Using Drosophila melanogaster in middle and high school classrooms

Transcription

1 Laboratory Handbook Using Drosophila melanogaster in middle and high school classrooms Drosophila Handbook page 1

2 Table of Contents This handbook was designed as a resource for classroom teachers interested in using Drosophila in laboratory experiments illustrating principles in genetics and evolution. First, basic techniques and resources are described. Then, I provide three handouts that could be easily modified into laboratory protocols. Many of the ideas in this handout have been adapted from Evolution Laboratory Notebook (Mueller, Long, and Rose 2008) for the 115L biology course at UC Irvine. Working with Drosophila Why Drosophila 3 Useful Drosophila facts 3 Drosophila life cycle 6 Fruit Fly Genetics and Biology Laboratory Part I 7 (Middle school, basic) Laboratory Part II 9 Insect Metamorphosis 10 Sexual Selection Introduction 11 (High school, intermediate) Methods and Procedures 12 Assessment Questions 14 Random Genetic Drift Introduction 15 (High school, advanced) Materials and Procedures 18 Assessment Questions 21 California Science Content Standards 22 Drosophila Handbook page 2

3 Working with Drosophila melanogaster Why Drosophila? All the experiments in this handbook involve the common laboratory fruit fly, Drosophila melanogaster. There are a number of reasons why we use fruit flies. The lab fruit fly has been used for research in evolution and genetics for the last 100 years, so we know a lot about it. The fruit fly is easy to raise in large numbers and it has a short generation time. The short generation time makes the fruit fly convenient for studying multi-generation phenomena, like evolution. Useful genetic mutants of Drosophila and specially created lines are already available. These genetic variations allow us to do experiments that could not be done with almost any other organism. As of the first decade of the 21 st Century, Drosophila is one of a select group of animals that has had entire genomes sequenced. This gives us a solid foundation of genomic information for specific studies of genetics and evolution. Useful Drosophila facts How to handle Drosophila The life cycle of Drosophila melanogaster is outlined in the figure on page 5. Experiments in this handbook involve the handling of adults only. Although it is possible to handle eggs and larvae, it is considerably more difficult than handling the adults. Because the adults can fly, they need to be knocked out before you count, sex, or genetically type them. There are several ways to do this: 1. Chilling Putting vials of flies on ice immobilizes them. Although this is the simplest way of knocking flies out, it is not the most effective. As soon as flies are taken off ice, they wake up, so this may not be practical in the classroom. 2. Carbon dioxide Most research labs at UC Irvine knock flies out using CO 2 anesthesia. It is a simple and non-hazardous method, but it does require a set-up with tanks, regulators, and tubing. Industrial suppliers deliver large cylinders of CO 2 for about $12, but regulators are very expensive. Drosophila Handbook page 3

4 3. Commercial anesthetics, such as FlyNap FlyNap is extremely easy to use in the classroom. It comes as a vial of fluid with about 8 wands that look like mascara applicators. These wands can be dipped in the fluid, then in the vial where flies are active. An exposure time of < 1 minute knocks flies out for at least 30 mins, although effects are not immediate. FlyNap is non-toxic (active ingredient = triethylamine), but it has a very strong smell that can be irritating. For this reason, you should always use FlyNap under a fume hood. How to sex Drosophila Males and females are easily distinguished (see below). In addition to being much smaller than females, males have a solid black patch on the tip of their abdomen. A ring of bristles surrounds their genitals (the genital arch ). Females lack the black patch at the tip of the abdomen, and have brown stripes across the back of their abdomens. In very young adults, the pigments in the bodies may be very light. This makes sexing the adults much more difficult. It is important to be aware of this, because the labs in this handbook sometimes require virgin flies that have not yet mated. To obtain virgins, one must separate males from females within 8 hours of emergence from the pupa. Drosophila Handbook page 4

