Student lab provided by George Wolfe, Director, Loudoun County Public Schools, Academy of Science, Sterling, VA

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1 Gall Size Effect on the Inhabitance of Parasites and Parasitoids in the Goldenrod Plant Abstract: We calculated volumes and examined species inside the galls in an effort to determine if gall size has any effect on the organism currently living in the gall. After analyzing the results and observing confidence intervals, we were able to conclude that the gall fly is more likely to be found in larger galls, Mordellistena unicolor beetles were more likely to be found in small galls, and both the obtusiventris and gigantea wasps had no statistically significant difference for what gall size they were found in. This most likely means that the gall fly larva is safer in larger galls that are harder for other species to penetrate, and that the beetle has an easier time getting into small galls. The wasps, on the other hand, do not care what gall size they inhabit, and were therefore found in a range of gall sizes. Introduction: Located across both the United States and Canada, goldenrod varieties such as Solidago canadensis are the ideal home for Eurosta solidaginis, or the gall fly, larvae (Abrahamson, Eubanks, Blair, & Whipple, 2001). With many species of Solidago existing due to crossbreeding, this plant was originally native to Europe. This perennial plant is often found along roadsides and in open fields, and as a goldenrod can grow from between three to five feet tall. With single wood stems and clusters of yellow flowers at the top, it is often mistaken for ragweed, which blooms at the same time, generally in the late summer. Goldenrod leaves may either have toothed and jagged edges, or smooth ones (University of Maryland Medical Center, 2010).

2 Defined as a growth on a plant, a gall is a reaction to the physical irritation produced from a chemical secreted from the gall fly larvae. This causes swelling of the plant stem, and the result can be any variety of shapes and sizes. Typically, in the late spring the goldenrod gall fly will lay an egg on the plant. Using an ovipositor, which is a pointed tube on the end of its abdomen, eggs are deposited on a goldenrod shoot (Weber, 2011). The egg is laid on the growing tips of the plants, or the terminal bud (Storey, n.d.). A little more than a week later, the egg will hatch and the larva begins to grow. The larva is white and vaguely barrel shaped, with a set of mandibles that look like a black speck. As the larva eats, the gall will thicken in response to a chemical stimulation. Once the gall is full sized, the larva is also full sized. It forms a tunnel with the mandibles in the fall that stops just short of the outermost shell, and in the spring it will use this tunnel to escape. Meanwhile, it spends the winter in the gall (Weber, 2011). The galls themselves are shown to mostly have either bilateral or radial symmetry (Raman, 2010). If the gall fly larva is successful and emerges in the spring, it does not eat and generally does not live for more than a week (Wise, Yi, & Abrahamson, 2008). The cycle continues in this way, unless interrupted by some type of predator. The gall fly has three main predators that frequently attempt to inhabit the gall, including two species of wasps and a species of beetle. The Eurytoma obtusiventris wasp, a parasitoid, uses the gall to lay its eggs. A parasitoid is an organism that preys on a host, and ultimately kills or consumes it. Generally the wasp oviposits, or lays eggs, into the host egg or larva while the gall fly larva is still maturing. Remaining quiet in the host larva until autumn, the parasitoid causes early pupation of the gall fly larva and begins its own growth (Abrahamson, & Heinrich, n.d.). It is for this reason that, as long as the gall is examined before the gall fly pupates on its own, anything in a pupa form is identified as an obtusiventris wasp. Since the wasp larva eats the gall fly larva from the inside,

3 not only does it have a place to live in the pupated shell, for easier development over the winter, but it has a source of food (Storey, n.d.). The other type of parasitoid wasp hatches inside the host larva, eats the entire thing, and then feeds on the gall (Weber, 2011). This wasp, the Eurytoma gigantea, uses a long ovipositor to penetrate the gall and lay its eggs in the gall chamber in a similar fashion to the other two species, the gall fly and the obtusiventris. Like the obtusiventris, it is thought that the success of the attack depends on the thickness of the goldenrod stem and of the ovipositor length. The gigantea s attack season may extend from June through August, and after deposition of eggs, the larva quickly gets to business (Abrahamson, & Heinrich, n.d.). When found inside the gall, this wasp is cream colored, like the gall fly larva. However, the obtusiventris wasp is occasionally smaller and has distinctive pointed ends, like a cone. Additionally, once the gall fly larva dies, the interior of the gall may discolor slightly as it starts to degenerate (Storey, n.d.). The last main type of predator for the gall fly, and the other wasp species for that matter, is a species of beetle, or Mordellistena unicolor. This stem burrowing beetle bores into the gall and develops on the tissue of the gall fly larva as well as the general gall itself (Abrahamson et al., 2001). Unlike the obtusiventris wasp, it eats the larva from the outside. It appears to be a small brown caterpillar, but with distinct legs (Storey, n.d.). The larva of this beetle is thought to attack galls of all sizes and therefore encounter galls with other species, such as the wasps, in them. Therefore the beetle eats whatever is in the gall, and inspection typically only reveals the remnants of the beetle s dinner inside the gall (Rosenheim, 1998). Some of the most recent work being done in the goldenrod gall area has to do with the observations of gall survival when the extremity of winter temperatures was varied. Although frequently hypothesizing that colder winters might have a detrimental effect on the spring flies, it

