Revised Lab

Isabel Rodriguez BIO 125 November 17, 2008 Abstract In Minnesota, Eurosta solidaginis inhabits two different host plants: Solidago gigantea and Solidago altissima. E. solidaginis does not tend to go back and forth between the two host plants; rather it tends to prefer one or the other (Waring et al. 1990). In order to determine if there was any allelic difference in E.solidaginis, we conducted an experiment involving protein electrophoresis and enzyme staining of the protein phosphoglucomutase (PGM). The results indicated that the P-value of <0.05 demonstrates great variation in the difference of alleles found in E. solidaginis that prefers S. altissima and that which prefers S. gigantea. Introduction Eurosta solidaginis, more commonly known as the gall fly, tends to inhabit two types of goldenrod in the state of Minnesota: Solidago gigantea and Solidago altissima. Gall maker E. solidaginis infects two host plants, S. altissima and S. gigantea growing adjacent to each other in Carleton College’s arboretum. Adult E. solidaginis life expectancy is of ten days. During this period, ovipositon occurs and the specie develops on the apical meristem of S. altissima or S. gigantea. Within the apical meristem of host plant, a thick spherical shaped tissue surrounds the developing larva as it undergoes three in star. The thick sphere of plant tissue around the larva serves as food and shelter for one E. solidaginis at a time. E. solidaginis larva is still susceptible to attacks from predators Mordellistean unicolor,Eurytoma obtusiventris,and Eurytoma gigantea (Deel et. al. 2004). However, attacks from predators are not significant to our study. 1

Research has been done in order to determine whether there is any allelic difference in E. solidaginis that chooses S. altissima to that which chooses S.gigantea. According to Waring et al. (1990), the gallfly chooses either one type of the goldenrod or the other, but it does not switch back and forth. The most common claim for the gallflies choosing the different plants in which to lay their eggs is that there is a variation of alleles in the species. Therefore, what Waring tried to determine was to see whether the populations of Eurosta solidaginis has a genetic recognition in host recognition, and if this genetic change increased its survivorship on the new host in multiple regions of northeastern United States (1990). In our experiment, we tried to determine whether there was variation in the population of gallflies found in the campus’ arboretum. Therefore, we looked at the protein, phosphoglucomutase (PGM), to verify if there was genetic variation, which form of the protein is more common. Materials and Methods After dissecting ten galls from Solidago alitissima and Solidago gigantean and removing the Eurosta solidaginis, students placed each larva in a 1.5ml microfuge tube that was kept in a prepared bucket of ice. In order to release the larva’s protein, a student then added 50µl of water and homogenized it with a pestle. Lab groups loaded a 10µl sample of each homogenized larva onto a sample plate. Following priming the gel applicator and blotting the gel, students loaded the samples onto the 2% agarose gel and placed it into the gel rig. Each group then ran the gel at 200 volts for 15 minutes in a buffer of 0.025 M Tris-glycine anda pH of 8.5. While the gel was running, at least one student in each lab group prepared a stain vial in order to help stain the molecules of phosphoglucomutase (PGM). Thus he/she mixed PGM stain of 9.1mM glucose-1-phosphate, 1.0mM NAD⁺, 1.3mM MTT, and 12mM MgCl₂ with 250µl 2

PMS and 20µl G6PDH. Once the gel stopped running, students mixed 2ml of melted agar into the vial containing the PGM stain. Before allowing the gel to dry, students quickly poured the agar stain solution over the top of the gel. The lab groups left the agar on the gel for approximately two minutes, after which one member of the group scraped the agar off the gel and rinsed it with water so as to prevent further staining. After the completion of staining the gel, students scanned and recorded the PGM results. Results Based on our population and on Waren et. al (1990), we used PGM enzyme to observe allele designations based on electrophoretic mobility. To determine variation between E. solidaginis developing in S. altissima and S. gigantea host plants, we used electrophoretic mobility of PGM to identify allele frequencies and to determine significant statistical differences between both species using the Chi Square Test for Independence. The PGM locus examined was polymorphic among E. solidaginis population. The PGM protein had three alleles, S for slow; M for medium; and F for fast present in both E. solidaginis populations. In the sample population, the most common allele was “M” in both S. altissima and S. gigantea. “S” and “F” alleles were less common (Figure 1). In Figure 1, S. altissima expresses more allele variation that S. gigantea. S. gigantea’s population mostly expressed the M allele. From our results, we can conclude that about 8.24% of the alleles found in Solidago altissima came from the Slow (S) allele. Similarly, about 73.08% came from the Medium (M) allele and only 18.68% came from the Fast (F) allele (Figure 1). As for Solidago gigantea, 6.6% of its alleles were Slow (S) alleles as well as Fast (F) alleles, and 86.79% were Medium (M) alleles (Figure 1). Therefore, we saw that there was more variety in the different types of alleles in Solidago altissima than that of Solidago gigantea. Based on the Chi Square Test for 3

Independence, we also concluded that the distribution of larvae found in Solidago altissima was significantly different than in Solidago gigantea (χ2=8.71, 2df, p<0.05).

Discussion Based on the research conducted by Waring et al. (1990) we know that the Eurosta solidaginis, which uses Solidago altissima, is different from that which uses Solidago gigantea as a host plant. The experiment we performed further supports this evidence, although on a smaller scale. Thus, looking at the p-value of <0.05, the probability implies that there is great Figure 1: Percentage of allelic frequency found in Eurosta solidaginis larvae from Solidago altissima and Solidago gigantea

variation between the alleles. Conclusively, we can tell that there has been a genetic host plant shift between the preference of S. altissima and S. gigantea. Here, our results corresponded to that of Waring et al. because they too determined that the gallflies had a host shift of plants and this within this shift, we could see some genetic differentiation between the gallflies. E. solidaginis developing in S. gigantea and S. altissima have different allele frequencies (Figure 1). Based on the results obtained from the chi-square test for independence, we can observe that E. solidaginis developing in S. altissima expressed more allele variation than S. gigantea. This variation may suggest a possible host shift as S. gigantea may be currently undergoing the founder’s effect as its variation has less genetic heterozygosity than that of S. 4

altissima as observer in the results obtained for Chi-Square Test for Independence. This variation is most likely in part to host plant preferences. However, we did not perform further experiment and analysis to determine the level of variation between both populations that would be necessary to assert accurately host- shifting relative to evolutionary development of E. solidaginis. It is likely that with further testing one can appreciate rapid evolutionary change in E. solidaginis developing in S. altissima than E. solidaginis developing in S. gigantea as it may currently be undergoing the founder’s effect, so its likely it evolved from E. solidaginis developing in S. altissima. This study is relevant, yet we had a relatively small population and we only analyzed genetic variation in PGM. For future experimentation, we will use more proteins and a relatively larger sample population also taking into consideration geographic variation. We will also analyze genetic variation by doing DNA analysis on the larva. We would sequence the DNA for E. solidaginis developing in S. altissima and S. gigantea and we would analyze genetic differences between both. Based on the results obtained in our experiment, there would likely be a high level of genetic variation. Therefore, we would perform In Situ hybridization on the larva to analyze variation in genes and gene expression.

Literature Cited Deel, S.Laboratory Manual for Biology 125: Genes, Evolution, and Development.2005 Carleton College Biology Department. Section 4,5. Waring, G.L., W.G. Abrahamson, and D.J. Howard. 1990. Genetic Differantiation Among Host-Associated Populations of the Gallmaker Eurostra solidaginis (Diptera: Tephritidae). Evol. 44: 1648-1655. 5

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