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Monarch butterflies (Danaus plexippus) inhabit islands and continents worldwide and form migratory and nonmigratory populations that differ according to local population density and exposure to the protozoan parasite, Ophryocystis elektroscirrha. Specifically, monarchs in non-migratory populations develop at higher densities and have higher parasite loads, whereas monarchs in migratory populations develop at lower densities and have much lower rates of parasitism. The goal of my study was to experimentally examine the effects of larval rearing density on the susceptibility and responses of monarch butterflies to infection with O. elektroscirrha. I predicted that monarchs reared at high densities would be more susceptible to infection and would experience more severe negative effects of disease. Monarch larvae were inoculated with calibrated parasite doses and reared in replicate containers at low (single larva), medium (5 larvae), and high (10 larvae) densities. Effects of density and parasite treatments were evaluated by measuring larval and adult survival, development rates, pupal mass, adult wing size, body pigmentation, and quantitative parasite loads. Results showed that rearing density significantly affected morphometric variables and development times. Parasite infection influenced monarch development rates, final parasite loads, and body pigmentation. There was an interactive effect of parasite infection and rearing density on pupal mass and total lifespan, and an interactive effect of density and sex on adult longevity. Thus, both rearing density and parasite infection significantly affected monarch development, survival, and final parasite loads.
Monarch butterflies are found in three main populations in North America. A population east of the Rocky Mountains breeds at low densities in the orange shaded region and migrates annually to central Mexico. A population west of the Rocky Mountains breeds in the yellow shaded region and migrates a shorter distance to coastal CA. A population in S. FL breeds year-round at high densities and does not migrate.
Figure 1. Wild monarch populations in North America
All monarch populations examined to date are infected with the neogregarine protozoan parasite, Ophryocystis elektroscirrha. Parasite development is highly tied to the life cycle of the host. Parasites are spread from infected adults to their progeny when dormant parasite spores are scattered onto milkweed leaves and ingested by larval hosts. After eclosion, infected adult butterflies emerge covered with thousands of spores on the outsides of their bodies.
Parasite prevalence varies dramatically among wild monarch populations. Only 8% of the eastern migratory population is heavily infected, about 30% of the western migratory population is infected, and nearly 80% of the resident S. FL population is infected.
Prediction
Larval rearing density could affect monarch susceptibility to infection. Two competing hypotheses arise from previous studies of insect-parasite interactions. If monarchs reared at high densities experience stress due to crowding (right side of diagram), they could be more susceptible to parasites and experience more severe effects of disease. If high densities trigger monarchs to invest more in immune defenses (left side of diagram) then monarchs reared at high densities will be more resistant to infection.
Measured Data
• Sub-lethal Effects
• Development Time
• Morphometric Measures
o Wing Density
o % Black on Wing
o Wing Area
o Pupal Mass
o Infection Status
o Quantitative Spore Measure
• Lethal Effects
• Larval Mortality
• Pupae Mortality
• Adult Survival
“Black Death”
During the course of the experiment 167 larvae and pupae died. The cause and source of death are unknown, but one possibility is that baculovirus was the cause. The larvae and pupae that were afflicted turned dark black and rapidly deteriorated. The “black death” affected the low and high density groups the most, while the medium group was the least affected (Figure 5). The unexplained deaths could mask any effect density may have had on the measured variables, by removing those individuals that are less fit. Figure 5 also shows that O. elektroscirrha did not affect susceptibility to the “black death.” Since not much is known about the “black death,” larvae and pupae dying from this were removed from statistical analysis.
[Figure 5. Number of deaths from the “black death” in each treatment and density group. Key: Orange= Monarchs exposed to parasites and Blue= Monarch not exposed to parasites]
Survival to adulthood
Monarchs reared at the high density treatment had lowest survival to adulthood, and those in the moderate density treatment had the highest survival to adulthood. Survival to adulthood was similar for monarchs exposed and not exposed to parasites.
