Emily Yixian Yueh
NIH Research Apprenticeship Program
University of Minnesota

Abstract  |  Introduction  |  Methods  |  Results  |  Discussion  |  Acknowledgements  |  Literature Cited  |  Research Projects

Abstract

This investigation examines the difference in flight endurance of healthy monarch butterflies, Danaus plexippus, versus ones infected with Ophryocystis elektroscirrha, a protozoan parasite.  This disease infects monarch populations across North American, and disease prevalence is negatively correlated with host migration distances.  Differences in flight endurance of healthy versus infected monarchs may be responsible for this pattern of observed prevalence.  I have therefore designed a flight apparatus which allows me to evaluate and compare flight duration, distances, and average velocities of healthy and infected hosts.  Results of this study show a significantly higher flight duration and flight distance for healthy hosts and equal flight speeds for healthy and infected hosts.

Introduction

Background
Upon entering into a high school summer research program, I was assigned to work with Dr. Karen Oberhauser in the department of Ecology, Evolution, and Behavior at the University of Minnesota.  Dr. Oberhauser studies the biology of monarch butterflies, including their interactions with a protozoan parasite, O. elektroscirrha.   Different monarch populations in North America have dramatically different percentages of infection, with populations that migrate the farthest distance having the smallest parasite burdens.  However, many factors such as migration distances, temperature, humidity, population density, and vegetation are all unique to each group.   The element that most interested me was the effect of O. elektroscirrha on the migratory abilities of monarch butterflies.  I therefore chose to test the effects of O. elektroscirrha on flight endurance of monarch butterflies, and from that draw an inference about effects of disease on migratory abilities.

First reported in 1971 in southern Florida monarchs, O. elektroscirrha today can be found in all three main monarch populations in the United States.  Monarchs east of the Rocky Mountains migrate to overwintering sites in Central Mexico.   Monarchs west of the Rocky Mountains migrate a shorter distance to overwintering sites along the coast of California, and a population in South Florida does not migrate, but breeds continuously throughout the year (Figure 1).  For the Florida resident, Western, and Eastern populations, infection percentages are respectively:  100%, 60% and 5% (Leong et al. 1997). 

Figure 1.  Map of the United States and Mexico with the three different populations labeled as 1, 2 and 3.  General migration routes are shown with arrows.

O. elektroscirrha infects its monarch host at the larval stage when the dormant spores of this parasite are ingested.  These spores are transmitted by infected females, who, while ovipositing, disseminate spores onto the milkweed where the eggs are laid.  The sudden pH change in the digestive acids of the larval gut serves as the initial catalyst which interrupts the dormancy and causes the spores to lyse and move through the gut wall towards the hypodermal layer of the larvae.  There, parasite cells undergo two cycles of vegetative reproduction.  After pupation of the larvae, haploid parasite cells round up to form gametes which pair to form diploid zygotes.  Following meiosis, spore formation is initiated about 3 days prior to the monarch emergence (Figure 2).  This active state of the parasite is what causes the most damage to the organism, and rapid parasite reproduction consequently may cause increased mortality in the larval stage and decrease the average adult life span by four days (http://www.monarchlab.umn.edu/Research/PNE/life.html and Altizer, S. pers. comm.). 

Signs of infection are black spots on the pupal wall, each consisting of thousands of spores.  Although some hosts suffer no visible effects once emerged, others have defects such as crumpled wings or limited use of legs.  The emergence of the monarch triggers the end of the active cycle of the parasite, and they return to the dormant spore form, embedded within the scales throughout the entire body of the monarch.  While the infection cycle can occur only once in a host's lifetime, transmission of parasite spores can occur each time a butterfly flaps its wings and sheds scales carrying spores.   Healthy females that mate with infected males can also pick up spores on their abdomens and transfer these to their offspring (http://www.monarchlab.umn.edu/Research/PNE/life.html).

Figure 3.  The life cycle of the protozoan parasite, Ophryocystis elektroscirrha (diagram by Sonia Altizer).

Several factors may influence the observed patterns of disease prevalence in North America.  Some of these are humidity, genetics, temperature, and migration.   However, migratory distances of these populations vary dramatically and are likely to be a key element affecting disease spread and infected population percentages.   While the least infected Eastern population of monarchs migrate some 2000 miles from Canada to Central Mexico, Californian monarchs which migrate some 400 miles are at a higher infection rate than the former.  South Florida monarchs don't migrate and are at the highest percentage of infection.  This migration is likely to be energetically costly, and infected hosts may be less likely to survive the journey.

Hypothesis
If differential migratory abilities of healthy and infected monarch butterflies are responsible for the observed patterns of disease prevalence across populations, then O. elektroscirrha should have a measurable negative effect on the flight distance, duration, or velocity of infected hosts.  Thus, if there are significant differences in the distance, duration, and/or flight speed in healthy versus infected monarchs, then host migration may be responsible for decrease parasite burdens in those populations that travel the greatest distance. 

To test this hypothesis, I evaluated healthy and heavily infected hosts using a flight mill apparatus described below.  I measured the distance of flight, flight duration, and average speed of flight for both healthy and infected monarchs.

Methods

Materials
I investigated the flight endurance of 20 healthy and 20 heavily infected monarch butterflies, by tethering the adults to a flight apparatus, described below.  All tests were performed in an enclosed 8 ft. cu. incubator at 26 degrees Celsius and 60% relative humidity.

