Introduction:

In the cornfields of the Midwest Corn Belt, pesticides containing a toxin produced by the bacterium Bacillus thuringiensis (Bt) are often sprayed on the corn as insect control. Although the pesticide is directed towards the corn, some of the treatment is dispersed onto nearby plants such as Asclepias syriaca (common milkweed), the main source of nourishment for Monarch butterfly larvae (Danaus plexippus). When consumed by Monarch butterfly larvae, this toxin causes sickness and eventual death. Past research has shown that the toxin has a definite negative effect upon the health of the larvae as shown in a study performed at Cornell University (Losey, Rayor and Carter 214). However, little is known about the Monarchs’ behavioral response to the toxin.

The naturally occurring soil pathogen, Bacillus thuringiensis (Bt) is a rod-shaped, aerobic, spore-forming bacterium. Unique to its own species, Bt has a distinctive parasporal body, which is a crystal found in mature and normal cells. In association with the parasporal body, metabolities (toxins and protoxins) and delta-endotoxins produced by the bacterium, Bt attains its insecticidal properties. These delta-endotoxins are not toxic until they have been activated within the stomach of the insect with the release of a protein from the parasporal. Once a host insect has digested the Bt, and the protein has been released into the insect’s stomach, the epithelial cells within the stomach burst causing the pH level within the stomach to lower and Bt spores to germinate quickly (Boberschmidt Bt-32). These cells destroy tissues and cause the insect to become sluggish, discolor, then shrivel, blacken and die. The mortality of the insect depends on size and dose of the bacterium eaten off of the plant material (Dipel DF Abbot Laboratories).

In order to understand the interaction of Bt with the Monarch butterfly, it is important to understand the life cycle of the Monarch. Like many other insects, the Monarch butterfly goes through a process called complete metamorphosis, which involves four distinct stages: egg, larva, pupa and adult (see Figure 1). Hormones circulating within the body cavity of the butterfly trigger each change in the process of metamorphosis (Oberhauser pers. comm. 1999). The first stage of metamorphosis, the egg, is the shortest of all stages, lasting approximately four days until it hatches and a larva emerges. This hatching is the beginning of the second stage of metamorphosis. It is during this stage that the butterfly undergoes most of its physical maturation, due to the vast amount of common milkweed the larva consumes. Because of this consumption, it is during the larval stage that the Bt has its greatest impact on Monarch butterflies. The larva eats until it reaches a point at which it has outgrown its skin and therefore, must undergo a process called molting. Each time the larva molts they enter what is called an instar. During the larval stage, the larva goes through five instars. One can distinguish different larval instars by comparing the size of the head capsule to the tentacle length. After about nine to fourteen days, the larva spins a silk mat from which its hangs upside down, forming a pupa, the third stage of metamorphosis. In the course of the next ten to fourteen days, the transformation from larva to adult occurs as the wings and other adult organs develop from clusters of cells present in the larva (Oberhauser pers. comm. 1999). At the conclusion of the pupal stage, the larva emerges as a full-grown adult butterfly, thus entering the terminal stage of its metamorphosis.

 The research performed sought to show the effects of Bt on the health and eating habits of the larvae, while determining if the larvae are able to detect Bt and subsequently avoid Bt. Based on the background information and the purpose of the study, a series of hypotheses were formulated. With knowledge of results from prior studies, it was hypothesized that the Bt would have an observable negative effect on the larva’s health status. It was hypothesized that the larvae would not be able, with their natural senses, to detect a difference between milkweed treated with a solution containing the bacterium Bacillus thuringiensis or the control of water alone. Without being able to detect the Bt when presented to the larva, it was also hypothesized that a larva would not be able to distinguish between different concentrations or dosages of the toxin, if placed in situation where a choice is given. Both of these hypotheses stem from the fact that larvae found in and cornfields have nothing inherent which allows them to detect the toxin. For many generations of Monarchs, Bt has not been in the environment, therefore, not exposing the larvae to the toxin. Without exposure to the Bt, the larvae could not build up a resistance or sensitivity to pass onto their offspring. For this reason, the following generations are unlikely to detect the toxin in the presence of their nourishment.

Experiment:

Bacillus thuringiensis Solutions

In order to accurately show the effects of the toxin on the feeding behavior of the larvae, the research was divided into two separate experiments: Larval Feeding Preferences Between Bt-Treated and Control Milkweed Squares and Larval Location on Bt-Treated and Control Milkweed Stems. Both used a pesticide called Dipel, which contains the strain of Bacillus thuringiensis called Kurstaki. At 100% concentration, 2.396 grams of the Dipel powder form was dissolved in 100 mL of distilled water and diluted from that point to 10% and 1% solutions. Asclepias syriaca (common milkweed) served as the host plant for both the bacterium and the control of distilled water.