Development Times
Larval development time, from oviposition to pupation, was longer for the parasite exposed monarchs in the medium and high densities, however it was longer for the control monarchs at low density (Figure 6). Total development time, from oviposition to adult eclosion, was longer for monarchs exposed to parasites relative to the control monarchs (Figure 7). Monarchs reared at the highest density had slower development times to adulthood. Monarchs reared at moderate densities developed the fastest overall (Figure 7). Males developed faster than females.
Figure 6. Time in days (log transformed) for larval development
Figure 7. Time in days (log transformed) for total development.
Total lifespan
Monarchs exposed to parasites had a significantly shorter lifespan than control treatment monarchs (Figure 8). Females exposed to parasites and reared at high densities had the shortest lifespan, and control males reared at high densities had the longest lifespan (Figure 8).
Figure 8. Time in days (log transformed) for total lifespan of monarchs. Purple= Control Male, Blue= Control Female, Orange= Infected Male, Yellow= Infected Female Morphometric measures
For monarchs exposed to parasites, pupal mass was greatest in the moderate density treatment and lowest in the high density treatment. Control monarchs were darker than infected monarchs in both the low and high density treatment (Figure 9). For the percent of the area of black pigmentation on forewings, females were darker than males, and monarchs exposed to parasites were lighter than control monarchs (data not shown).
Figure 9. Percent black (melanization) on the forewing of the monarch.
Parasite loads
Most monarchs exposed to parasites emerged with many parasites spores (Figure 10); control treatment monarchs showed very low contamination. Spore loads on adult butterflies increased with increasing larval rearing density (Figure 10).
Figure 10. Mean number of spores per micro liter from monarchs in the infected treatment. Four control individuals that were infected with spores were removed from statistical analysis.
Table 2. Analysis of variance results based on model simplification procedures, where AICC was used to determine minimum adequate model. Values were averaged within each rearing container prior to analysis (n=102 to 112 for all traits). X represents P<0.05 and ~ represents parameters that remained in the minimum adequate model but were not statistically significant.
Table 3. Correlations between dependent variables. Pearson’s correlations between all the measured data two-tailed significance. n=111 to 124 for all traits. *P<0.05; **P<0.01 Table 3. Correlations between dependent variables. Pearson’s correlations between all the measured data two-tailed significance. n=111 to 124 for all traits. *P<0.05; **P<0.01
• Density significantly affects on all morphometric measurements (except wing color) and all development times.
• Treatment significantly affected area black on wing, larval and total development, spore load and adult mortality.
• Sex had an effect on the all morphometric measures, adult development and total development. Treatment by density significantly affected pupal mass and total lifespan. Density by sex significantly affected total lifespan.
Our results do not conform to either the density dependent prophylaxis (DDP) or the crowding hypotheses. However, there are components that independently support both hypotheses specifically: Spore load fits well with the idea that crowding increases stress, because those in the medium and higher densities were found to have higher spore loads. Survival rate for the uninfected and infected males fits the DDP hypothesis because total lifespan increased for the above groups.
We would like to thank these individuals for helping us implement and carry out this project: Catherine Bradley, Zack Baumann, Andrew Davis, Laura Gold, Liz Harp, Jaap de Roode and Nick Vitone.
This material is based upon work supported by the Emory University SIRE (Scholarly Inquiry and Research at Emory) Grant and the Howard Hughes Medical Institute under Grant No.52003727.
In most species crowding affects the probability of infection, rate of infection, the development of the individual and other traits. Our study focused on the effects of crowding on monarch butterflies. This study is important because in the wild, monarchs are found to live in different population densities and because of large scale habbitat destruction, they are being forced to live in more crowded areas. To better understand this, we reared monarch larvae at different densities: low (single larvae,) medium (5 larvae,) and high (10 larvae) in same sized containers. We infected half the larvae with a parasite in order to understand how infection plays a role. We found that density and infection influenced the developments and health of the monarch.
Spore Removal, Spore Counting using a hemocytometer and Digital Image analysis
Dannus plexippus, neogregarine, Ophryocysitis elektroschirrha, density
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