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Danielle YNA 2012

Danielle

Grade: 12 | State: California

The Prevalence of Ophryocystis elektroscirrha Infections in the Monarch Butterfly (Danaus plexippus): A Study of the Protozoan Parasite in a Wild Population of Western Monarchs

Introduction

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Figure 1: Tagged Monarch


High in the eucalyptus trees in coastal southern California, a cluster of migrating monarch butterflies formed huddled masses, as if to brave the chill. I could appreciate their need for warmth as my own wind-chilled fingers refused to move, making the delicate task of handling one of the netted butterflies even more challenging. As I carefully placed the small white tag on the butterfly’s right hindwing (Figure 1), I considered its significance. This small aggregation of western migrants may have traveled from as far south as Arizona or as near as my own backyard, stopping at autumnal wintering sites like this along the way. The promise of this tiny tag on the seemingly fragile butterfly could tell us of that journey.

It was during these tagging sessions that I learned about the protozoan parasite Ophryocystis elektroscirrha, which infects monarch butterflies (Danaus plexippus) in North America. The western monarch migratory population has an approximate infection rate of 30% (Leong et al., 1992) and overwinters along the central and northern California coast (Urquhart 1978, Brower 1995). As I gained a deeper understanding of the influence that migration has on the fitness and abundance of wild populations, I began to consider the health of monarch butterflies in our gardens, where breeding is observed throughout the year.

For years I've reared or observed monarch butterflies, witnessing firsthand how parasitic infections are a natural form of population control in the ecosystem. Unlike parasitoids that result in host mortality in the larval or pupa state, O. elektroscirrha infections in adult monarchs can be undetectable to the naked eye, spread among hosts by spores on the abdomen (McLaughlin & Myers, 1970). I wondered how the infection rate for a year-round population compared to the overall prevalence of the parasite estimated for the entire population. Also, as O. elektroscirrha is most commonly transmitted maternally, from heavily infected females to their offspring (McLaughlin & Myers, 1970; Leong et al., 1997b), I wondered if the prevalence of the infection in sample gardens would be directly influenced by fluctuations in the population of infected females.

Background

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Figure 2


The monarch butterfly (Danaus plexippus) is native to North and South America. As specialist herbivores, monarch larvae eat only plants in the milkweed family (Asclepiadacea). They typically have three to four generations annually. In the fall, the fourth generation migrates to climates that will allow them to sustain diapause through the winter (November to March). Non-migrating populations breed year-round in some southern states. This important biological phenomenon occurs in close proximity to wintering sites in Mexico and California, and is not well monitored (Davis et al., 2009). The life span for adults varies depending on whether they are migratory (up to nine months) or non-migratory (two to five weeks). Among the three populations, the eastern population migrates the greatest distance, from Canada to Central Mexico (Brower 1991). The western population migrates shorter distances to winter along the coast of California (Nagano et al., 1993; Brower 1995). The third represents non-migratory populations that breed year-round in southern Florida, Hawaii, the Caribbean Islands, and Central and South America (Knight 1998, Altizer 2001). 

Ophryocystis elektroscirrha

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Figure 3: Ophryocystis elektroscirrha


Discovered in 1966, O. elektroscirrha (McLaughlin & Myers, 1970) is an obligate protozoan parasite whose primary host is the monarch butterfly. A protozoan is a single-celled organism that shares many of the same characteristics as animals. As an obligate parasite, O. elektroschirrha requires a live host to complete its life cycle and spread infection. An O. elektroscirrha (Figure 3) infection is spread between hosts vertically (parent to offspring) and horizontally (via unrelated adults to the environment) by transmission of infective spores (Vickerman et al., 1999; Altizer et al., 2004). Maternal transmission occurs when the infected female scatters spores onto the eggs during oviposition. The contaminated shell is eaten by the newly hatched caterpillar, which ingests the spores (De Roode et al., 2007). While in the gut, the parasite replicates asexually before completing sexual reproduction during pupation. After eclosion, the monarch secretes the parasite’s spores through its midgut lining , and they appear on the hypoderm of the abdomen (McLaughlin & Meyers, 1970; Leong et al., 1992). Paternal transmission occurs during mating. Researchers found that nearly 95% of the offspring from heavily infected captive females became infected, whereas 75% to 90% of the offspring were infected following paternal transmission (Altizer et al., 2004). Horizontal transmission involves the transfer of spores to milkweed foliage during contact. The contaminated leaves are ingested by unrelated caterpillars, creating new infections (De Roode et al., 2007).

