1. Biology and life cycle of Oe

2. What is Oe? Ophryocystis elektroscirrha (Oe)
Protozoan parasite that infects Monarch butterflies in NZ

3. What is Oe? Protozoans: single celled organisms
Share many characteristics with animals (often called animal-like protists)
Protozoans are single celled organisms, living things that have many of the same characteristics as animals. In fact, protozoans are often called animal-like protists.
This slide has several protozoans pictures at the bottom that you may be familiar with. The first two are ‘free living’ protozoans called Euglena and a ciliate called Paramecium. The third is a parasitic protozoan called Plasmodium – this is a blood parasite that causes Malaria in humans
Euglena
Paramecium
Plasmodium

4. Obligate parasite: must live within a host to grow and multiply.
Monarch (and queen butterflies overseas) are the only known hosts of Oe
Monarch Butterfly (Danaus plexippus)
Queen Butterfly (Danaus gilippus)

5. Oe produces spores on the outside of Monarchs
Highly Magnified Image
Spores: dormant cells that can resist harsh environmental conditions
Found on the outside of infected Monarchs
Between infections Oe survives as spores that are resistant to extreme environmental conditions.
Photo of Oe spores is a scanning electron micrograph taken by Chip Taylor.
Oe spores
Monarch scales

6. Oe Spores
Greatest concentration of Oe spores is on the abdomens of infected Monarchs
Abdomen
These tiny spores are sandwiched in between the scales that cover a butterfly’s body.

7. Oe Spores Spores are ~ 100x smaller than Monarch scales
Need to view under x power
Spores are much smaller than scales. In fact, a Monarch scale is about 100 times larger than an Oe spore. You must use a light microscope set at 40 to 100X to see a spore. This slide shows a researcher using a stereomicroscope at 60 times magnification to examine Oe spores from an infected Monarch
Looking for spores using a microscope

8. Oe Spores Spores appear as small, brown or black lemon-shaped objects.
Monarch scales
Oe Spores
Even at this magnification spores look like small, brown or black lemon-shaped objects.

9. Oe Spores
At 400x
Here is a picture of Oe spores at 400X.

10. Life cycle of Oe is closely related to the life cycle of the Monarch butterfly.

11. Oe Life Cycle Oe can only reproduce inside living Monarchs
Infected females pass the parasite to their offspring when they lay eggs
Dormant spores on the outside of the female’s abdomen are scattered on the eggs and milkweed leaves
Infected females
lay eggs and
scatter Oe
spores on eggs
and leaves
Dark spots are Oe spores

12. Oe Infects the Caterpillar
When a caterpillar emerges, its first meal is the egg shell
It ingests Oe spores along with the shell and milkweed
Newly emerged caterpillar
A caterpillar eating the egg shell and
any Oe spores on the shell’s surface

13. Oe Moves to the Midgut
The dormant spores move through the larva to the midgut
Digestive chemicals break open the spores releasing the parasites
The parasites then pass through the intestinal wall to the hypoderm (underneath caterpillar’s skin)

14. Oe Reproduces in the pupa
Most damage to the butterfly happens during the pupal stage
Where Oe reproduces asexually
Each Oe parent cell divides many times, greatly increasing the number of parasites
The Oe parasite then goes through sexual reproduction, followed immediately by meiosis

15. Oe cells in the pupa Merozoites: vegetative, replicating stage of Oe
Single parent cell  many hundreds of daughter cells
Oe merozoites in the hemolymph of a 5-day old Monarch pupa

16. Spores Form in the pupae
About three days before the adult emerges from the pupa, Oe spores begin to form
Spores allow Oe to survive outside of the Monarch’s body
The spores can be seen through the integument or outside layer of the pupa
Oe Spores

17. Adult Emerges with Spores
Infected adults emerge covered with spores
Once butterflies are infected, they do not recover
By the time adults emerge with parasite spores, all physical damage by the Oe parasites has been done
The parasites do not grow or reproduce on the adults
The spores are inactive or dormant until they are eaten by another caterpillar

18. Parasitised emerging Monarchs
Monarchs that are heavily infected with Oe can have difficulty emerging from their pupal cases
Infected Monarchs are covered with millions of tiny Oe spores

19. Where in the World do you Find Oe?
Monarchs have a wide geographic range
Oe occurs in all Monarch populations examined to date
Monarch geographic range:
N. America
Central and S. America
Caribbean Islands
Pacific Islands
Australia
New Zealand
Oe was first discovered infecting Monarch and queen butterflies in Florida in the late 1960s. Since then this parasite has been found in all other Monarch populations world-wide. Monarchs and Oe have an extensive range that currently includes North America, Central America, central South America, Cuba and other islands of the Caribbean, Bermuda and Canary Islands of the Atlantic. During the 1800s, Monarchs were introduced to New Zealand, Australia, Philippines, south Pacific Islands, and Hawaii. Because Oe parasitises Monarchs all over the world, all indications are that this parasite has coevolved with Monarchs.