The first portion of the experiment involved pinning 3 squares of milkweed coated with the 3 concentrations of Bt listed above and a fourth control square into the bottom of a plastic tub equidistant from the center and each other (Figures 2-3). All four squares were of equal size at the onset of the experiment with a measured area of 294 cm² (Figure 4). Each trial included 20 replicate tubs, with one larva placed in each. The larvae in this portion of the experiment were

either 3^rd or 4^th instars. The initial direction at which the larva was placed in the tub rotated between the four squares to prevent a bias. After all the tubs housed a larva, the larvae’s initial attraction to one of the four pieces was recorded. Following the first observation, the larva’s location in the tub was recorded hourly along with whether or not the larva was eating, resting, or molting. After a 7-hour period of time, the larvae were removed from the tubs and their health status was evaluated as either healthy, healthy and molted, unresponsive, or dead. This process was repeated for four trials.

At the conclusion of the four trials, the individual leaves were examined to determine the amount of each eaten. The leaves that showed evidence of consumption were scanned into a computer in order to be inputted into a program that would analyze their area. This software, titled Fractal Dimensions, took the individual leaf squares and scanned the leaf to determine the amount of boxes of a unit size covered the entire area. Before the leaves with evidence of consumption were analyzed, a leaf with its original area was scanned for its box count to use as a guide for finding the proportion of area eaten for the other leaf squares. Once an initial box count was determined, the rest of the leaf squares were scanned and a proportion for each was found.

To analyze patterns discovered during the collection of data concerning the larvae’s location in the tub, chi-square statistical tests were performed on a variety of data sets to support these patterns. A chi-square test was applied to data concerning the initial direction the larvae were facing when placed in the tub compared to what piece they were initially attracted to. The chi-square test was also applied to data corresponding to the amount of each square eaten., concerning both the larva's health status at the end of the test, compared to what square they ate the most from, as well as what square they ate the most from compared to what square they first ate from. All of these separate tests emphasized patterns which aided in drawing conclusions regarding the larvae’s relationship with the Bt found on their food source.

Experiment 2: Larval Location on Bt-Treated and Control Milkweed Stems

The second experiment utilized twenty plastic water bottles of equal height and volume. At the onset of the trial, one stem of common milkweed was placed in each bottle filled to the top with distilled water (Figure 4). A piece of parafilm was placed on the opening of bottle to lock the piece of milkweed in an upright position while making sure that the larva would not fall into the bottle. The condition of the milkweed, including yellowing or browning and damage, number of leaves and stem height was recorded before larval contact. Ten of the twenty bottles were chosen at random to be sprayed from two inches above with 100% concentration of Bt while the other ten were sprayed with the same amount of distilled water. Each of the twenty bottles had a number written on the side for identification of the larva that was to be put on the stem. Once the stems had dried, one 5^th instar was placed on the bottom-most leaf of each stem. And the larva’s initial position was recorded, including what pair of leaves they were on and if they were on the right or left leaf of the pair. Every half-hour after the initial recording, the larva’s location on the stem was recorded.

After ten observations over a period of five hours, the larvae were removed from the stems. The larval feeding patterns were assessed by determining the percentage of damage on the top, middle and bottom of each stem of milkweed. In order to determine which part of the plant was the top, middle and bottom, the number of leaves were counted and then divided by three. If the number of leaves was divisible by three, a third of the total was the top, middle and bottom. If the number was not divisible by three then the extra leaf would be added to the top first and then the middle section if needed.

Discussion:

During the course of the research, information concerning the effects of Bt on the health and eating habits of Monarch Butterfly larvae was sought, while determining if larvae are able to detect the presence of the Bt and avoid it. Through the data attained and various statistical tests, many conclusions were drawn.

Chi-Square analysis on the data shown in Table. 1 illustrates that there was a relationship between where the larvae first ate and whether or not they moved on to eat from another square. The results show that none of the larvae that initially ate from the squares covered with 100% Bt proceeded to eat from another square. Since the concentration of the Bt was so high on the 100% square, it is likely that the Bt had an almost immediate effect on the larvae’s appetite, therefore suppressing any desire to continue eating. Furthermore, the larvae that initially ate the control square were the most likely to continue on and eat from one of the three squares soaked in a concentration of Bt. The larvae that ate from the control first were not exposed to the toxin and, therefore, nothing suppressed their appetite and they readily proceeded to continue feeding on another square.

As Losey (1999) demonstrated, the Bt toxin has a negative effect on the overall health of the larvae. This conclusion, derived from a chi-square analysis, is directly supported by the data showing that there was a direct correlation between which square the larvae ate the most from and whether or not they were unhealthy or dead at the conclusion of the trial (see Table 2). The data show that the highest proportion of larvae that were unhealthy or dead at the conclusion of the 7-hour test period were those that fed primarily on the 100% Bt squares. This suggests that the Bt quickly affected their health, causing them to become discolored, shriveled and/or dead. However, this part of the study also exemplifies the fact that the larvae must actually consume plant material contaminated by the toxin for the toxin to have a negative effect upon their health. None of the larvae that ate the greatest percentage of the nourishment they consumed from the control square were unhealthy or dead at the conclusion of the trials. Therefore, it is clear that being in the presence of Bt within an enclosed container is not enough to harm the health of the larvae.