Ophryocystis elektroscirrha is a naturally occurring parasite distributed throughout the diverse monarch populations (Leong et al., 1997a). Parasite prevalence is known to vary among host populations relative to the length of migration. For example, more than 80% of the non-migratory populations in southern Florida and Hawaii are heavily infected (Leong et al., 1997b; Altizer et al., 2000). In contrast, the eastern population has less than 5% heavily infected (Leong et al.,1997b; Altizer et al., 2000). In 1992, a study of two wintering sites in California found that 30% of the migratory population was heavily infected (Leong et al., 1992; Altizer et al., 2000).

Hypothesis

Question 1: How does the prevalence of O. elektroscirrha in the sample population compare to the average rate of infection for the migratory population?

I predict the prevalence of O. elektroscirrha in the sample population will be the greater than in the migratory population. The sample population that occupies coastal southern California is a subset of the greater western population. However, in the southern range, monarchs are observed to breed year-round in gardens, with larvae and adults present during winter (December to March). Therefore, I suggest that O. elektroscirrha will more readily spread among hosts, to offspring, and build up in the garden environment, having a greater influence on parasite virulence due to behavioral differences between the populations.

Question 2: Does the incidence of heavily infected female monarchs in the sample population positively correlate to the monthly average of O. elektroscirrha infection for the entire sample?

I anticipate there will be a positive correlation between the percentage of heavily infected females each month and the subsequent fluctuation in the infection rate per month. Previous studies on methods of transmission of O. elektroscirrha in captive monarchs revealed that heavily infected females (with spore loads of 100 or more) produce infected offspring 95% of the time (Altizer et al., 2000; Altizeret al., 2004). I expect my field study will demonstrate a similar result using the wild sample population.

Sample Population

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Over the course of a year (December 2010 to November 2011), I collected 746 scale samples from wild-caught monarch butterflies in 17 test gardens located within the coastal city of San Clemente in southern California (Figure 4). The selected sample represents the observed year-round breeding population of monarchs in the southern range of the western population. The gardens encompassed five square miles, with the farthest distance inland being fewer than 1.5 miles from the Pacific Ocean. They ranged in size from small to large (100 milkweed plants). The milkweed species grown in 94% of gardens was Asclepias curassavica, a non-native evergreen common to gardens in this region. Sample gardens included homes, elementary schools, a cultural center/botanic garden, and planted nature areas in city and state parks. In order to conduct a field study of this duration and size, I enlisted help from two adults whom I trained to assist with collecting some of the samples from their own test gardens.

Materials

  • Butterfly net, 14” diameter with an extending pole (30” to 69” length)
  • Plastic field study box, size 12” x 6”, to contain items in the field study kit
  • Plain white index cards, size 4”x 6”, for recording sample data (Figure 5)
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    Figure 5


    Clear Avery envelope seals, size 1”, for collecting scale samples (Figure 5)
  • White circular identification tags (Figure 5) (Monarch Alert, California Polytechnic State University, San Luis Obispo) to differentiate specimens by tag number and track their movements
  • Pen for recording data and two Sharpie markers, one blue (male), one red (female) to differentiate sampled butterflies from a distance by a wing symbol (Figure 2)
  • Pocket caliper (gauge) to measure the specimen’s wing size
  • Hand sanitizer for use between specimens, to prevent cross-contamination
  • Digital LCD biological microscope with objective lens 40x-100x-400x

Procedure

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Figure 7


The test gardens were visited multiple times, some on a weekly basis, to obtain the greatest number of samples by month and season. Depending on the site, scale samples were taken from either wild-caught monarchs or confirmed garden enclosure.

To obtain scale samples in the field:

  • Capture the specimen. For monarchs in flight, I used a 14” diameter butterfly net. Once netted, monarchs can be held between the thumb and index fingers, gently holding the forewings and hindwings together near the thorax to prevent injury.
  • Sex the specimen. Males have claspers (for holding females during mating) at the end of their abdomens as well as symmetrical scent glands on the hindwings.
  • Measure wing length. Using the caliper, measure the distance from the thorax to the wing tip. 
  • Record the data. Write the date, test garden name, sex, and wing length on the index card. Record the Monarch Alert tag number to be applied.
  • Apply identification tag. While holding all four wings, remove the circular white Monarch Alert tag from the sheet, being careful to touch a minimal amount of the sticky underside. Apply it to the mitten-shaped discal cell in the center of the right hindwing. Press gently with the right thumb and index finger, holding for several seconds.
  • Mark the wing with study symbol. Re-position the butterfly with the left hindwing facing up. Gently draw the study symbol (Figure 2) with the marker. 
  • Obtain a scale sample. While holding the butterfly with the abdomen facing up, use a clear envelope seal to swipe the abdomen to collect the scale sample. Press gently to avoid injury but ensure a complete sample (Figure 7). Press the clear seal onto the index card next to the left edge, in line with the tag number.
  • Release the specimen. Once complete, release the monarch.
  • Sanitize between samples. Use antibacterial lotion to limit cross-contamination in the field.