4 was discovered that a low temperature is important to maintain a high productiveness (University of Maryland Medical Center, 2011). Many studies along these lines have been conducted to validate this conclusion. However, this work, although done on galls, is not directly relevant to the experimental problem here. Some hypotheses have already been made about an organism s preference of gall size, as shown above. It is though that the wasps might target smaller galls because of the ovipositor length. If the ovipositor is short, it might not be able to penetrate a thick gall, and therefore the larva will not survive. Also, the beetles are thought to target any range of gall sizes, because it can just as easily get through a thick gall as a thin one (Rosenheim, 1998). However, while conclusions like these were considered logical, they lacked any supporting evidence. Our experiment varies from this because actual concrete data and proof will be provided to back up any conclusions we draw. Our experimental setup is important in this field because it will tell not only may it tell the advancement level of some of these species, but it will help expand the field of research in this area, by allowing other researchers to make educated guesses about what species are found in what sized galls. For example, if our conclusions are valid, a researcher looking for a certain species inside the gall might be able to use these conclusions to hone in on what galls were picked. Our experimental setup was based around our quest to discover what gall size had to do with the type of organism found inside. It is possible that some organisms prefer one size gall over another, yet there is an equal possibility that there is no preference at all and that the selection is random. By measuring the diameters of several axes, the volume of each of a large quantity of harvested galls was calculated. By cutting them open to record the species found inside, the data was organized and analyzed to draw conclusions about the relation of gall size to the species inside. Using further statistical methods, we were able to calculate what our

5 comparatively small trial (in relation to the number of galls in existence) had to do with the gall population as a whole and how our data could be interpreted to represent this. Using this experimental design, the independent variable was the gall size, and the dependent variable was what organism, or in some cases, organisms, were found inside. The goldenrod species was kept constant, as was the general place (field) that the galls were harvested from. For this experiment, there was no control group. Unlike some experiments, we had no one variable that was changed so that we could compare those results to the original. I predicted that more gall fly larvae will be found in large galls (large relative to the ones harvested), because it is harder for other parasites, like the gigantea wasp, to lay eggs through the thicker gall. I then hypothesized that More gigantea and obtusiventris wasps would be found in medium sized galls because they may be easier to locate in a field of goldenrod than the small galls and yet easier for the ovipositor to inject eggs into than the large galls. Lastly, I hypothesized that the Mordellistena unicolor beetle would be evenly distributed throughout the various sized galls due to a lack of preference and need to isolate one particular sized gall. Materials and Methods: After obtaining the galls, they were divided up evenly among the 65 students. A paper towel of adequate length (from one to two feet long) was placed under the galls at all times to prevent dirtying the tables. We all designated and numbered a small section of the paper towel for each gall to prevent the galls from getting mixed up and for ease of recording the results later. Starting with gall number one, we used a vernier caliper to measure the major axis, or the longest section of the gall from one end to the other, as well as the largest and smallest axis, which both run perpendicular to the major axis. All axes are considered to be diameters. Since the galls were not perfectly three dimensional ellipse shaped figures, getting these measurements gave us the

