Drosophila Lab Handout

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Drosophila Melanogaster (Fruit Fly) Laboratory Project Read and study this document in its entirety! I. Pre-Lab Preparation  Refresher on the genetics of autosomal and sex-linked traits  Drosophila melanogaster basics II. In-Person Laboratory - “ Hands-O n” Crosses  Prepare culture medium to maintain fruit flies  Anesthetize fruit flies  Sex and count fruit flies  Perform crosses with fruit flies  Assignments: o Canvas quiz o Grading sample lab reports o Lab project report (draft and final version) III. Remote Laboratory - Virtual Crosses  Perform virtual crosses with fruit flies  Analyze the fruit fly cross outcome with the Chi-square statistical test  Assignments: o Canvas homework ______________________________________________________________________________ I. Pre-Lab Preparation Introduction This laboratory project is an opportunity to study the basic mechanisms of transmission genetics using the fruit fly, Drosophila melanogaster . The objective of the project is to determine the mode of inheritance of a single mutation of the eye color. To do so, you will learn to maintain and manipulate the fruit flies, to make genetic crosses, and to collect, analyze, and interpret data. To refresh your understanding of the genetic transmission of autosomal and sex -linked traits, read your genetics textbook and also watch this summarizing video 1 . The life cycle of the fruit fly is shown in Figure 1. An interesting “microscopic journey” into the Drosophila life cycle is shown in video 2 . You can also check the YourGenome website for two “ short and swee t ” stories on Drosophila melanogaster ’s long history in genetic research.

Day 0: Female lays eggs Day 1: Eggs hatch Day 2: First instar (one day in length) Day 3: Second instar (one day in length) Day 5: Third and final instar (two days in length) Day 7: Larvae begin roaming stage. Pupariation (pupal formation) occurs 120 hours after egg laying Day 11-12: Eclosion (adults emerge from the pupa case). Females become sexually mature 8-10 hours after eclosion Figure 1. The life cycle of Drosophila melanogaster (C. Ong et al. 2015. Drosophila melanogaster as a model organism to study nanotoxicity, Nanotoxicology, 9:3, 396-403) II. In-Person Laboratory - “ Hands-O n” Crosses Organization This handout provides a summary of the fruit fly culturing and crossing procedures. A complete guide for maintaining and studying Drosophila melanogaster , from Carolina Biological Supply Company, is available on Canvas. The class will be divided into teams of two students each. However, each student will work independently at their own table. The two members of each team will study the same mutation. One member of each group will undertake a set of crosses designated the “A” set, while the other membe r will do a [gender] reciprocal cross, the “B” set. Data from the “A” and “B” sets will be used to determine the mode of inheritance of the mutation. Stock Cultures You and your lab partner will receive two cultures of fruit flies. One culture breeds true for the normal eye color, commonly described as “ brick-red ”; this culture is the “wildtype” stock. The other culture breeds true for a mutant eye color, white eye. One of you will be responsible for sexing and counting the pure breeding (parental) wildtype stock, while your lab partner will be responsible for sexing and counting the pure-breeding (parental) mutant stock. Watch an overview of the stepwise procedure for culturing and sexing fruit flies in video 3 .

CULTURING PROCEDURES 1. Preparing a Culture Vial to Maintain the Fruit Flies Materials Needed:  Sterile glass tube or vial  Cotton/foam plugs  Fruit fly medium  Dry viable yeast Procedure: Watch the first 3 minutes of video 3 to observe the culture medium preparation.  Place 1 plastic cup of fly medium in the tube/vial.  Add 1 cup of cool tap water. o Allow the vial to sit for a few minutes, adding additional water, if necessary, until the media is completely hydrated. o The surface should be moist with a shiny appearance and there should be no spaces in the media. (If you turn the tube/vial upside-down, no liquid should run out of the medium.)  Add 3-4 grains of yeast. Note: A vial with medium should be plugged at all times, except when adding or removing flies, to prevent contamination by stray flies (and mold spores). If the medium appears too dry (after you added the flies), add a few more drops of water using a transfer pipette. Make sure to avoid dropping water onto the flies or along the side of the tube. Cultures should be maintained at 20-25 o C (room temperature). 2. Anesthetizing the Fruit Flies Materials Needed:  Anesthetizer solution (FlyNap from Carolina Supplies Co.)  Plastic anesthesia chamber/vial with foam plug  Anesthesia “wand”  Stock cultures of Drosophila  Dissecting scope

