Genetic Variation Between Four Mosquito fish (Gambusia holbrooki) Populations in Miami

Transcription

1 Genetic Variation Between Four Mosquito fish (Gambusia holbrooki) Populations in Miami By: The Fishsquitos Students: John Benton Amy Diaz Sarah Greenhaus Carl Honorat Graham Murza George Trudeau Teachers: Ms. Marie Glemaud Mr. Q. Thompson Undergraduate Facilitator: Ms. Lisa Del Valle Graduate Mentor: Ms. Tiffany Plantan Faculty Mentor: Dr. Michael Gaines July 31, 2005 Research in Ecology Program, University of Miami 1

2 Abstract Variation in Fast (F) and Slow (S) allele frequencies at a locus for glucose-6- phosphate isomerase (GPI) was examined among four mosquito fish (Gambusia holbrooki) populations in Miami using protein electrophoresis. Based on pairwise comparisons of allele frequencies, we found that both Tamiami Canal and Kendall Lakes are significantly different from Fleming s Pond. Two of the four populations, FIU and Kendall Lakes, are not in Hardy Weinberg Equilibrium. No conclusions could be made regarding the direction of changes in allele frequencies over the past seven years. Introduction The organism under study is the mosquito fish (Gambusia holbrooki). Male mosquito fish are approximately 1.6 inches long and females are 2.6 inches long. Mosquito fish are found in fresh water environments. They feed mostly on mosquito larvae, as their name implies. The goal of the study is to determine the genetic differences among four Mosquito fish populations in South Miami using the method of protein electrophoresis. The four field sites were: Tamiami Canal, Florida International University Pond (FIU), Kendall Lakes, and Fleming s Pond. The Tamiami Canal and Kendall Lakes populations are connected, allowing fish to travel from one population to the other and exchange genes, while FIU Pond and Fleming s Pond populations are isolated. Our null hypothesis is that there are no significant genetic differences between the four populations under study. Our alternative hypothesis is that there are significant genetic differences between the four populations under study. We predict that Tamiami Canal and Kendall Lakes will be more genetically similar than either one is to FIU Pond and Fleming s Pond, which are isolated. We expect the connected mosquito fish populations not to show significant genetic differences. On the contrary, the isolated mosquito fish populations should look more genetically different. The enzyme studied was Glucose-6-Phosphate Isomerase (GPI). This was use to look at the allele frequencies in the four populations. Methods In this experiment, we collected 30 mosquito fish from four places on Friday, June 24, 2005: Tamiami Canal (TC), FIU Pond, Kendall Lakes (KL) and Fleming s Pond (FP). On the day of collection, it was sunny and partly cloudy. We caught all the fish in nets and then placed them in jars, one jar per site, with each jar containing water from that particular site. Our first stop was Tamiami Canal, a human-made canal 15.6 miles from The University of Miami. The water was murky and at a high level, and there was a lot of vegetation around the canal. The temperature was about F. It took five minutes to catch all thirty fish. Next we traveled 7.5 miles to FIU pond, which is an isolated man-made pond. There was a lot of algae in the water and fish of other species were caught. It was really shady. It took about 10 minutes to catch all 30 fish. At 2