5 How to feed Drosophila In nature, fruit flies eat fermenting fruit. Most recipes for laboratory fly food are based on some fruit type, commonly bananas or grapes. Other ingredients include corn syrup or molasses for extra sugar, ethanol, yeast, and gelatin for solidification. Although it is easy to prepare this food, media is readily available from the Developmental and Cell Biology Media Facility in McGaugh Hall at UCI. This facility provides food in plastic 8-dram vials. How to obtain Drosophila Many educational supply vendors sell a large variety of stocks of Drosophila at reasonable prices. The drawback to this is that they come in units of 1-2 vials, which only house about 100 flies. While it is easy to build up numbers of the next generation (one female lays up to 70 eggs per day!), this takes time. Most university labs that use Drosophila for research are more than happy to provide large numbers of flies to teachers for free. But, individual labs may not have mutant stocks you d like to use in classroom experiments. How to maintain Drosophila Fruit flies are extremely low-maintenance, which is of course one reason they are so ubiquitous in research. Once they are in a vial with food, they do not need additional food or water. But, since adults can reproduce throughout their life, untended vials do get messy. So, the best way to maintain flies is to move groups of adults to fresh vials, let them lay eggs overnight, and then dispose of the adults. This is easily done by making a morgue consisting of a jar filled with soapy water, putting a funnel over the opening, and emptying flies into the water. It s fine to pour the morgues down the sink, but be careful that large number of flies could clog pipes. Since it takes flies to metamorphose from egg to adult in at least 10 days, most university research labs find it easy to transfer flies in this way every two weeks. Flies like warm temperatures, and if an incubator is available, it is ideal to keep them at a constant 25 C. However, the southern California climate, especially in the spring and summer, is usually plenty warm enough to maintain flies. If you keep flies in your classroom, try to keep them in a part of the classroom that stays at a relatively constant temperature. If the temperature dips below 22 C or so, flies may slow their development. Viability will start to become affected at about 15 C. Drosophila Handbook page 5

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7 Fruit Fly Genetics Part I In this lab, you will use the model organism Drosophila melanogaster (the fruit fly) to study genetics. You will make crosses between fruit flies with different eye phenotypes. The R allele codes for red eye color and is dominant. The r allele codes for white eye color (no pigment) and is recessive. Based on your knowledge of genetics, you will need to make predictions about what the offspring s phenotypes will be when a homozygous RED MALE is crossed to a homozygous WHITE FEMALE. I. Purpose The purpose of this experiment is to observe a living Punnett square. You will cross fruit flies with genotypes you know, and predict what the offspring s genotypes and phenotypes will be. Then, after offspring have actually been produced, you will compare your expectations to your results. II. Hypothesis 1. Draw a Punnett square that shows your predictions as to what the offspring genotypes will be after a cross between a white-eyed male (rr) and a red-eyed female (RR). Put the male s alleles on top of the Punnett square and the female s alleles to the left. 2. What phenotypes do you expect to observe in these offspring? Drosophila Handbook page 7

8 III. Materials Vial of red-eyed flies, vial of white-eyed flies, microscope, paintbrush, FlyNap, four vials of fresh fly food, cotton balls, paper towels, Petri dishes, tape IV. Procedures (you will work in groups of four) ** You may split the work within your group so that one pair of students separates the whiteeyed flies at the same time the other pair separates the red-eyed flies. 1. Use FlyNap to put your vials of flies to sleep. Carefully place the wand into the vial without removing the cotton plug. Leave the wand in the vial for about 5 minutes. 2. Empty the vial of red-eyed flies into a Petri dish and place the dish under the microscope at low power. 3. Using a paintbrush, separate the males from the females. 4. Place one male in each of the four fresh vials. Plug the vial with a cotton ball. 5. Empty the vial of white-eyed female flies into a Petri dish and place the dish under the microscope. 6. Using a paintbrush, place one female in each of the four vials with a male in it. Replug the vial. 7. Label each vial using take with your group number and period on it. If your flies wake up at any time, let Ms. Burke know. V. Observations Using color, draw what each of the four types of flies look like under the microscope at low power. Red-eyed female Red-eyed male White-eyed female White-eyed male You will record your observations of offspring phenotypes in two weeks! Drosophila Handbook page 8