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 Camel-hairbrush  3 x 5 index cards  Sharpie pen  Fly “morgue” - flask with vinegar covered with perforated Saran Wrap Procedure: Watch video 4 to observe how to anesthetize fruit flies using the FlyNap anesthetic from Carolina Biological. This can be done either after transferring the flies to an empty tube (the safest way) or directly in the stock culture tube. This procedure is also demonstrated in video 3 (min. 6:11 to 11:11). Anesthetize after transfer. Remove the foam plug from the anesthesia chamber. Prepare to transfer flies from a culture vial to the anesthesia chamber by lightly tapping the stock vial against your hand or the table. This will knock the flies away from the cotton plug at the top of the vial. Caution : If you tap too hard, the flies may get stuck in the medium at the bottom of the vial. As you continue knocking the flies away from the cotton plug, ease the plug toward the top of the vial but do NOT remove it yet. When the last flies have been knocked down, quickly remove the cotton plug from the culture vial and invert the open vial over the anesthesia chamber. Encircle the top of the chamber and the bottom of the culture vial with the fingers of your left hand to seal the gap created by the difference in diameter between the two containers. (Otherwise, the flies will escape from the vial!) Lightly tap the culture vial so that the flies fall from the vial into the anesthesia chamber. Caution: If you tap too hard, not only the flies, but the medium, will fall into the chamber. When the flies have been transferred, quickly remove the culture vial and plug the chamber. Immediately plug the culture vial. Alternatively, the fruit flies can be transferred to the anesthetizing chamber with the help of a small funnel as shown in video 5 . Take an anesthesia wand and bend the wire end so that you create a small hook. Dip just the tip of the anesthesia wand into the FlyNap and gently remove excess by wiping it across the inner edge of the lip of the FlyNap bottle. Place the wand hanging into the anesthesia chamber without disturbing the plug too much. Keep the chamber in an upright position and observe the flies. If the flies are to be kept alive, remove the flies from the chamber as soon as they stop moving. Transfer the flies onto an index card. If the flies are to be terminated, leave them in the anesthesia chamber for 10 minutes before removing them, and place the dead flies into the fly “morgue . ”

Note: Never add FlyNap to the stock culture vials or your mating vials! The FlyNap may affect the viability of the eggs in the culture vials. Now that your live fruit flies are sleeping on the index card, you may manipulate the flies with your camel-hairbrush. Determine their gender and transfer them to a new tube/vial. Never add a sleeping fly into an upright culture tube. (The sleeping flies get caught in the medium and cannot extricate themselves.) Always transfer sleeping flies into a tube that is lying on its side or in a 45-degree angle (vial top down). Tubes can remain on their sides for several days without harming the culture. Sleeping flies should be carefully brushed into the horizontal tube, safely far enough away from the medium and far enough away from the top of the tube so that when you add the plug, the flies are not crushed. Some fruit flies will “escape” ! Try to minimize this event, but if that happens, there are fly traps located around the laboratory (a container of apple cider vinegar, covered with perforated Saran Wrap). Watch video 6 and video 7 (starting at min. 5:06) to observe the anesthetizing procedures by ice cooling and by CO 2 generated with Alka Seltzer water. What are some of the advantages/disadvantages of using each of these procedures compared to the FlyNap? 3. Determining the Gender of Fruit Flies Here are some characteristics that can be used to distinguish male from female adult fruit flies: Males:  Males are generally smaller than females (but this is not a reliable way to identify them).  With your brush, turn the fly over on its back so that you can examine its abdomen. In the male, the posterior ventral portion of the abdomen appears black. (Imagine that your male fruit flies are wearing little black bikinis.)  Males have a small black, comb-like structure (the sex comb) located about 1/3 of the way up each foreleg (front leg). This is difficult to see without a microscope, but easily seen with a dissecting scope. Females:  Females are generally larger than males (but this is not a reliable way to identify them).  With your brush, turn the fly over on its back so that you can examine its abdomen. In the female, the entire abdomen appears light tan. Sometimes, if the abdomen is full of eggs, it may even appear whitish. You may see a very fine line of black around the very edge of the abdomen, but not a “bikini . ”  Females do not have sex combs on the foreleg.