3 Kendall Lakes, which is ~5 miles from FIU pond, the water was clear and it took the longest amount of time (~15-20 minutes) to catch 30 fish at this site. The last site, Fleming s Pond, is closest to The University of Miami (5 miles) and has a lot of vegetation and algae. The pond was small and it, too, was human-made. It took us 5-10 minutes to catch all 30 fish. We brought the fish back to the laboratory in Cox Science Center. We wrapped them in paper towels and froze them. Later we removed the fish from the freezer and let them thaw out before processing them. We put each fish in a 1.5 ml eppendorf tube and labeled and numbered the tubes: KL for Kendall lakes, FP for Fleming s pond, FIU for FIU pond, and TC for Tamiami Canal. We got a spatula and ground the fish, making sure to use a clean spatula each time we ground a new fish. Then we put 100µl (or 200µl of water if the fish was larger than average size) into each of these tubes. We vortexed each tube for 10 seconds, and then put them in a centrifuge for 8 minutes at 12,000 rpm. The centrifuge rapidly spins liquid samples and separates out the particles by their density. This allowed us to then remove the proteins with a micropipetter and transfer them into new, labeled eppendorf tubes. We soaked a gel plate in Tris glycine buffer for 20 minutes, drying it in filter paper when it finished soaking. We transferred 6µl of each fish proteins into the well plates, placed the soaked buffer plate shiny side down on the base plate, and then stamped the protein samples onto the gel plate. We put the gel plate shiny side up into the electrophoresis tank for 50 minutes at 205 volts. We removed the gel plate from the tank, placed it shiny side down in a petri dish, poured glucose-6-phosphate isomerase (GPI) stain onto the gel plate, and covered it with foil. We waited five minutes for the gel plates to finish and then we read the genotypes of each of the fish samples. This procedure was repeated for all of the fish in each of the four populations. After this we conducted a Chi- square test to see if the populations were in Hardy-Weinberg equilibrium. We used the Homogeneity test to conduct pairwise comparisons in order to compare each of the four populations to one another and to determine the similarities between them. Results During our study, we found two alleles at the GPI locus: Fast (F) and Slow (S). Genotypes for each individual at the GPI locus from each of the four sites are listed in Appendix 1. In the Tamiami Canal mosquito fish population, the frequency of the F allele was 0.75 and the frequency of the S allele was In the Kendall Lakes population, the frequency of the F allele was 0.7 and the frequency of S allele was 0.3. In the FIU population, the frequency of the F allele was 0.8 and the frequency of the S allele was 0.2. In the Fleming s Pond population, the frequency of the F allele was 0.91, and the S allele was The allele frequencies are compared with data from prior years in Table 1. and Figures 1. & 2. Using a Chi-square test, Tamiami Canal and Fleming s Pond were found to be in Hardy-Weinberg equilibrium. FIU and Kendall Lakes were not in Hardy- Weinberg equilibrium. After performing a Homogeneity test, we found a significant difference in the genotypes between the Tamiami Canal and Fleming s Pond populations and also 3

4 between the Fleming s Pond and Kendall Lakes populations (Table 2.). Based on our results, Tamiami Canal and Kendall Lakes are the most homogeneous populations. Discussion Our null hypothesis that there was no genetic difference among the four mosquito fish populations, was not supported by our data and was rejected. There is a significant difference between the allele frequencies among some of the populations in this study. We predicted that the two connected populations, KL and TC, would be genetically similar because fish can travel from one population to another and interbreed, allowing gene flow to occur. The results of the Homogeneity test were consistent with this prediction. These two populations were the most homogeneous of all the populations. We found significant differences in the allele frequencies between TC and FP and between KL and FP, which we predicted since FP is an isolated pond. However, not all of our predictions were supported by the data collected. We found no significant genetic differences between TC and FIU, between FIU and KL, or between FIU and FP. We expected to find differences since FIU and FP are isolated ponds. However, perhaps we did not find significant genetic variation between these populations because evolution takes a long time and these populations haven t had enough time to evolve. We expect that after several more years the genes in FIU Pond will significantly differ from those of TC, KL, and FP due to the effects of genetic drift and gene flow. Two of the four populations, FIU and KL, were not in Hardy-Weinberg Equilibrium, which means they are undergoing evolutionary change at the locus examined. This result is consistent with data from This means that at least one of the five conditions necessary for a population to be in Hardy-Weinberg equilibrium (No mutation, no migration, no natural selection, large population size, and random mating) is not being met. There has not been enough data collected to accurately conclude whether the frequency of the F allele has increased or decreased over the past seven years (Figure 1.). Other factors that may have affected our results include those associated with human error. Spatulas may not have been rinsed properly between samples, or new tips may not have been placed on the micropipetters before transferring new samples, leading to cross-contamination of fish proteins. Acknowledgements We would like to thank: Dr. Krempels for her encouraging work with us; Dr. Gaines for just about everything; Tiffany Plantan for teaching us how to do the experiment; Lisa Del Valle for keeping the peace. Mr. Q. Thompson and Ms. Marie Glenmaud for being there to help; Ms. Pappas for teaching us the statistical tests; Dr. Theodore & Marcia Fleming for use of their pond; and Ms. Deneesha Wilson for all her help in power point. This program was funded by the Howard Hughes Medical Institute. 4

