Effects of a protozoan parasite, Ophryocystis elektroscirrha, on the flight
endurance of its lepidopteran host, the monarch butterfly
Emily Yixian Yueh
NIH Research Apprenticeship Program
University of Minnesota
Abstract | Introduction |
Methods | Results | Discussion
| Acknowledgements | Literature Cited
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/Lab/Research/Topics/Enemies/LifeCycle.aspx
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/Lab/Research/Topics/Enemies/LifeCycle.aspx).
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/Lab/Research/Topics/Enemies/LifeCycle.aspx