Figure 2. Anatomical characteristics of adult flies. Left: Sex combs of the male fruit fly. Right: Ventral abdomen of a female (left) and male (right) fruit fly. In the photos below, are you able to identify the male and the female individuals? A. left fly _______ right fly _______ B. top fly _______ bottom fly _______ A. B . Figure 3. Adult males and females . A - dorsal view; B - ventral view. 4. Collecting Virgin Females When crossing fruit flies of distinct phenotypes , virgin female flies are crossed with an adult male. The reason for this is that female fruit flies store sperm and fertilize their eggs with it over time. Virgin flies are needed to make sure that that the crosses are being appropriately made with the females using the desired sperms to fertilize their eggs. Virginity of the male flies is irrelevant. Female Drosophila are considered virgin 8-10 hours after they hatch from their pupa because, during that time, they are not receptive to male companionship and mating. Although females are able to

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lay eggs as virgins, they will be sterile and no larvae will be produced. Below are four ways to obtain virgins, with the “removal method” being most encouraged for beginners. Removal Method From a culture vial where mature/dark pupae are present, remove all adult flies 8-10 hours before collecting. Visually inspect surface of food to ensure complete removal of flies. After 8 -10 hours, collect all females that are present. All will be virgins. Place them in a fresh culture vial and wait 2-3 days to look for larvae (indicating that some females were not virgin) in the initial culture vial. Virgin females can lay eggs, but they will be sterile. Since they are photoperiod sensitive, females tend to eclose early in the morning. Therefore, early collections will ensure the greatest number of virgins for experimentation. However, collection is possible later in the day. Visual Method for Adult Flies Being able to recognize virgin females removes the necessity of emptying culture vials on a timely basis and allows students to collect their own without the necessity of coming to class at odd times of the day. Note that virgin females are much larger than older females and do not have the dark coloration of mature females. In addition, in the early hours after eclosure, there will be visible a dark greenish spot (the meconium, the remains of their last meal before pupating) on the underside of the abdomen. Figure 5. A) A newly eclosed female. This is the “wet” stage where the fly is sticky to the touch. The wings and body have a wet appearance. B) Virgin female showing the meconium (arrow). The meconium is a dark green area and is the remains of larval food. Figure 6. A) Comparison between a mature (top) and a virgin (bottom) female. This is not long after eclosure. After 4+ hours it becomes more difficult to tell the difference between the two. Note the meconium on the virgin (bottom) female. B) Comparison between a mature (top) and virgin (bottom) male. The coloration is similar to virgin females, however, the genitalia are distinctly different. The meconium is also found in young virgin males as in females.

Visual Method for Pupae Select a fly culture of the required type in which the pupae are beginning to hatch. Remove all adult flies from the vial. These can be temporarily placed in an anesthesia chamber (without the FlyNap) and later put back in the original culture vial. The sex of the pupa is more easily identified if it is still attached to the wall of the culture vial (once on the index card, too much glare of a pupa makes it difficult to identify the male sex combs). Place the vial on the stage of your dissecting scope. Examine the ventral side of one of the mature pupae. Be patient and adjust the light source so that a minimum amount of glare is on the pupa. Look at mature pupae only. These will be very dark when seen with the naked eye. Mature pupae are within a day or two of “hatching” (eclosing). If you inspect the ventral side of a mature pupa, you should see the eyes, wing buds, and legs of the developing fly inside the pupa case. But if the pupa is dorsal side up or lying on its side, you will not be able to determine its gender while it is attached to the wall of the culture vial. Identify a foreleg (front leg) of the innermost (most medial) of the 3 pairs of legs of the developing fly within the pupa case and determine if a sex comb is present. If you cannot see the legs, that means that the pupa is still immature and its gender cannot yet be determined. A sex comb, present only in males, is a small, black structure located about 1/3 of the way up the foreleg, near the anterior portion of the wing bud. Once you have identified a pupa that you think is a female, make a circle around the pupa on the outside of the vial with a Sharpie pen. Once the gender of the pupae has been determined, transfer the female pupae to a mating tube and then add the adult males. Some people use a dissecting needle to transfer the pupa, while others have more success using the handle -end of your brush. Very gently nudge the pupa case from the wall of the vial. Be sure that you do not puncture or crack the pupa case. Keep the mating tube on its side and add the female pupae to the inside wall of the tube. Leave the tube on its side until the pupae hatch. (If the pupae fall into the medium, they will not usually hatch – ever!) And a cracked or damaged pupa case will not hatch. 5. Making Subcultures to Maintain a Wildtype or Mutant Stock (only done when necessary) Your team (you and your partner) may need to make subcultures of your stocks each week. This is usually necessary if the medium in your vials becomes very moldy or very dried out. Your instructor will tell you if that is necessary. Prepare a new culture vial with medium. Be sure the vial is kept plugged except when you are adding/removing flies. Transfer a sample of ~20 flies from the stock into an anesthesia chamber and lightly anesthetize them. Select 5 to 10 males and 5 to 10 females from the sample and put them in the newly prepared vial with medium. Be sure the flies are placed in the vial while it is

