Tested Studies for Laboratory Teaching Proceedings of the Association for Biology Laboratory Education Vol. 32, 195-210, 2011 Evaluating the Effects of Genetic Drift and Natural Selection in Drosophila melanogaster Elizabeth Welsh Biology Department, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, B3H 4J1 ([email protected]) This laboratory provides a “hands on” experimental approach to illustrate evolution in a semester-long study us- ing red-eye and white-eye phenotypes of Drosophila melanogaster. Students set up and maintain small and large fly populations for several generations to observe the effects of genetic drift and natural selection. They record the phenotypes, calculate allele frequencies and at the end of the semester, submit a formal laboratory report on this experiment which includes chi-square tests, and graphs of allele frequencies, heterozygosity and Fst values. They gain valuable practical, analytical, and writing experience from this experiment. Keywords: evolution, Drosophila melanogaster, natural selection, evolution, genetic drift, population size, hetero- zygosity Introduction Introduction This laboratory has been adapted from one developed Timing at the University of Toronto (Goldman, C., 1991). It is cur- One thing to consider is that because it is an ongoing se- rently used in the second year Evolution class at Dalhousie mester -long experiment, it does take up a bit of time, and University, which is a required class for all Biology Majors. other labs need to be organized around the fly lab schedule. Drosophila melanogaster has proven to be an ideal organ- Our labs are two hours long. We do an introductory lab dur- ism for an undergraduate laboratory in evolution because it ing which we go over administrative details of the laborato- is relatively easy to maintain and has a short life cycle. It is ries but also use the time for the students to get acquainted often difficult to find “hands on” experimental laboratories with the flies, using the dissecting microscope and aspirator, to illustrate concepts for evolution. However, because of the and making a practice culture. This only takes about 60 – short generation time of Drosophila, this experiment works 90 minutes in total. The second scheduled lab time is spent in one semester, and is an effective means of observing evo- entirely on setting up the fly cultures. In alternating weeks lution as it is occurring. Although the methodology and ob- the removal of the adult flies should take about five to ten jectives are preset, this lab offers students a chance to have minutes at most. I usually have students do this as they are a bit of “real” laboratory research experience. Flies wake up coming in so that most groups have this done before the and escape, cultures get contaminated, and yes, you do have scheduled lab begins. Two weeks from the initial set up of to come in over the study break to look after your flies. At the cultures, the entire lab is spent on the first transfer. Some the end of the experiment, the students have to analyze their groups are much quicker than others. The second and third data and write a formal paper on their experiment. They gain transfers require between 30 and 60 minutes for the students experience in finding and using research articles on which to to complete. Students get much faster as the semester goes base their expectations and to evaluate their results. I have along and as their confidence and experience increases. The broken up the due dates for components of the report. Stu- final fly lab is spent doing the phenotype counts of the F dents submit the Methods and Materials the week that they 4 generation, and recording the allele frequencies and hetero- set up their fly cultures, the Introduction a week or two after zygosities for the small groups in Excel and discussing how that, and the rest at the end of the semester. The students to do the final report. I then post the data on the class website seem to appreciate a more manageable assignment at the end for students to use in their results. of the semester, and I find they have a greater depth of under- standing of the study while they are doing it, instead of after they write the report at the end. © 2011 by Dalhousie University, Halifax, Nova Scotia 195 Welsh Student Outline The purpose of this study is to provide you with an opportunity to perform and evaluate a long term experiment to illus- trate evolution using living organisms. You will track the phenotypes (allele frequencies) of your populations and compare the results with other groups in your lab. You will look for evidence of evolution in the form of genetic drift or natural selec- tion by the changes in allele frequencies and further data analysis. You should be able to see which evolutionary forces have a greater effect on populations that differ in size. Read through the appropriate sections in your textbook and lecture notes for more background information, and look up research articles in the library for the more detailed understanding required in writing your laboratory report. Genetic Drift Unlike other forces of evolution, genetic drift is non-directional. It is the result of the random sampling of alleles at each generation, and its effects are most prevalent in small populations, or populations that are below an effective population size. This has been demonstrated in the following examples. The Flour Beetle (Tribolium confusum) has become a standard subject for laboratory experiments and has a body color of either brown or black, which is governed by two alleles at a single gene locus. The results of an experiment by Rich et al (1979) demonstrate the effects of genetic drift in these insects. In the experiment, populations of 10, 20, 50 and 100 individuals were set up. Each population began with an equal ratio of black to brown alleles. A change in allele frequencies occurred in all population sizes, but with the most extreme fluctuations (fixa- tion at a frequency of one or elimination of the allele) occurring in the smallest population size of 10. In the second example, Buri (1956) performed an experiment using small populations of Drosophila melanogaster having equal frequencies of the two alleles bw and bw75 at the brown locus. In many of the populations the alleles became randomly fixed for one or the other allele. Interestingly, if you took the average, the overall allele frequency of all the populations was close to the starting frequency of 0.5, but the individual population allele frequencies varied widely. The results for both of these experiments are consistent with the expectations for genetic drift. Natural Selection For natural selection to occur there must be variation among individuals that is inherited and allows some of these indi- viduals to be more successful at survival, mating, or reproducing than others. Unlike genetic drift which is random, natural selection will tend to produce a directional change in allele frequencies from generation to generation. If one allele consis- tently experiences greater success, then that allele will become more prevalent over time in that population. It is important to remember that while drift can behave differently in each population, the results of natural selection are consistent from population to population. Dawson (1970) observed an example of natural selection while studying a recessive lethal allele in Tribolium beetles. The recessive allele caused death in homozygotes and was therefore not passed on to offspring as often as the dominant viable allele. In repeated experiments this caused a steady decrease in the lethal allele and increase in the viable allele in succeeding generations. Geer and Green (1962) observed evidence of natural selection in colonies of Drosophila melanogaster. Their study sup- ported previous experiments that concluded that mate selection was visually dependent, but they also found that when red- eyed male flies were competing with white-eyed males for mates, the wild-type males had greater mating success. Materials and Methods Study Organism: Drosophila melanogaster The common fruit fly Drosophila melanogaster is often used in laboratory studies. The fruit fly is easily cultured and its generation time (egg to adult) is only two weeks at 21-23oC. It makes an ideal organism for laboratory studies because cultures occupy little space and the fly is big enough to easily observe the phenotypic traits under a dissecting microscope. We will be working with eye-color phenotypes for this lab. Life Cycle of Drosophila There are four distinct stages in the life of the fruit fly: egg, larva, pupa, and adult. A fresh culture ofD. melanogaster can produce new adults in two weeks: 2-3 days for the embryo stage, 6 days in the three larval stages, and six days in the pupal stage. The adult fruit fly is fertile after 12 hours but reaches maximum fertility between days 3 and 7, although the adult fly may live for several weeks and remain fertile during that time. 196 Tested Studies for Laboratory Teaching Major Workshop: Genetic Drift and Natural Selection in Drosophila Figure 1. Drosophila melanogaster Life Cycle (K. Weigmann et al, 2009) The larva hatches one day after the egg is laid. The larva molts twice shedding its cuticle, mouth hooks, and spiracles. The larva is called an instar during the periods of growth before and after molting and there are three such instars in Drosophila. Finally the cuticle hardens and darkens to become the puparium, inside which metamorphosis occurs. After metamorphosis, the adult fly emerges from the puparium. It is important to note that newly emerged flies are light in color with folded wings and a longer appearing abdomen. Within a few hours the coloring darkens, the wings expand, and the abdomen rounds up a bit more.