6 closest number to the actual volume. To measure the smallest minor axis, we slid the caliper around the middle of the gall and tightened the caliper as we slid the gall around, until the caliper would not slide any more, letting us know this was the smallest axis of the gall. To find the biggest minor axis, we slid the gall around inside the prongs of the caliper, letting the caliper adjust itself, until it would get no wider. Lastly, we found the major axis by taking a precise measurement of the longest axis between the caliper prongs. All three of these measurements were then recorded in a table. This process was repeated for all the galls. After measuring all the galls, we cut open our individual ones to see what organism inhabited them. I started with gall number one, and held it firmly by the ends. Using a typical kitchen knife, I applied pressure to the middle of the gall, with the blade on the one of the minor axes, slicing into it but not all the way through it. Cutting all the way through it would have most likely severed the organism inside. I worked slowly around the gall with the knife, slicing until the entire axis was cut. Then the gall was easily broken open by applying pressure to both halves created by the cuts. The hole inside of the gall was then visible, along with whatever organism, or lack thereof, was present. After I examined the contents of the gall and identified them according to the guidelines explained previously, I recorded them all in a data table. Occasionally it was difficult to differentiate between two species, specifically the gall fly larva and the gigantea, because they were both whitish larva. In these cases we used dissecting microscopes to get a closer look at the organisms, and then it was clear to see which was which. After everyone s individual data was recorded, all the information for all the galls was then recorded in on chart. Some results were calculated purely on individual results, but for the most part, conclusive results were drawn from the entire groups data. Once the data table for the entire group was created, it was sorted by what organism was found in it for ease of calculation.

7 Safety precautions: Knives were used in this experiment, so we were all extra cautious of what was near the blade when we were cutting the galls. Additionally, closed toe shoes should be worn at all times to prevent something from falling on your feet. Data: Table 1: Diameter Measurements Organism Minor Axis Large (cm) Minor Axis Small (cm) Major Axis obtusiventris wasp beetle obtusiventris wasp gall fly beetle Results: To calculate volume, we simplified the equation (4/3)(!) (D 1 /2)(D 2 /2)(D 3 /2) to equal (!/6)(D 1 )(D 2 )(D 3 ), where D 1, D 2, and D 3 are the axes, or diameters, of the gall. In table 3, mean volume was calculated by adding all the volumes for a given category and then dividing by how many were added. We found standard deviation by using the function STDEV in Excel. The sample size was simply how many galls fell into a given category, and we calculated standard error by taking using the formula standard error=(standard deviation/sqrt(sample size)). SQRT in Excel is equal to the square root function. Table 2: Volume Measurements Organism Volume (cc) obtusiventris wasp 5.93 beetle obtusiventris wasp 7.67 gall fly 6.01 beetle 5.21

8 Table 3: Calculations on Group Data Category Mean Volume (cc) Standard Devition (cc) Sample Size Standard Error (cc) Beetle Anything but beetle gall fly Anything but gall fly gigantea Anything but gigantea obtusaventris anything but obtusaventris Graph 1: Gall Fly vs. Not Gall Fly Normal Distribution Graph Relative frequency Mean Gall Volume (cm 3 ) Gall Fly Not Gall Fly Gall Fly: Left bound of 95% confidence interval Gall Fly: Right bound of 95% confidence interval Not Gall Fly: Left bound of 95% confidence interval Not Gall Fly: Right bound of 95% confidence interval

9 Graph 2: Beetle vs. Not Beetle Normal Distribution Graph Relative frequency Mean Gall Volume (cm 3 ) Beetle Not Beetle Beetle: Left bound of 95% confidence interval Beetle: Right bound of 95% confidence interval Not Beetle: Left bound of 95% confidence interval Not Beetle: Right bound of 95% confidence interval

10 Graph 3: gigantea vs. Not gigantea Normal Distribution Graph Relative frequency Mean Gall Volume (cm 3 ) gigantea Not gigantea gigantea: Left bound of 95% confidence interval gigantea: Right bound of 95% confidence interval Not gigantea: Left bound of 95% confidence interval Not gigantea: Right bound of 95% confidence interval

11 Graph 4: Obtusiventris vs. Not obtusiventris Normal Distribution Graph Relative frequency Mean Gall Volume (cm 3 ) obtusiventris Not obtusiventris obtusiventris: Left bound of 95% confidence interval obtusiventris: Right bound of 95% confidence interval Not obtusiventris: Left bound of 95% confidence interval Not obtusiventris: Right bound of 95% confidence interval Calculating statistical error was vital to ensure our results could still be credited. One source of error comes with the identification of the species in the gall. If the gall fly is found, we can be certain none of the three other organisms, the two wasps and the beetle, were ever in there. However, if one of three organisms was found inside the gall, there are some gray areas as to what species had ever been in there. We know that the gall fly must have been in the gall at