lying on its side and place the flies in a dry spot. Plug the vial and keep it on its side until the flies recover. Label the new vial (generation, wildtype or mutant, date, your initials) and record the appropriate information fro this new subculture in your lab notebook. Check the vial every few days for signs of larvae and, later, pupae. Larvae are expected to appear after 7 days, pupae after 12 days, and adult flies will emerge after 2 weeks from the subculture date. Remove and discard the parent when offspring pupae begin to appear. Keep the old vial and look for pupae within the next week; if no pupae emerge, you can discard the old vial. Canvas Quiz. Your understanding of the fruit fly culturing and crossing procedures will be assessed with a multiple-choice quiz that you will take on Canvas. ______________________________________________________________________________ PERFORM CROSSES One member of each team will do Set A crosses and the other member will do Set B crosses. Set up one vial of each type of cross. Be sure to record the gender and phenotype of all offspring from both the Parental and F 1 X F 1 crosses that emerge during the first 10 days of hatching. Student 1 - Set “ A ” Crosses (Direct) P cross: female wildtype (virgin) X male mutant  F 1 offspring AND F 1 cross: F 1 X F 1 (females need not to be virgin)  F 2 offspring Student 2 - Set “ B ” Crosses (Reciprocal) P cross: female mutant (virgin) X male wildtype  F 1 offspring AND F 1 cross: F 1 X F 1 (females need not to be virgin)  F 2 offspring

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Making Parental (P) Crosses to Obtain F 1 Offspring Prepare cross. You will receive the stock culture and a new culture vial with medium from your instructor. Be sure the vials are kept plugged except when you are adding/removing flies. You will need 1 vial for each cross. Label the new vial with the type of cross you are making, the date, and your initials. Virgin females are mandatory for this cross. Inspect the stock culture for potential pupae and notify the instructor if you find any (you may be instructed to select female pupae). Anesthetize your flies and sex them as per the procedures described above. If you have wildtype flies, make sure to retain the wt females (for a direct cross with mutant males) and give your partner the wt males. If you have mutant flies, make sure to retain the mt females (for a reciprocal cross with wildtype males) and give your partner the mt males. After placing the anesthetized flies into the newly prepared vial, be sure the vial is on its side and the flies are placed in a dry spot. Plug the vial. The vial must remain on its side until the flies awaken. At the next opportunity, return the vial to an upright position to prevent the “melting” of the medium, which results in the medium oozing on to the side of the vial and covering the flies/pupae. F 1 Generation Counting Check the vial every few days for signs of larvae (offspring) and, later, pupae. Remove the parents as soon as the larvae begin to appear (6-7 days after mating). Either transfer the parents to a new vial with medium (making another subculture) or discard them in the “morgue . ” Beginning on the first day that the pupae begin to hatch (12-14 days from mating - Day 1), count, sex, and phenotype all F 1 offspring emerging over the next 10 days. Keep a record of when larvae and pupae were observed and when flies emerged. Make all efforts to count all of the F 1 flies before the new generation (F 2 ) emerges – no later than 10 days from the day the first F 1 flies hatched. Anesthetize all of the adults over those 10 days and determine gender and phenotype of each fly. Record the data. Some of the F 1 offspring should be placed in a new mating vial to make the F 1 X F 1 crosses. Do not forget to count these flies as part of your F 1 generation. Making F 1 X F 1 Crosses to Obtain F 2 Offspring