5 Literature Cited Bader J.M Measuring genetic Variability in Natural Populations by Allozyme Electrophoresis. Case Western Reserve University, Department of Biology: Cleveland, Ohio. Bloodsuckers Genetic differences of three mosquito fish (Gambusia holbrooki) populations in South Miami. Research in Ecology Program, University of Miami. Fearless Fishers Genetic differences between mosquito fish (Gambusia affinis) populations in South Dade. Research in Ecology Program, University of Miami. Herbert P., and Beaten M Methodologies for allozyme analysis using cellulose acetate electrophoresis. University of Guelph, Department of Zoology: Guelph, Ontario. Parasites Population genetics of four populations of Mosquito Fish (Gambusia holbrooki) in central and southern Dade County. Research in Ecology Program, University of Miami. Piranhas. Genetic differences of three mosquito fish (Gambusia holbrooki) populations in South Florida. Research in Ecology Program, University of Miami. 5

6 Table 1. Fast (F) and Slow (S) Allele Frequencies for GPI in each of the four mosquito fish populations from N/A=Not Available; TC= Tamiami Canal, FIU=Florida International University; KL=Kendall Lakes; FP= Fleming s Pond. Fast Allele Frequencies Year TC FIU KL FP N/A 1999 N/A N/A N/A N/A N/A N/A N/A Slow Allele Frequencies Year TC FIU KL FP N/A 1999 N/A N/A N/A N/A N/A N/A N/A

7 Frequency of Fast Allele Allele Freqency (F) TC FIU KL FP Year Figure 1. Frequencies of Fast (F) allele in each of the 4 mosquito fish populations from Data from 1999 is unavailable. 7

8 Frequency of Slow Allele Allele Frequency (S) TC FIU KL FP Year Figure 2. Frequencies of Slow allele (S) allele in each of the 4 mosquito fish populations from Data from 1999 is unavailable. 8

9 Table 2. Pairwise comparisons using Chi-square (x 2 ) Homogeneity tests. * Indicates significant at 95% Confidence Interval. Pairwise Comparisons Using Chi-square Homogeneity Tests F S TC vs. FIU observed observed Total x2 df TC FIU Total F S TC vs. KL observed observed Total x2 df TC KL Total F S TC vs. FP observed observed Total x2 df TC * FP Total F S FIU vs. KL observed observed Total x2 df FIU KL Total F S FIU vs. FP observed observed Total x2 df FIU FP Total F S KL vs. FP observed observed Total x2 df KL *7.5 1 FP Total

10 Appendix Appendix 1. Genotypes of each fish sample as determined by allozyme electrophoresis. Site Sample # GPI TC 1 FS TC 2 FF TC 3 FS TC 4 FF TC 5 FF TC 6 FS TC 7 SS TC 8 FF TC 9 FF TC 10 FS TC 11 FS TC 12 FF TC 13 FS TC 14 FS TC 15 FF TC 16 FF TC 17 FF TC 18 FF TC 19 FF TC 20 FS TC 21 FS TC 22 FS TC 23 FS TC 24 FF TC 25 FS TC 26 FS TC 27 FS TC 28 FF TC 29 FF TC 30 FS FIU 1 FF FIU 2 FF FIU 3 FS FIU 4 FS FIU 5 FS FIU 6 FF FIU 7 FS FIU 8 FF FIU 9 FS FIU 10 FF FIU 11 FF FIU 12 FS FIU 13 FF 10

11 FIU 14 FS FIU 15 FF FIU 16 FF FIU 17 FF FIU 18 FF FIU 19 FS FIU 20 FS FIU 21 FS FIU 22 FS FIU 23 FS FIU 24 FF FIU 25 FF FIU 26 FF FIU 27 FF FIU 28 FF FIU 29 FF FIU 30 FF KL 1 FF KL 2 FF KL 3 FS KL 4 FS KL 5 FS KL 6 FF KL 7 FS KL 8 FF KL 9 FS KL 10 FF KL 11 FF KL 12 FS KL 13 FF KL 14 FS KL 15 FF KL 16 FF KL 17 FF KL 18 FF KL 19 FS KL 20 FS KL 21 FS KL 22 FS KL 23 FS KL 24 FF KL 25 FF KL 26 FF KL 27 FF KL 28 FF KL 29 FF KL 30 FF FP 1 FS FP 2 FF FP 3 FS 11

12 FP 4 FF FP 5 FS FP 6 FF FP 7 FS FP 8 FF FP 9 FF FP 10 FF FP 11 FF FP 12 FF FP 13 FS FP 14 FF FP 15 FF FP 16 FF FP 17 FF FP 18 FF FP 19 FF FP 20 FF FP 21 FF FP 22 FF FP 23 FF FP 24 FF FP 25 FS FP 26 FF FP 27 FF FP 28 FF FP 29 FF FP 30 FF 12