To determine infection in the lab:

  • Examine samples for evidence of infection. After field collection, use the microscope to determine O. elektroscirrha infections. Scale samples on the index cards are examined under 100 x magnifications. Spores are identified as the regular, dark, football- or oval-shaped objects. Spore loads estimated at more than 100 (Figure 3) are recorded as “heavily infected” (Altizer et al., 2000).

DATA ANALYSIS AND DISCUSSION

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Figure 8: Collection Data (12/1/2010-11/30/2011)


Monarchs were observed in the gardens year-round,illustrated in Figure 8: Collection Data and Figure 9: Monthly Sample Size.Males in the sample population outnumbered females by 2:1. The greatest breeding months, indicated by sample size, were July to October, which is consistent with peak summer and fall generations.

This graph represents the monthly fluctuations of O. elektroscirrha infections in the year-round breeding population and the respective average rate of infection, 40.8%, in comparison to the average rate of infection, 30%, for the western migrant population (Leong et al., 1992). The sample population is 36% higher than the average for the migratory population.

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Figure 9: Monthly Sample Size


This graph demonstrates the relationship between the overall monthly infection rate and the influence of infected females. In the sampled population, each span of about a month and a half represents a new generation, and the offspring of the previous one. The highs and lows of infected females lead to the subsequent monthly infection rates.

This graph demonstrates the relationship between the overall monthly infection rate and the influence of infected males. In comparing this to the female graph (Figure 11), the correlation between the male infection rate and the overall average is not as strong because males transmit the infection to their offspring only 75% of the time (Altizer et al., 2004).

 
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Figure 10: Average Infected Per Month: Comparing Sample Population to Migrant Population


 
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Figure 11: Average Infected Per Month as Influenced by Monthly Female Infection Rates


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Figure 12: Average Infected Per Month as Influenced by Monthly Male Infection Rates


Conclusions

Question 1: How does the prevalence of O. elektroscirrha in the sample population compare to the average rate of infection for the migratory population?

The data collected for the sample population supports my hypothesis, as demonstrated in Figure 10. The average infection rate for O. elektroscirrha over the 12-month period for the sample population was 40.8%. This represents a 36% increase over the 30% average infection rate estimated for the western migratory population (Leong et al., 1992). Although the populations sampled were subsets of the western population, there are variables that differentiate each data set. My results are based on a 12-month field study, whereas the prior research was conducted over the migratory season alone. My study sampled multiple generations where year-round breeding is observed, as compared to the 1992 study that sampled only fourth-generation migrants. It would have improved the quality of the comparison to have used a more recent migratory sample from the southern region, but this is not an area of frequent study. As the graph in Figure 10 demonstrates, the monthly rate of infection for the year-round breeding sample continues to climb upward, beginning in July/August and ultimately reaching 81% by November. This is in significant contrast to the migratory population sampled in winter.

Now that a baseline average infection rate and seasonal population statistics have been established for the southern region, additional collection years may correlate monarch cycles and migration with climate change. Further research needs to be done to verify whether or not monarchs from the southern region migrate. If not, are the causes the environment or human behavior?

Question 2: Does the incidence of heavily infected female monarchs in the sample population positively correlate to the monthly average of O. elektroscirrha infection for the entire sample?

As the graph in Figure 11 shows, there is a positive correlation between the monthly infection rate of heavily infected females and the subsequent fluctuations in the overall infection rate for the population. This is consistent with my hypothesis and the results of prior lab research. For example, each span of about a month and a half (32-45 days) represents the potential progeny of the heavily infected females (depending on season and climate), and their subsequent offspring. The peak infections for May can be assumed to be in response to the highly infected females in March; the continual rise in July/August may be in response to infected females in May, combined with peak reproductive season. The dips in the graph in April and late June can therefore be assumed to be the result of an influx of spring migrants who traditionally have lower rates of infection (Leong et al., 1992). Over time, the influence of migrants is diluted substantially as the season continues and the year-round populations combine. 

However, the dominance of males in the sample population invariably contributed to the overall infection rate through paternal transmission. Males outnumbered females 2:1 in the sample population. The graph in Figure 12shows that although there were more males than females and their monthly infection rates often surpassed the females, the correlation of the male infection rate and the overall infection rate isn’t as close. According to prior research, males transmit the infection paternally at a rate of 75% to 90%, which is less than the 95% transmission rate for females (Altizer et al., 2004). This research is also consistent with the data in the graph.

In future repetitions of this experiment, I would prefer to use a more accurate and even representation of the data through a truly random sampling of the data collected. This mathematical method would reduce the male to female ratio to 1:1 while still accurately representing the data. I was limited in my understanding of how to accomplish this. Additional education and guidance could improve the results.