Equipment

• Flight mill
• Larvae and butterfly cages
• 6 ft X 6 ft netted cage
• Inoculation equipment
• Spore checking equipment
• Incubated room with adjustable temperature, lighting, and humidity
• Stainless steel single strand wire
• Timer
• Colored tape
• Rubber cement

Procedure
Infected larvae were inoculated in the laboratory, and groups of healthy and infected larvae were reared separately.  On the day of emergence, parasite burdens were assessed on all monarchs.  Masses were recorded the second day, and wire attachments were rubber cemented onto the dorsal thorax of each subject.  Monarchs were then placed into a 6 ft x 6 ft x 6ft netted cage on the roof of the Ecology Building.   Monarchs were fed a 25% honey-water solution on cellulose sponges each morning.   Deaths were recorded each day.  Each day an average of 5 butterflies were removed from the cage, massed, and put onto the flight mill for flight tests. 

The flight mill consists of a graphite arm that was attached to a low friction pivot on top of a vertical support.  A small light-weight piece of wire was fused onto the end of this arm, and the wire (from the backs of monarchs) was then attached to this wire to force the monarchs to fly in one direction only.  Thus, upon attachment to the apparatus, the distance (number of rotations) and time spent in flight was measured for each monarch (Figure 1).

Butterflies were removed when they exhibited signs of exhaustion based on the criteria of Hocking (1989).  If a butterfly paused, I waited 30 seconds and then blew on the butterfly to attempt to disturb it into flight.  If a monarch did not resume flight for more than 15 seconds after 3 consecutive attempts to disturb it, the butterfly's run was terminated.

Figure 1.  Butterfly flying attached to flight mill.

Results

Although monarchs were run at different ages, I found no relationship between age and flight duration, distance, or velocity.  Based on statistical analyses done on unweighted least squares linear regression of flight distance versus age of subject at time of flight, there appear to be no significant effects of age on one's flight distance (T = 0.041, p = 0.6807).  These results were also true for flight duration and flight velocity.  Seven of the 40 monarchs did not fly, or flew for less than 25 meters before stopping, and could not be induced to continue.  I therefore assumed that these monarchs were not flying due to some factor other than physical exhaustion, and I removed these individuals from the analysis below.

The flight duration of a monarch was measured as the total time it took for the subject to fly its total distance capacity.  This included all "stopping intervals" between the initial starting time and final ending time (Hocking 1989).  The average flying time of healthy monarchs was 2233 seconds, while the average flight duration of heavily infected monarchs was 1288.8 seconds (Figure 3a).  Statistical analysis was done using a two-sample T test for flight endurance by spore load (T=2.09, p = 0.0453).   Thus, healthy subjects displayed a significantly longer flight duration than to infected subjects.

Healthy subjects also displayed a significantly greater flight distance (T + 2.09, p = 0.0451) than that of heavily infected subjects (Figure 3b).  The average flight distance for healthy monarchs was 2275.3 meters, while infected monarchs achieved 1234.2 meters on average.  However, the mean flight velocity for healthy monarchs was 0.9622 meters/second, and for infected it was 0.9345 meters/second (Figure 3c).  Statistical analyses done on a two-sample T test showed no significant differences in the two velocities (T = 0.29, p = 0.7738).

       
Figure 3 (a-c).  Graphs of average (a) flight distance, (b) flight duration, and (c) flight velocity flown by healthy (spore load = 0) and heavily infected (spore load = 5) monarchs.    

Discussion/Conclusion

This study showed a significant effect of the protozoan parasite, O. elektroscirrha, on the flight duration and distance of its host, D. plexippus, with infected monarchs flying shorter times and distances on a flight mill apparatus.  However, both healthy and infected butterflies showed similar flight velocities.  Therefore, the longer flight distances of healthy butterflies is not due to greater speed, but to their longer times spent in flight.

These results allow me to draw some inferences pertaining to the effect of flight endurance during a seasonal migration.  If infected hosts are unable to successfully migrate in both the fall and spring journeys, then disease prevalence may decline as migration distances increase.  Thus, the effects of O. elektroscirrha on the migratory abilities of monarchs may lead to the dramatically different parasite burdens in migratory versus non-migratory populations.

Acknowledgements

I would like to express my gratitude to Sonia Altizer for her constant guidance, continuous support, and keen interest in my research.  Thanks also to Dr. Karen Oberhauser for her excellent advice, Don Alstad for his brilliance in building the flight mill apparatus, David Herr for his kindness in building two extra flight mills, Kari Geurts for proof-reading of my paper, and all other members involved in the process of this experiment.  I would most of all like to thank my parents for their support and encouragement in this and all things I do.  Without them, this would not have been possible.

Literature Cited

Hocking, B. 1989.  The intrinsics of range and speed of flight of insects studied using measurements of efficiency measured by using a insect tethered flight method. 

Leong, K. L. H., H. K. Kaya, and M. A. Yoshimura.  1997.  Occurrence of a neogregarine protozoan, Ophryocystis elektroscirrha (McLaughlin and Myers), in populations of monarch and queen butterflies.  Pan Pacific Entomologist 73:  49-51.

Monarch Lab website:  http://www.monarchlab.umn.edu/Research/PNE/life.html