20. Where in the World do you Find Oe?
Prevalence of Oe
Population
Eastern NA
Rockhampton,
Australia
Northern
S America
Western
Sydney,
S Florida
n = 27
n = 25
n = 39
n = 1103
n = 717
n = 152
Prevalence: Measures proportion of Monarchs infected with Oe
# infected / total # sampled (n)
To see what fraction of Monarchs in different populations were actually infected, researchers have done extensive sampling of Monarchs collected throughout NA both in recent years and from past historical collections dating back to the early 1970's. An important measure of infection for any population is the fraction or proportion of animals that are infected. This is called prevalence. This graph shows the prevalence of Oe in 6 different wild Monarchs populations. Researchers went out, caught the butterfrlies, and examined them for Oe. …
What they found is that the prevalence of this parasite is highly variable among populations, and declines with increasing host migration distances. As you go from L to R in this graph, you are seeing populations that go from migrating the longest distance to those that migrate short distances or are resident breeders. Highest prevalence in pops that breed continuously in the same area (such as Florida and Hawaii) and lowest in Eastern NA.
To understand what might be going on here requires some working knowledge of Monarch butterfly migration

21. Monarchs in North America Summer Ranges and Migratory Routes
Eastern migratory
population
There are three Monarch populations in North America. Two populations migrate and the third one, which is in South Florida, does not migrate. The Rocky Mountains separate the two migratory groups of Monarchs.
2. Western migratory
population
3. South Florida resident
population

22. Eastern Migratory Monarchs
The eastern migratory population is the largest and most famous
These Monarchs spend the winter in the transvolcanic mountains of central Mexico
Transvolcanic Mountains

23. Monarchs Overwintering in Oyamel Fir Trees in the Mountains of Central Mexico

24. Eastern Migratory Monarchs
Spring: After mating, migrate north to their summer breeding grounds in the United States and Canada
Summer: Several generations of Monarchs inhabit lands east of the Rocky Mountains from Alberta to Maine

25. Eastern Migratory Monarchs
Fall: tens of millions of Monarchs return to Mexico in a spectacular migration

26. Western Migratory Monarchs
Shorter, less dramatic migration to their roosting areas on the coast of California
Overwinter in much smaller groups than the ones in Mexico
Spring: migrate north and east
Rocky Mountains
Summer Range for Western Monarchs
Overwintering Sites for Western Monarchs

27. Monarchs of South Florida
Do not migrate
Milkweed plants grow here all year round, the butterflies do not need to leave the area
Resident Monarchs reproduce throughout the entire year
Monarch larva feeding on tropical milkweed, a common but non-native host plant species in S. Florida

28. How common is Oe in North America?
1. Eastern migratory
population
– Less than 10%
heavily infected
3. South Florida resident population
– Over 70% heavily infected
Oe is found throughout North America, but the prevalence (fraction of the population infected) varies dramatically among populations. Fewer than 10% of Monarchs in eastern N. America re heavily infected, about 30% of Monarchs on the west coast are infected, and over 70% of Monarchs in S. Florida are heavily infected.
2. Western migratory
population
– 30% heavily infected

29. Differences between populations have persisted for many decades
Western migratory
By examining historical collections of Monarchs, scientists have found that differences in prevalence between populations have persisted for up to 30 years. This graph shows the % heavily infected Monarchs for each year where collections were available from the 3 populations, including samples from the most recent 5 years. Despite the inter-annual variations, the eastern migratory population has been less than 8% infected for the past 30 years, with a sign of some recent increases in the past 5 years, whereas more limited data suggests that the western population has been 30% infected, and the S. Florida population near Miami has had high parasite loads, % heavily infected.
Eastern migratory

30. Do my Monarchs have Oe? What are the symptoms?
Butterflies can become sick for many different reasons
Monarchs infected with Oe have a variety of symptoms
Caterpillars may have damage to their gut walls
Infections may be fatal
Monarch caterpillars often turn brown within a few hours of death.

31. Damage to the pupa
Infected pupae may develop dark spots or blotches 2-3 days before the butterfly emerges
These abnormal dark areas are parasite spores
Spores form on the eyes, antennae, wing veins, but mostly on the abdomen
You can see the spores through the outside layer of the pupa a day or two before pigments that color the butterfly normally darken the pupa

32. Dissected pupa Showing Oe Spores
The dark spots are Oe spores
Head
Abdomen
Infected pupa
before dissection
Before a butterfly emerges from the chrysalis, pigments are laid down coloring the scales that cover the butterfly. This normal change in the color of the pupa is symmetrical. The color change of an infected Monarch happens earlier and does not create a balanced pattern on the pupa.
Wings
Dissected pupa

33. Damaged Adult Monarchs
Heavily infected adults are weak and often have difficulty emerging from the chrysalis
Some Monarchs die before emerging
Others emerge, but are too weak to cling to the pupal case
They fall to the ground before fully expanding their wings
These severely deformed Monarchs do not survive long

34. Mild infections also harm butterflies
Infected adults are often smaller than healthy Monarchs
They weigh less and have smaller wings and shorter lifespans than normal Monarchs
Parasites also damage the cuticle or outside layer of the Monarch’s abdomen
This causes the butterfly to dry out and lose weight faster than normal.
Especially a problem if there is a shortage of nectar or water

35. Parasite infection hinders Monarch flight ability
Studies have shown that Monarchs infected with Oe can not fly as far or as long as healthy butterflies
Flight mill
A flight mill is used to measure
a Monarch’s flight endurance

36. Monarchs with mild infections can transmit disease
Infected adults that survive to reproduce will pass the infections on to their offspring

37. Many infected Monarchs look the same as healthy butterflies
These are all symptoms of Oe, but many infected Monarchs look healthy
They emerge normally and are not deformed
The only way to really know if your Monarch is infected is to check for parasite spores on its body.
Easy way to test for infections: swabbing

38. How to test for Oe

39. Use sterile protocols when handling Monarchs
Oe spores are difficult to destroy
Use latex, nitrile or vinyl gloves when touching butterflies
Sterilise all tools and surfaces that contact Monarchs (counter tops, rearing containers) with 20% bleach solution

40. Can You Tell Which Monarchs are Infected with Oe?

41. Can You Tell Which Monarchs are Infected with Oe?
ALL OF THEM!
Admittedly, the one in the lower right had a mild infection.

42. Adapted with thanks from an original presentation of the Monarch Health Project, Odum School of Ecology, University of Georgia Athens, GA, USA.