Acknowledgements

I would like to thank my parents, who are my inspiration; David Marriott of the Monarch Program; Bob Allen (or "Bug Bob"), who taught me how to tag my first monarch at San Clemente State Beach and introduced me to Ophryocystis elektroscirrha; Bill Schafer (a citizen scientist); and all the wonderful participants who gave up some privacy to invite me to traipse through their gardens each week in search of my elusive target. I truly appreciate how enthusiastically the community supported my research project.

Bibliography

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Altizer, S.M. “Migratory behaviour and host-parasite co-evolution in natural populations of monarch butterflies infected with a protozoan parasite.” Evolutionary Ecology Research 3 (2001): 611-632.

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

Altizer, S. and K. Oberhauser. “Effects of the protozoan parasite, Ophryocystis elektroscirrha, on the fitness of monarch butterflies (Danaus plexippus).” Journal of Invertebrate Pathology 74.1 (July 1999): 76-88.

Brower, L.P. “Understanding and misunderstanding the migration of the monarch butterfly in North America: 1857–1995.” Journal of the Lepidopterist's Society 49 (1995): 304-385.

Brower, L.P., and S.B. Malcolm. “Animal migrations: endangered phenomena.” American Zoologist 31 (1991): 265-276.

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Davis, A.K., S.M. Altizer, and E. Friedle. “A non-destructive, automated method of counting spores of Ophryocystis elektroscirrha in infected monarch butterflies (Lepidoptera: Nymphalidae).” Florida Entomologist 87.2 (2004): 231-234.

De Roode, J.C., A.J. Yates, and S. Altizer. “Virulence-transmission tradeoffs and population divergence in virulence in a naturally occurring butterfly parasite.” PNAS 105 (2008b): 7489-7494.

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Knight, A.J. “A population study of monarch butterflies in North-Central and South Florida.” Unpublished M.S. thesis, University of Florida, Gainsville, Florida, 1998.

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Leong , K.L.H., M.A. Yoshimura, H.K. Kaya, and H. Williams. “Instar susceptibility of the monarch butterfly (Danaus plexippus) to the neogregarine parasite, Ophryocystis elektroscirrha.” J.Invertebrate Pathology69 (1997b): 79-83.

Leong, K.L.H., H.K. Kaya, M.A. Yoshimura, and D.F. Frey. “The occurrence and effect of a protozoan parasite, Ophryocystis elektroscirrha (Neogregarinida: Ophryocystidae), on overwintering monarch butterflies, Danaus plexippus (Lepidoptera: Danaidae), from two California winter sites.” Ecological Entomology 17 (1992): 338-342.

Marriott, David. Executive director of the Monarch Program, Encinitas, CA. Interviewed by Danielle Ethington, 24 Jan 2011.

McLaughlin, R.E. and J. Myers. “Ophryocystis elektroscirrha sp.n., a neogregarine pathogen of the monarch butterfly, Danaus plexippus (L.), and the Florida queen butterfly, Danaus gilippus berenice Cramer.” Journal of Protozoology 17 (1970): 300-305.

McLaughlin, R.E. and J. Myers. “Monarch Alert.” California Polytechnic State University, San Luis Obispo. Retrieved from http://monarchalert.calpoly.edu/.

“Monarch Health.” University of Georgia, Athens, GA. Retrieved on 25 Nov 2010 from http://monarchparasites.org/.

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“Monarch Net.” University of Georgia, Athens, GA. Retrieved on 28 Dec 2011 from http://monarchnet.uga.edu/.

“Monarch Watch.” Retrieved on 25 Nov 2010 from http://www.monarchwatch.org/tagmig/tag.htm.

Nagano, C.D., et al. “Spring migration of monarch butterflies in California.” In Biology and Conservation of the Monarch Butterfly, S.B. Malcolm and M.P. Zalucki (eds). Los Angeles: Natural History Museum of Los Angeles County, 1993: 219-232.

Urquhart, F.A., and N.R. Urquhart. “Autumnal migration routes of the eastern population of the monarch butterfly (Danaus plexippus L.;Danaidae; Lepidoptera) in North America to the overwintering site in the Neovolcanic Plateau of Mexico.” Canadian Journal of Zoology 56 (1978): 1759-1764.

Vickerman, D., A. Michels, and P.A. Burrowes. “Levels of infection of migrating monarch butterflies, Danaus plexippus (Lepidoptera: Nymphalidae) by the parasite Ophryocystis elektroscirrha (Neogregarinidae: Ophryocystidae), and evidence of a new mode of spore transmission between adults.” Journal of the Kansas Entomological Society 72 (1999): 124-128.

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