunnels made in the food as the larvae eat their way through it are usually visible. The larvae grow rapidly, and within about two days, the first instar will molt into the second instar. These larvae will eat, grow, and molt again to become the third instar. After the third instar crawls out of the food and onto the surface, the larvae begin to pupate. In the pupal stage, the larval body shortens and the cuticle hardens and becomes pigmented, developing into the pupal case. Metamorphosis occurs within the pupal case; dormant localized tissues that originated during the embryonic stages develop into their adult forms, while the remaining larval tissue is broken down to furnish both raw material and the energy needed for adult development. After three days, the adult emerges. The adult flies reach sexual maturity and begin producing new gametes about eight hours after emerging from the pupae, and then the cycle can begin again.

2 Phenotyping mutant and wild-type flies

In next week’s lab about the Hardy-Weinberg Model, we will examine Drosophila for alleles of the Bar gene, which affects eye morphology. An allele is the name for any of the alternate forms or copies of a gene that an organism possesses. We will study the patterns of inheritance of the Bar gene by examining the phenotype of our flies, or their observable characteristics, in order to determine their genotype, or genetic characteristics.

Figure 1: Comparison of three eye phenotypes. [Images: Bixler and Schnee 2005]

The Bar gene is useful to us because (a) the gene is sex-linked, located on the X chromosome; (b) there is a clear phenotypic effect of mutant alleles for this single gene; and (c) the mutant allele shows incomplete dominance (i.e., the heterozygotes are phenotypically different from either homozygote)—which allows us to make the jump from genotype to phenotype. Females that are homozygous for Bar (X^BX^B) and males that are hemizygous for Bar (X^BY) have eyes that are reduced to a slit or bar shape (Figure 1, right). Bar-heterozygous females (X^BX⁺) have a kidney- or heart-shaped eye (Figure 1, center). Wild-type females and males (X⁺X⁺, X⁺Y ) have round eyes (Figure 1, left).

We will also need to distinguish male flies from female flies using several visible anatomical features. Below (Figure 2) are images that can guide you in distinguishing male flies from female flies. One of the easiest methods for distinguishing flies is to look at the ventral side of the tip of the abdomen. In the genital region, the male has a dark pigmented genital disc with anal plates, genital arch, claspers and a penis (Figure 2a). In contrast, females have an unpigmented genital disk with anal and ovipositor plates (Figure 2b). Additionally, males have dark brown-black sex combs on their forelegs (Fig. 2c). The males use sex combs during courtship, and as such they are lacking in females.

Figure 2: Comparison of D. melanogaster by sex. [Images: Carolina Biological Supply Company 2005]

During lab, your instructor will provide you with flies to sort by sex and Bar status and the materials necessary to do so. The flies that your group is counting today are the parents of the flies that you will be working with next week.

We will be sedating the flies using FlyNap, an anesthetic that immobilizes flies for about 50 minutes. To anesthetize your flies, first, you will need to get them into the empty vial provided. See your instructor’s demonstration on how to accomplish this feat. Next, dip the pipe cleaner end of the black wand into the small bottle of FlyNap, being careful to get rid of excess FlyNap by briefly running the wand through the constriction at the neck of the bottle. If any flies are close to the top of the vial, knock them to the bottom by gently tapping the bottom of the vial against your lab notebook (or another padded surface). Then, quickly, put the foam stopper with wand, in place. Avoid letting the wand tip touch the sides of the vial. Leave the wand in the vial until all of the flies have stopped crawling. Finally, spill the flies onto the card provided and separate them by sex and Bar status. Use the table below to record the number of flies of each genotype observed in your group’s population.

Population # ________

Phenotype	wild-type female	heart-eye female	Bar-eyed female	wild-type male	Bar-eyed male
Genotype	X+ X+	X+ XB	XB XB	X+ Y	XB Y
# of Flies

After you finish counting your flies, begin the Mitosis and Meiosis demonstration, after which you may begin the post-lab assignment.

3 Meiosis and mitosis demonstration

By this point in the semester, you’ve been thinking a good bit about meiosis and mitosis – weeks 2 and 3 of class and readings have covered these topics. As the completion of this exercise, you must explain these processes to your instructor in order to verify that you understand them.

Using the snap beads that your instructor will provide, work with your group and practice demonstrating the processes of mitosis and meiosis. Make sure you have the appropriate number of chromatids / sister chromatids / homologs at each step in each process. Remember the differences in how chromatids behave in mitosis, compared to meiosis, and be sure to use the correct terminology to describe the chromosomes and each stage of the process.

Next, think about how meiosis relates to Mendelian inheritance, which uses parental genotypes to predict the expected frequency of each genotype in offspring. Using a differently colored bead to represent the mutant Bar allele, demonstrate the inheritance pattern of the Bar gene in Drosophila.

Imagine that the cell undergoing meiosis is from a Bar-heterozygous mother. Incorporate the new beads into your demonstration of meiosis and answer the questions in the Post-lab Assignment (due next week in lab; can find it HERE).

4 Pre-lab Exercise: Due at start of lab period

Read 1 Background on Drosophila and Life Cycles and 2 Phenotyping mutant and wild-type flies before completing the questions below.