12 some point, or else the gall would not have formed. However, for example, if beetle waste was found, either of the wasps could have chosen that gall for a home and then been eaten by the beetle. If the gigantea wasp was found, the obtusiventris wasp might have been in the gall prior to the gigantea entering. There was no way to be certain when we conducted our experiment, so we assumed that whatever organism found was the only one ever in there, even though that may or may not be true. An additional source of error was the lack of gigantea found in our data. We only found a small number of them, and therefore it is hard to determine if our conclusions on this parasitoid are as accurate as they could be. To fix this, we could gather more galls to hopefully widen our knowledge on gigantea s preference on gall size. Discussion: Our results only partially supported my hypothesis. For the gall fly larvae, I predicted that more of them would be found in the large galls, because it would be harder for other predators to penetrate the gall. Our conclusions supported this hypothesis, because there is a statistically significant difference between galls with and without gall fly larva. There was also a statistically significant difference in gall size for galls with and without beetles, although this counteracts my original hypothesis. Our results show that the Mordellistena unicolor beetles prefer small gall sizes. This is likely due to the ease of penetrating the gall, for the small galls have less material to go through. For both of the wasps, there was no statistically significant difference in preference of gall sizes. Both the obtusiventris and gigantea were found in a variety of sized galls, showing that they had no preference of one sized gall over another. By just looking at table one, which contains one set of individual volumes from my data, it is extremely hard to draw any conclusions whatsoever. There isn t enough data to confidently say that it represents the whole goldenrod gall inhabitant population, and that is why the group

13 data was used instead. Furthermore, it would have been impossible for me alone to draw conclusions for the gigantea wasp, since none of my five galls contained one. The gall fly larvae seemed to be found in bigger galls when compared to the rest of the parasitoids. When looking at the normal distribution graph one, it is key to observe the upper and lower ranges of the 95% confidence interval. The 95% confidence intervals in this case to not overlap, and therefore there is a significant change. This is based on the assumption that I repeated this single trial many times (because the standard error was used), and I would expect that 95 times out of 100, these confidence intervals would capture the true mean value of each distribution. Therefore we can conclude that since they don t overlap, there is a statistically significant difference between the two distributions. The beetle, using the same logic as for the gall fly, also had a statistically significant difference concerning which galls they were found in and which galls they weren t. They are more likely to be found in smaller galls, which tells something about their nature. They may have an instinct to be able to tell which galls are easiest to penetrate, rather than going to any random gall like I previously hypothesized. Both wasp species showed no preference, as can be seen from graphs 3 and 4, where the 95% confidence intervals either mostly overlap or totally do. However, the small sample of gigantea may have affected this conclusion. Since the obtusiventris lays its eggs before the gall forms, it is logical that it has no preference as to gall size. However, this completely reversed my opinion that wasps would have a preference of gall size and beetles would not. Rosenheim s (1998) theory about goldenrod predators was not supported by this data either. He drew seemingly logical conclusions that ended up not being supported by our actual data. Since our classes results clearly do not match Rosenheim s, it may be a good next step to

14 look into what he either did differently or wrong to come to this conclusion. Assuming our results are correct, this benefits other researchers in this field as well, because they would now know where to find a certain species. Additionally, it opens up further research opportunities. Others may be able to use a combination of this research plus additional data to conclude what the likelihood of multiple species having been in the same gall would be. For instance, perhaps there was a gall fly larva, then a gigantea wasp larva, and then a beetle in one gall. These statistics may help draw these conclusions.

15 References Abrahamson, W. G., & Heinrich, P. (n.d.). Eurosta parasite and predator page. Retrieved from Abrahamson, W. G., Eubanks, M.D., Blair, C. P., & Whipple, A. V. (2001). Gall flies, inquilines, and goldenrods: A model for host-race formation and sympatric speciation. American Zoology, 41, doi: /icb/ Raman, A. (2010). Morphogenesis of insect-induced Plant Galls: Facts and Questions. Flora, 206, doi: /j.flora Rosenheim, J. A. (1998). Higher-Order Predators and the Regulation of Insect Herbivore Populations. Annual Reviews, 43, Storey, K. B. (n.d.). Cold Hardy Insects-Invertebrate Cold Hardiness. Retrieved from server.carleton.ca/~kbstorey/insects.htm University of Maryland Medical Center. (2011). Goldenrod. Retrieved from Weber, L. (2011). Young naturalists: The curious world of galls (Nov. - Dec. 2000). Retrieved from Wise, M. J., Yi, C. G., & Abrahamson, W. G. (2008). Associational Resistance, Gall-fly preferences, and a Stem Dimorphism in Solidago altissima. Acta Oecologica, 35, doi: /j.actao