The data generated from the first experiment of the study supports the conclusion that the larvae did not purposefully avoid the milkweed squares coated in Bt. Figure 5 shows that there was no apparent pattern between the number of milkweed squares the larvae consumed and did not consume based on treatment. The larvae, at times throughout the four trials, actually ate more, based on the count of squares with evidence of consumption, of those treated with Bt. This leads to the conclusion that the larvae were just as likely to feed off of the toxin soaked squares as the control squares as shown by c ² test in Table 3 and 4. This experiment provides strong evidence that the larvae had no instinctive aversion to Bt and, therefore, showed no preference for control squares over the Bt squares.

Based on the second experiment concerning the individual stems of milkweed treated either with 100% Bt or the control of distilled water, data once again supported the conclusion that the larvae did not purposefully avoid the Bt. Considering the Bt was sprayed on the stems from above, a higher density of the toxin settled on the top few pairs of leaves. This is because the pairs of leaves on the common milkweed plant are alternating. For this reason, one might expect that the larvae would avoid the top pairs of leaves if they were able to detect the Bt. The chi-square analysis of the treatment sprayed on the milkweed stems versus what part of the stem the larvae spent most of their time also supports the conclusion that the larvae did not avoid the top leaves covered in Bt, because the majority of the time was spent on the uppermost part of the stem for both treatments (see Table 5). The graph, relating to the second part of the experiment, provides additional support to the conclusion that the larvae did not detect the Bt and, consequently, did not avoid the top leaves with a denser amount of the toxin (see Figure 6). A great majority of all the larvae ate 100% of all of the milkweed they consumed from the top of the plant for both of the treatments. An explanation for this is that the leaves on a plant tend to be fresher towards the top, where the leaves are younger and less damaged (Solensky pers. comm. 1999). Larvae appeared to detect that, migrate towards the top and eat from the fresher leaves. Even though they were able to detect that the food source was better towards the top, they were unable to detect that the top of the stem also would simultaneously have a negative effect on their health because the concentration of the Bt was higher where the food source was better.

The conclusions reached in this study provide significant evidence that Bt overall is an extremely harmful toxin to the Monarch butterfly. If the larva does not have an aversion to the Bt, they will not refrain from eating milkweed plants interspersed in a cornfield that has been sprayed with Bt, as exemplified in this research. Though the death of the Monarchs induced by the consumption of Bt will not immediately bring a significant drop in population size of the species, generations in the future could possibly be reduced as an effect of the toxin. For this reason, Bt should be used in limited quantities to ensure that the Monarch butterfly species continues to flourish.

Besides expanding on the sample size and the questions already examined in this study, a future direction that could be addressed is the effect of transgenic corn containing pollen genetically altered to included Bt, on the eating behavior of larvae. This would determine whether or not pollen, found in genetically spliced corn, would have a different effect than that of the liquid form of Bt used in this experiment. Considering that the liquid form is not found as frequently as the pollen form of the toxin in nature, expanding the project to include pollen would simulate a more natural setting for future research.

Conclusion:

This research, concerning the feeding behavior of Monarch larvae, demonstrated that the larvae’s health was negatively affected by the consumption of Bacillus thuringiensis, especially when the concentration of the toxin reached levels as high as 100% concentration. Although the toxin was shown to have a negative effect on the larvae, evidence collected during the course of the experiments supports the hypothesis that the larvae did not purposefully avoid the toxin.

Acknowledgements:

Throughout the course of the research performed, there was a lot of help given in order for the project to be completed in such a successful manner. I would like to thank Dr. Karen Oberhauser, Assistant Professor at the University of Minnesota Department of Ecology, and Michelle Solensky, a graduate student at the University of Minnesota, for their tremendous amount of help in the execution of this project and aid in the compilation of project ideas and data. I would also like to thank Ms. Lois Fruen for her help in finding a laboratory site and a professor to work with me. Dr. Jacob Miller is also deserving of many thanks for his excellent guidance and willingness to help and always make me question. Thanks to everyone who aided me in any form during the course of my studies.

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Statements. Volume IV. Mclean: The MITRE Corporation, 1989.

Dipel DF. Chicago: Abbot Laboratories, 1996.

Losey, John E., Linda S. Rayor, and Maureen E. Carter. "Transgenic Pollen Harms Monarch

Larvae." Nature. May 1999: 214.

Oberhauser, Karen. Monarchs in the Classroom. Minneapolis: University of Minnesota

Publications, 1997.

Oberhauser, Karen. Personal Interview. Starting May 1999-Aug. 1999.

Solensky, Michelle. Personal Interview. Starting May 1999-Aug. 1999.