9 Fruit Fly Genetics Part II Two weeks ago, you mated one female white fruit fly to a male red fruit fly. Their offspring have grown up. What do you observe? Results 1. What were you expecting the offspring to look like? 2. Collect data: record the number of offspring you observe that are male, female, whiteeyed, and red-eyed. Red-eyed male Red-eyed female White-eyed male White-eyed female 3. Do your results fit with your hypothesis? Use the Punnett square you drew in your lab notebook to answer this question. 4. Ms. Burke lied to you during your last lab. Eye color in fruit flies is not a completely dominant trait it is sex-linked. That means that females can have two copies of the trait, but males can only have one copy. Make a new Punnett square that shows what should happen when you mate a white female (X W X W ) to a red male (X R Y). Does this explain your results? Drosophila Handbook page 9

10 Insect metamorphosis Fruit flies go through a developmental process called complete metamorphosis. That means that they look very different at different stages of their lives. Go to the station Ms. Burke has set up with a different developmental stage under a different microscope. Draw each developmental stage that you see in detail. Egg Stage How long does the stage last? 1 day Drawing Larva 5-6 days Pupa 4-5 days Adult Up to 2 months Drosophila Handbook page 10

11 Sexual Selection Introduction In animals that have two sexes, there is often a difference in the time and energy each will devote to reproduction. Usually, the female will make the larger investment of time and energy since she will produce eggs that require more energy to produce than sperm. Also, for some animals, females also care for the offspring after birth, while males do not. But in many cases among the fish species, the most abundant vertebrates, the male invests most in the care and feeding offspring. In fact, in seahorses, males get pregnant and incubate their offspring until they give birth, at which time their offspring emerge from their large brood pouch. In either case, it usually happens that the parent that invests more energy into reproduction ends up choosing when to mate and who to mate with. As a result of this asymmetry in the decision making process, the sex which invests less energy in caring for offspring, which in insects is usually males, will compete among themselves to be chosen as mates by females. This type of competition is called sexual selection. If there are characteristics that are inherited by males that give them some advantage in this competition for mates, such as structures or behaviors that females find attractive in a prospective mate, then we can expect sexual selection to favor these characteristics. In some species, like peacocks, males have elaborate coloration, long tails or build complex structures to attract females. All of these characters are thought to result from evolution driven by sexual selection. In some cases, sexual selection may have produced awkward male morphology or dangerous competitive male behavior that can actually reduce male fitness, compared to the fitness that males might have achieved if females did NOT discriminate among them. Therefore, there is potential for conflicts between the effects of sexual selection and natural selection. Natural selection may successfully oppose sexual selection, preventing the evolution of extreme morphology or behavior. For instance, brightly colored males may be more attractive to females but may also be more conspicuous to predators. It is interesting to note that birds are generally more colorful than mammals of similar size, suggesting that the greater ability of birds to flee from predators may have tilted the balance toward less camouflaged sexual plumage. Thus, the evolutionary dynamics of sexually selected traits may be quite complicated. Laboratory sexual selection will be studied by measuring a component of male fitness called virility. In biology, the term virility refers to the relative success of males in being chosen as mates by females, when multiple males are striving to mate with the same female(s). Drosophila Handbook page 11

12 Materials Fly anesthetic (NOTE: for this experiment, ice will work best), paintbrushes, 10 vials of fresh fly food, cotton balls, supply of red-eyed males, supply of white-eyed males, and at least 10 unmated (virgin) red-eyed females. Procedures Each laboratory group will have a population of experimental males that are wild type (red-eyed). The virility of these males will be tested against males that carry the white (w) allele on the X-chromosome. This experiment allows females to choose between males with red eyes and males with white eyes. 1. Place one red-eyed male and one white-eye male in each of ten vials. Make sure that you do not expose males of one eye color to more anesthetic than males of the other eye color. 2. After both males have recovered from anesthetic, about minutes, take 10 red-eyed virgin females and place each one in each vial. By letting both males recover fully you ensure that neither has an advantage over the other. 3. Carefully watch the females. When a female has mated for more than 30 seconds record the type of male she mated with. It takes male fruit flies more than a few minutes to transfer sperm, unlike most mammals, so a mating that only lasts likely not a successful fertilization. 4. Stop recording data after 20 minutes. Sexual Selection Experimental Data Number of matings Male Your group Entire class Red-eye White-eye Total Drosophila Handbook page 12

