Monarchs in the Classroom

What is the difference in spore load of Ophryocystis elektroscirrha in monarch butterflies likely reared on the non-native milkweed, Asclepias currasavica, and monarchs likely reared on other native milkweeds?

Monarch butterflies face a detrimental infection caused by the protozoan parasite known as Ophryocystis elektroscirrha (Altizer and Oberhauser 1999). These spores are spread throughout the monarch population in several ways. One of the ways is by physical contact. When a pair of monarchs are mating, if one of them is infected, they can spread their spores by brushing some of the spores off during abdominal contact. Another common way spores are spread is during the laying of eggs. When an infected female monarch lays her eggs on a milkweed plant, she will more than likely drop off some of the spores on the egg and milkweed plant. When the larva hatches, it eats its eggshell, or chorion, and begins to feed on the milkweed plant. In the feeding process it consumes the spores on the plant, and becomes infected, which starts the cycle all over again. Although not proven, many scientists believe that the spores may also be spread during physical contact when the monarchs clump together and roost to keep warm during the migrating season.

In researching articles concerning this problem, we found that although a large number of monarchs (14,790) had been examined (Altizer, Brower, and Oberhauser 2000), focus had been on the monarch and transmission by insect to insect. However, the link of transmission by the milkweed plant was not emphasized, particularly the possible relationship between the spread of the spores as connected to specific milkweeds.

A dense spore population on the non-native milkweed, Asclepias currasavica, in Texas could have a tremendous effect on the whole eastern population of monarchs, because Texas is in the middle of the monarch migratory route, and is home to at least two generations of monarchs. We are curious about the availability of A. currasavica as a larval food source and spore habitat. Since the A. currasavica does not senesce in southern Texas due to the mild Texas winters, the spores can remain on the milkweed, possibly threatening future monarch populations.

H_o: There is no difference in spore load of Ophryocystis elektroscirrha in butterflies likely reared on Asclepias currasavica and those monarchs reared on other native milkweeds.

H_a1: Monarchs that eat non-native milkweed, A. currasavica, as larvae will have a greater spore load of O. elektroscirrha.

H_a2: Monarchs that eat native milkweeds as larvae will have a greater spore load of O. elektroscirrha.

1) Our team collected butterflies in four locations: urban, rural, coastal, and hill country areas. The actual collecting technique involved sweeping the net forward, then quickly flipping the net to prevent the escape of the monarch butterfly. We caught as many specimens as time and effort permitted.

First, the butterflies were held by all four wings in a vertical position. Then a clear piece of scotch tape was pressed lightly against the back of the abdomen thrusting it forward. Next, the scotch tape was placed on a laboratory slide. Lastly, the date, specimen's sex, location, and number was placed on the slide. Slides were then brought to a laboratory and examined under a microscope at a 400x magnification to determine the presence or absence of spores for analysis. Finally, with a permanent marker, a colored dot was placed on the right upper wing to ensure that the butterfly would not be sampled again.

3) Many groups and individuals aided us in collecting monarchs (Table 1, Figure 1).

Name of Cooperator	Organization	Location of Collection	Habitat	Assumed Milkweed Type	Sample Size
Mary Kennedy	Texas Military Institute	San Antonio	Urban	non-native	100 wild, 48 lab-reared
Dr. William Calvert	Texas Monarch Watch	Bandera	Hill Country	native	34
Dr. Karen Oberhauser	Monarchs in the Classroom	Selah/Bamberger Ranch	Hill Country	native	101
Harlen Aschen	Victoria Christian School	Port Lavaca and Victoria area	Coastal	non-native	180
Joylynn Woodruff	MITC/SMM Monarch Monitoring Project	Burnet	Urban	non-native	30
Westview research team	MITC/SMM Monarch Monitoring Project	Taylor, Georgetown	Rural	native	16
Westview research team	MITC/SMM Monarch Monitoring Project	Austin	Urban	non-native	3

4) This data was then used in Chi-square tests to determine if there was a correlation between whether the monarch was likely reared on native or non-native Asclepias and the spore load, as well as a correlation between locations where native and non-native milkweeds are found. From October through the first of December we collected data and got a total of sixteen rural, one hundred eighty coastal, one hundred eighty-one urban, and one hundred thirty-five Hill Country monarchs.

There were significant differences among the collection categories. Therefore, we were able to reject our null hypothesis which states that there is no difference in spore load of Ophryocystis elektroscirrha in butterflies likely reared on Asclepias currasavica and those monarchs reared on native milkweeds.

Our data showed that the percentage of coastal and urban spore loads were greater than the spore loads of rural and Hill Country monarchs (Figure 2a and Table 2). Coastal butterflies had the greatest infection levels; over 40% of the butterflies collected near the coast were infected with O. elektroscirrha while the others had less than 15% infection levels. The comparison in Figure 2b, the percentage of infected monarchs assumed eating native milkweed versus those that ate non-native milkweeds, show that 3.3% of monarchs that likely ate native milkweeds were infected with O. elektroscirrha, whereas 28.5% of the monarchs that likely ate non-native milkweed were infected with O. elektroscirrha. Since it is likely that the coastal and urban monarchs fed on A. currasavica, we can then conclude that our hypothesis, H_a1, is correct, which states that monarchs that eat non-native milkweeds, A. currasavica as a larvae will have a greater spore load of O. elektroscirrha (Table 3).