If you could use a refresher on mitosis and meiosis, we suggest one of the following resources:

The course content videos on Mitosis and Meiosis (from week 3)
Chapters 10 and 11 of the online text Biology 2e
Mitosis vs. Meiosis: Side by Side Comparison video by the Amoeba Sisters

For each of the following Drosophila life history stages, mark whether that stage is haploid or diploid:

Stage	Sperm	Egg	Zygote	1st Instar	2nd Instar	3rd Instar	Pupa	Adult
Haploid
Diploid

For each of the following Drosophila life history stages, mark whether meiosis or mitosis is occurring:

Stage	Mitosis	Meiosis
1st instar larvae grow rapidly
2nd instar larvae grow
3rd instar larvae pupate
pupa develops into adult
mature adult flies produce gametes

When the cells of a Bar-heterozygous female undergo meiosis, what types of gametes are produced, with respect to the Bar gene? How many of each type are produced by meiosis of a single cell? What is the percentage of gametes with each allele?

How would the percentages of gamete types differ if…
1. the female was Bar-homozygous?
2. the female was wild-type?

Remember that a female parent only provides half of her offspring’s genotype – the male parent contributes the other half. Predict all possible genotypes for offspring (male and female) of a cross between a Bar-heterozygous female and a wild-type male. What are the expected proportions of each genotype in their offspring?

5 Post-lab Assignment: Punnett squares and populations

Note that this assignment is due at the start of next week’s lab.

One way you can simulate inheritance is through Punnett squares, which provide a graphical representation of the alleles each parent can contribute to their offspring. Punnett squares are an alternative way to carry out the same calculations that you did in the pre-lab exercise.

We’re going to use a Punnett square to predict the genotypes that would result from a cross of our mutant flies, but first, let’s work through a simple cross for a gene encoding a protein that affects body color in Drosophila.

For this example: each parent possesses two alleles for a body color gene, in which B, brown body color, is dominant and b, black body color, is recessive. Both the female and the male parent in this cross are heterozygous for the gene (this gene is not sex-linked).

Draw your Punnett square, following the steps below, and fill in the percentage of offspring with each phenotype.
1. Parental genotypes in this cross:
2. Punnett Square:
3. What percentage brown-bodied offspring do we expect? __________
4. What percentage black-bodied offspring do we expect? __________

The inheritance of Bar works a little differently than the example of a gene encoding a protein affecting body color. As you’ll remember from section 2 Phenotyping mutant and wild-type flies, the Bar allele is inherited on the X chromosome, making it sex-linked in this species.

In the example about body color, each parent has two alleles for the gene that they could contribute to their offspring. Is this situation true for the Bar gene? If not, then how many alleles (at most) might the female parent have for the gene? The male parent? How would these numbers change if the gene were on the Y -chromosome (which is another way that a gene can be sex-linked)?

How would you set up a Punnett square to accommodate sex-linked alleles? Below, work through a Punnett square to determine the genotypes produced in a cross between a Bar-heterozygous female and a wild-type male.

For a population with those 3 alleles of this sex-linked Bar gene (X⁺, X^B, and Y , which is like a “null” allele here):
1. How many genotypes are possible (remembering that a genotype is determined by how many of which alleles are present in an individual)? List them.
2. How many eye phenotypes are possible? List them.
3. Which, if any, genotypes are found in both sexes?
4. Which, if any, eye phenotypes are found in both sexes?
So, you’ve already completed a Punnett square for one possible cross that could occur in a population with Bar mutants! In the “Crosses” column below, write out the five remaining possible crosses. Draw out Punnett squares (in the space below) for each of the additional crosses, and then calculate expected genotype frequencies. Below the columns for each of the genotypes, write the proportion in decimal form (not percentage and not fraction) of offspring with that genotype that you expect to result from each cross. Be careful about calculating the frequencies - remember that Bar males and Bar females have the same phenotype but NOT the same genotype. The sum of all genotype frequencies for a cross will be 1 (with half of that from males and half from females).

a	b	c	d	e	f
_	Expected genotype frequencies
_	Female genotypes			Male genotypes
Cross	X+ X+	X+ XB	XB XB	X+ Y	XB Y
X+ XB x XB Y
a
b
c
d
e

Note that offspring have a 50% chance of inheriting each allele from each parent. How could you use this knowledge to write an equation to calculate the probability of a given offspring having a genotype without physically counting the frequency of each genotype in the offspring population? Below, write and clearly label an equation calculating the probability that a particular offspring from a cross between a Bar-heterozygous female and a Bar-hemizygous male will be a Bar-homozygous female. Explain how you created the equation.

Remember that since males have the chromosomes XY , a male has to get his only X chromosome from his female parent (in contrast to his sisters, which get X chromosomes from both parents). Given this information, and what you know about how Bar is inherited, what general rule could you devise to predict the frequencies of the male genotypes of any particular cross? Note that this should be one rule that applies regardless of the female’s genotype (not a rule listing every specific possible outcome).

You’ve predicted the genotypes of the F₁ generation, or offspring, of a cross between 2 flies. However, in the coming lab, we’re going to be working with populations of flies, not just one set of parents. Remember that genotypes result from the inheritance of alleles, and that when you have one set of parents with two alleles each, the offspring have a 50% chance of inheriting either allele from either parent.

But when you’re looking at a population of individuals instead of just one set of parents, the chance of one offspring chosen at random from the population inheriting a particular allele may not be 50%. Consider a population of Drosophila in which 80% of the alleles (not the phenotypes) for a non-sex-linked gene are wild type, and there is only one other allele for that gene.
Using a Punnett square, how would you use this information to calculate the frequency of each genotype in the F₁ generation of the above population? Write out your plan, and then show and clearly label all of your calculations.

Lab 3. Inheritance in Drosophila

February 20 & 21, 2024