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14 Assessment Questions 1. In your experiment do the females show a preference for white-eyed or red-eyed males? 2. Summarize the results of the entire class. Calculate the average proportion (HINT: denominator of each group s proportion will be 10) of successful matings for white-eyed and red-eyed males. Are the average proportions different? 3. Given your results, what do you think would eventually happen to the number of whiteeyed flies in the class population of flies if they were allowed to continue mating for many generations? Use the terms fitness and sexual selection in your answer. 4. Imagine that in nature, there are equal numbers of white-eyed and red-eyed males. If sexual selection is favoring one type of male, this shouldn t happen. Explain how this could happen, and use the term natural selection in your answer. Drosophila Handbook page 14

15 Random Genetic Drift Introduction Gene, or allele frequencies fluctuate in populations due to random effects; meiosis and recombination result in random joining of gametes to create the fertilized eggs of the next generation. This does not happen because of external factors, such as the environment. It is just like the fluctuation in the amount of money you have when you play poker or blackjack. Sometimes the cards favor you. Sometimes they favor another player. In the same way, luck sometimes favor one allele over another. This process is called genetic drift. Genetic drift is particularly strong when the population size is small. But on average the effects of drift are not biased. It is just as likely that random drift will cause a particular allele to increase OR decrease in frequency. We can t predict what will happen to any one allele s frequency over a long period of time, as a result of drift. But we can predict that drift will tend to cause populations that were initially the same in allele frequency to become different, over a long period of time. The best way to see the effect of drift is to monitor allele frequencies in a large number of populations that are made up of a small number of individual organisms. Because the effects of drift are not directional, we expect the average frequency of a particular allele over all the populations to remain about the same. But the variance of the allele frequency, among populations, should increase as evolution proceeds. You can think of the variance as a measure of how much allele frequencies vary among populations. High variance among populations means that the allele frequencies of different populations are quite different from each other. Low variance means that the allele frequencies are quite similar. For example, if we monitor ten populations for the frequency of allele A at a locus, then if there is low variance the ten allele frequencies might be: 0.12, 0.11, 0.13, 0.11, 0.09, 0.10, 0.08, 0.11, 0.12, 0.11 Alternatively, if there is high variance, the ten allele frequencies might be 0.01, 0.34, 0.42, 0.03, 0.22, 0.67, 0.32, 0.12, 0.00, 0.55 Our theoretical prediction is that, if you start with the ten low-variance allele frequencies, genetic drift for many generations might produce the ten high-variance allele frequencies. However, this prediction does not allow us to say exactly which population will evolve to which particular allele frequency. Our ability to predict what will happen with genetic drift is not that strong. Drosophila Handbook page 15

16 The Wright-Fisher Model The major goal of our genetic drift experiment will be to observe the effects of random genetic drift on the mean and the variance of allele frequencies over several generations. Basic Concept of the Wright-Fisher Model Sewall Wright, an American biologist, and Ronald A. Fisher, an English statistician, independently developed a very simple model that shows how genetic drift works. We will describe this model in general terms now. Suppose that we have N diploid individuals in a population. Then there are a total of 2N alleles at a diploid genetic locus in this population. If we assume that these individuals mate by shedding their gametes into a common pool, like some fish do, then the frequency of gametes that bear a particular allele from a particular individual is on average just 1/2N. If the frequency of a particular allele (say the allele is called A) in the population is given by p, then on average there will be p times 2N gametes of that allele. But sometimes a heterozygous parent will not generate gametes that are exactly fifty-fifty, or even, frequencies of the two alleles that it carries. This is a principle that is used all the time in Mendelian genetics. If a heterozygous (Aa) parent generates two gametes, half the time they will be A and a gametes, one quarter of the time they will be two A gametes, and one quarter of the time they will be two a gametes. In the same way, chance is involved in the combination of gametes in a population of size N. Each fertilization event involving two gametes can have a variety of outcomes: both gametes can have A alleles, both can have a alleles, or one A and one a allele-bearing gamete can combine. In a population with both alleles, it is mathematically possible that all offspring will end up AA homozygotes. Or they could all be aa homozygotes. That is, genetic drift can accidentally fix one allele or the other, even if the previous parental generation is genetically polymorphic. In addition to this extreme possibility, the same sort of sampling effect can cause the allele frequency of a population to rise or fall, even though there is no directional evolutionary mechanism, like selection, acting on the population. The mathematics that underlies the Wright-Fisher model is that of combinatorics. But don t let this term impress you. You use combinatorics every time you play a game of chance. Combinatorics tells us that getting dealt a bridge hand of thirteen cards all of the same suit (Hearts, Spades, Diamonds, or Clubs) is very rare compared to getting a mixture of two or more suits. Similarly, the changes of being dealt all four Aces and a King in five-card stud poker is very rare. In the same way, the accidental fixation of the A allele in one generation is an improbable (but not impossible) event in a population of ten individuals if there are only ten (out of a maximum number of 20) copies of the A allele in the parents of the preceding generation. Drosophila Handbook page 16