Prepare cross. Prepare a new culture vial with medium. Label the vial and record the appropriate information for this new F 1 X F 1 cross in your lab notebook. Be sure the vial is kept plugged except when you are adding/removing flies. Virgin females are NOT mandatory for this cross since you self-cross them anyway. Sex the female and male adult flies from an F 1 culture as described for the P cross. Lightly anesthetize the F 1 offspring from a parental (P) cross that has been hatching less than 10 days. You should select at least five flies of each gender to transfer to the new culture vial. Be sure the flies are placed on the side of the vial in a dry spot. Plug the vial and keep it on its side until the flies recover. When the flies awaken, stand the vial upright near a lamp. F 2 Generation Counting Check the vial every few days for signs of offspring (larvae) and, later, pupae. Remove and discard the parents when the pupae begin to appear. When the pupae begin to hatch (Day 1), count, sex, and phenotype all adult F 2 offspring emerging until Day 10. Record the data in your lab notebook. Ideal Cross Schedule Student Workdays Lab Day – Day 1 Day 2 Day 3 Day 4 Day 5-6 Week 1 Set up parental cross for F 1 generation. Place vial on its side Set vial upright. Observe culture for viable flies Inspect culture for presence of larvae Inspect culture for presence of larvae Lab Day – Day 7 Day 8 Day 9 Day 10 Day 11-12 Week 2 Remove adult flies (parents) if larvae are present Inspect culture for presence of larvae Inspect culture for presence of pupae Inspect culture for pupae/hatched flies Lab Day – Day 13 Day 14 Day 15 Day 16 Day 17-18 Week 3 Flies hatched. Start counting F 1 flies - Day 1 of 10 day counting period F 1 counting - Day 3 F 1 counting - Day 5 Lab Day – Day 19 Day 20 Day 21 Day 22 Day 23-24 Week 4 F 1 counting - Day 7. Set up F 1 x F 1 cross for F 2 generation (follow parental cross schedule) F 1 counting - Day 9 F 1 counting - Day 11

Data Analysis You will record the results of your crosses. In addition, you will collect data from the class for the rest of the crosses. The F 1 and F 2 generations lab project report (at the end of this handout) will assist you in organizing and analyzing the data. When appropriate, the data will be tested using Chi-square analysis to determine if the observed results fit the expected ratio. An explanation of the Chi-square test will be presented in class and it is also summarized on Canvas as a lecture slide presentation. Conclusions Based on the analysis of your data you are to determine if the mode of inheritance of your mutant gene is:  Autosomal dominant  Autosomal partial (incomplete) dominant  Autosomal recessive  X-linked dominant  X-linked partial (incomplete) dominant  X-linked recessive

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LAB PROJECT REPORT Detailed guidelines on project format/content/style, the lab report grading rubric, and the grading system is available On Canvas. You will include in your lab report: Cross # 1:  The phenotype and genotype of the parents (P)  The expected and observed phenotype/genotype of F 1  Punnett square demonstrating the cross  Data obtained: o Total number of offspring in 10 days o Number of males vs. females o Number of each phenotype observed Cross #2:  The phenotype and genotype of the parents (F 1 )  The expected and observed phenotype/genotype of F 2  Punnett square demonstrating the cross  Data obtained: o Total number of offspring in 10 days o Number of males vs. females o Number of each phenotype observed  Chi-square test on phenotype ratio. Clearly show your calculations based on expected vs. observed phenotypes and number of degrees of freedom. Indicate both the Chi-square number and the corresponding probability value ( p ). Refer to your instructor for additional information about what is required for your report.

III. Remote Laboratory - Virtual Crosses Before starting this lab, watch the Canvas slide presentation titled “A Chi -Square Goodness-of-Fit Test” on the statistical analysis of genetic crosses . For this part of the laboratory, you will use the Virtual Fly Lab to perform virtual crosses with flies and analyze their outcome using the Chi-square test. In this demonstration video , you are instructed on how to use the Virtual Fly Lab to design the fruit flies that you will cross. The steps for analyzing the cross outcomes with a Chi-square test, in the Virtual Fly Lab, are described in the “Virtual Drosophila Lab Handout ” file av ailable on Canvas. Practice Cross To become familiar with the Virtual Fly Lab application, first replicate the cross presented in the “Virtual Drosophila Lab Handout. ” Then perform your own practice cross between a wildtype female for the eye color and a mutant male with brown eyes. Based on the outcome of this cross, determine the nature of the brown eye mutation (autosomal vs. sex-linked, dominant vs. recessive). Perform a Chi-square test to verify whether the difference in the number of observed phenotypes and the predicted (expected) phenotypes is due to chance. Compare your results to the “guided” example of the sepia eye mutation presented in the “Virtual Drosophila Lab Handout. ” Canvas Homework: In this assignment, you will be asked to design the flies to perform two separate crosses, whose outcomes you will analyze by Chi-square testing.

Drosophila Lab Handout

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