Observed Results:
Sites	No spores	Spores	Total
Rural	15	1	16
Coastal	103	77	180
Urban	155	26	181
Hill Country	131	4	135
Total	404	108	512

Expected Results:
Sites	No spores	Spores	Total
Rural	12.63	3.38	16
Coastal	142.03	37.97	180
Urban	142.82	38.18	181
Hill Country	106.52	28.48	135
Total	404	108	512

Table 2. Observed and expected counts of monarchs with and without O. elektroscirrha spores. Results of a Chi-square test showed that the calculated Chi-squre value was greater than the critical Chi-square value (calculated Chi-square=86.55, df=3, p<0.01). We are 99% confident that we can reject the null hypothesis.

Observed Results
Milkweed type	No spores	Spores	Total
Native	146	5	151
Non-native	258	103	361
Total	404	108	512

Expected Results
Milkweed type	No spores	Spores	Total
Native	119.15	31.85	151
Non-native	284.85	76.15	361
Total	404	108	512

Table 3. Observed and expected counts of monarchs with and without spores from native and non-native milkweed locations. Results of the Chi-square test showed the calculated Chi-square value to be greater than the critical Chi-square value (calculated Chi-square=40.68, df=1, p<0.01). We are 99% confident that we can reject the null hypothesis.

(a)	(b)
Figure 2. Percentage of Ophryocystis elektroscirrha infection in monarchs from (a) four different locations and (b) regions with native and non-native milkweeds.

When we decided to do this project, we did not realize the problems we would have in collecting the monarchs. We had difficulty in obtaining specimens due to the following:

Collecting larvae would have been preferable because we would have known what they had eaten. With the adult monarchs, we were only able to assume what they had eaten as larvae. Our urban collection of monarchs may have had a mixture of migrants and possibly ate native milkweed. Plus, any monarchs that might have been local may have eaten non-native milkweed.

Future studies could include controlled laboratory studies using large numbers of monarch larvae feeding on Asclepias currasavica, and cutting back the non-native milkweed during the fall and determining its effect on spore infestation. More extensive collections of larvae throughout the season would be helpful in determining the effects of A. currasavica on spore load. Looking for a possible relationship between monarch characteristics (wing size, sex, weight, wing condition) and spore load might be interesting.

By increasing public awareness in this area, nuseries selling A. currasavica could recommend to customers that they cut back the plant in the fall to possibly reduce spore infestation.

We would like to thank Dr. Karen Oberhauser and her staff for collecting migrating monarchs at the Bamburger Ranch, Dr. William Calvert for collecting Hill Country monarchs, Harlen Aschen for his expertise on Asclepias currasavica and his thoughts concerning the infestation of monarchs and for the data that he shared with us. We would like to thank Mary Bishop Kennedy and Carol Johnson for sharing their data, Dr. John Abbott of the University of Texas for the use of his dissecting microscope, Leticia Flores and Cindy Petersen for all of their help. A special thank you goes to the National Science Foundation and the Science Museum of Minnesota for funding this project. We would also like to thank Pflugerville ISD and Bonifacio Durán, our principal, for providing vans and for their support of this project.

Altizer, S.M. and K.S. Oberhauser. 1999. Effects of the protozoan parasite Ophyrocystis elektroscirrha on the fitness of monarch butterflies (Danaus plexippus). Journal of Invertebrate Pathology, 74: 76-88

Altizer,S.M., K.S.Oberhauser, and L.P.Brower. 2000. Associations between host migration and the prevalence of a protozoan parasite in natural populations of adult monarch butterflies. Ecological Entomology 25: 125-139.

Monarch Lab website. University of Minnesota Monarch Parasites and Natural Enemies Research Projects: Interactions between monarch butterflies and the protozoan parasite, Ophryocystis elektroscirrha. http://www.monarchlab.umn.edu/Research/PNE/soniaresearch.html

Adam Arceneaux is an eighth grader at Westview Middle School who is an excellent student and plays the trombone in the wind ensemble. Adam puts his athletic abilities to good use by playing football and basketball. He will also be running track this spring. He enjoys hanging out with his friends and listening to music.

Christopher Corales is an eighth grader at Westview Middle School. He is an honor student who plays the basoon in the top band of the school, as well as playing the violin in the University of Texas music program. Chris enjoys surfing the net, playing video games, reading, drawing, bing outdoors, and spending time with his family.

Emily Torok is a bright fourteen year old who enjoys all subjects in school and has a deep passion for writing, and many, if not all fields of science. She is very artistic and creative which carries over into all aspects of her life. Emily has a deep desire to learn and her true passion resides in working her mind. She is also an eighth grader at Westview.

Carole Minnix is an eighth grade science teacher at Elgin I.S.D. in Elgin, Texas. Carole is completing her thirty first year in education. She has taught both at the elementary and middle school levels. Her favorite pasttimes include anything to do with animals and nature photography.

Beverly Zamponi is a seventh grade science teacher at Westview Middle School and a driver's ed instructor. She has been in education for twenty-four years. Beverly enjoys being outdoors, reading suspense novels, and spending time with her Daschund and her three grown children.