17 Quantitative Predictions of the Wright-Fisher Model There are several important theoretical results that have been mathematically derived from the Wright-Fisher model of genetic drift that will be illustrated by our genetic drift experiment. Here we will give the major theoretical results for the Wright-Fisher model, results that supply us with predictions for our experiment. Suppose that, in a small population with effective population size N, the initial frequency of an allele is p 0. At some time in the future, t, the frequency is p t. The Wright-Fisher model predicts the following: Let E represent the expected or mean value for a variable. Then the symbol E(p t ) stands for the expectation of the random variable p t. This is similar to the mean of the random variable p t. E( pt+ 1 ) = E( pt ), (1a) In words, this means that genetic drift does not, on average, change the frequency of an allele. The variance ( Var ) is a measure of dispersion about the mean value. The greater the variance, the more individual values deviated from the mean or expected value for a variable. There are two variances that we can predict when genetic drift occurs. The first of these variances is the variance in allele frequencies that arises from a single generation of random sampling of gametes in the creation of the next generation. Let Var(p t+1 p t ) represent the variance in allele frequencies at time t+1 given that the allele frequency was p t at time t. This is the variance of allele frequencies due to just a single generation of drift, which is given by the following equation. pt ( 1! pt ) Var( pt+ 1 pt ) =, (1b) 2N Notice from this equation that, if N is huge, there will be virtually no genetic drift in a single generation, because N appears in the denominator of the right-hand side of the equation. The second variance that we are interested in is the accumulated variance over the entire sequence of generations in which genetic drift occurred. Let Var(p t ) represent the variance of the allele frequency at time t. It is given quantitatively by the following equation. 1 t [ 2 N ] Var( p ) = p q 1! ( 1! ). (1c) t 0 0 Bear in mind that equations 1(a-c) are theoretical predictions from the Wright-Fisher model. It is possible that the actual mean and variance in this experiment may be different from these predictions. One goal of this experiment will be to compare the observed mean and variance in allele frequencies from our experiment with the expected values from the Wright-Fisher model. We will discuss methods of estimating the variance of allele frequencies from actual population samples next. Drosophila Handbook page 17

18 Materials Procedures 10 vials of fresh fly food (x 3 generations = 30 total vials), cotton balls, paper towels, fly anesthetic (e.g. Flynap), paintbrushes, supply of red-eyed males, red-eyed females, white-eyed males, and white-eyed females (females do not have to be virgins). Population Maintenance 1. Each group of students will be responsible for ten populations. Each population will initially consist of four males (two red-eyed and two white-eyed) and four females (two red-eyed and two white-eyed). 2. These 8 adults will be put into a single fresh vial and allowed to lay eggs for 2 days, as shown in the figure. 3. You will have a total of ten vials, one for each population. 4. After 2 days of egg-laying, discard adults by disposal in a morgue or paper towel days after eggs were laid, count and record the number of flies with different eye-color phenotypes. 6. Repeat the procedure for two more generations, as depicted in the figure. 7. Make sure to keep your vials labeled 1-10 each generation. Don t mix up vials! Scoring Genotypes This experiment uses two alleles at the white (or w) locus, a gene that affects eye color. The gene white is located on the X chromosome of Drosophila melanogaster. One allele is referred to as w. Females homozygous for w have white eyes, males with one copy of w (a condition that is called hemizygous ) also have white eyes. The second allele, +, is wild type and is dominant to w: females heterozygous (w/+) or homozygous (+/+) for the wild allele have red eyes. Males hemizygous for the + allele also have red eyes. The frequency of the w allele is most easily estimated by counting the total number of white males divided by the total number of males. Compute allele frequencies from all the male data. For example, if you have 10 w males and 30 + males, the frequency of the w allele is 0.25 and the frequency of the + allele is The frequency of the male phenotypes gives the allele frequency in the current generation for each vial s population. The frequency of the red allele is just 1 the frequency of the white allele. Record your data in the table given. Drosophila Handbook page 18

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20 Drift Experimental Data Population Genotype/Sex Generation 1 Generation 2 Generation 3 1 w-male r-male w-female r-female 2 w-male r-male w-female r-female 3 w-male r-male w-female r-female 4 w-male r-male w-female r-female 5 w-male r-male w-female r-female 6 w-male r-male w-female r-female 7 w-male r-male w-female r-female 8 w-male r-male w-female r-female 9 w-male r-male w-female r-female 10 w-male r-male w-female r-female Drosophila Handbook page 20

21 Sample Variance Equation 1c gave the expected variance due to drift. This equation shows how the population size and the allele frequencies affect the variance that is generated by drift. At the end of this experiment, you will have data from ten populations in the form of allele frequencies for each population at each generation. Estimating the variance in this sample is different than computing the theoretical variance expected from drift alone. This is because actual experimental data almost always differs at least slightly from the predicted results. Suppose that in one generation the allele frequencies that you observe in the ten populations are represented by, 1 p, 2 p,.., 10 p. Then the sample variance, s 2 is computed as, where, i 10( ) (10 1)! = s = " i p " p, i= 1 i! = 10 1 p 10 i i= 1 p =. Assessment Questions 1. Record your group s mean allele frequency for the white allele, each generation. 2. What happened to the mean allele frequency of your group s ten populations over time? Is this consistent with the theoretical prediction? 3. What is the variance in your group s data for the mean allele frequency of the white allele, each generation? 4. Equation 1b predicts that the variance should be 1/64 after one generation (convince yourself that this is true, and remember that N = 8 in this experiment). Are your actual data close to this prediction? If not, can you think of reasons why there might be a difference between the observed variance and the variance predicted by equation 1b? 5. What happened to the observed variance in allele frequencies in your ten populations over time? Compare the observed variances to the theoretical predictions of equation 1c, generation-by-generation. Drosophila Handbook page 21

22 Relevant California Science Content Standards Fruit Fly Genetics Laboratory Activity: Grade 7 California Science Content Standards Genetics 2b. Students know sexual reproduction produces offspring that inherit half their genes from each parent. 2c. Students know an inherited trait can be determined by one or more genes. Genetics 2d. Students know plant and animal cells contain many thousands of different genes and typically have two copies of every gene. The two copies (or alleles) of the gene may or may not be identical, and one may be dominant in determining the phenotype while the other is recessive. Investigation and Experimentation 7a. Students will select and use appropriate tools and technology (including calculators, computers, balances, spring scales, microscopes, and binoculars) to perform tests, collect data, and display data. 7c. Students will communicate the logical connection among hypotheses, science concepts, tests conducted, data collected, and conclusions drawn from the scientific evidence. 7d. Students will communicate the steps and results from an investigation in written reports and oral presentations. Grade 9-12 California Science Content Standards Sexual Selection Laboratory Activity & Random Genetic Drift Laboratory Activity: Genetics 2c. Students know how random chromosome segregation explains the probability that a particular allele will be in a gamete. 2f. Students know the role of chromosomes in determining an individual's sex. 2g. Students know how to predict possible combinations of alleles in a zygote from the genetic makeup of the parents. 3a. Students know how to predict the probable outcome of phenotypes in a genetic cross from the genotypes of the parents and mode of inheritance (autosomal or X-linked, dominant or recessive). 3b. Students know the genetic basis for Mendel's laws of segregation and independent assortment. Evolution 7a. Students know why natural selection acts on the phenotype rather than the genotype of an organism. 8a. Students know how natural selection determines the differential survival of groups of organisms. 8c. Students know the effects of genetic drift on the diversity of organisms in a population. Investigation and Experimentation 1c. Students will formulate explanations by using logic and evidence. 1d. Students will recognize the usefulness and limitations of models and theories as scientific representations of reality. 2b. Students will recognize the issues of statistical variability and the need for controlled tests. Drosophila Handbook page 22