How many monarchs in natural populations are infected with O. elektroscirrha?
(see also: Altizer, S.M., Oberhauser, K.S., and Brower, L.P. 1999. Associations
between host migration and the prevalence of a protozoan parasite in natural populations
of adult monarch butterflies. Ecological Entomology. In press)
Abstract
Monarch butterflies are susceptible to infection by the obligate protozoan parasite,
Ophryocystis elektroscirrha (Apicomplexa: Neogregarinida). Monarchs form migratory
and non-migratory populations world-wide, and within the migratory populations not
all generations migrate. Therefore, this host-parasite system provides the opportunity
to examine how geographical variation in parasite prevalence relates to host migratory
patterns.
In collaboration with Karen Oberhauser, Lincoln Brower, and other colleagues, I
evaluated parasite prevalence using 14,790 adult monarchs captured between 1968
and 1997. Comparison of 3 populations in North America indicates that parasite prevalence
is associated negatively with monarch migration distances. A non-migratory population
in southern Florida shows high prevalence (over 70% heavily infected). The western
population migrates moderate distances to overwintering sites on the Pacific Coast
and has intermediate prevalence (30% heavily infected). The eastern migratory population,
which travels the longest distance to Mexican overwintering sites, has exhibited
less than 8% infection throughout the past 30 years.
I investigated changes in prevalence within North American migratory monarchs to
determine whether parasite prevalence declined during migration or overwintering.
Average parasite loads of summer-breeding adults in western North America decreased
with increasing distance from overwintering sites. This suggests that heavily infected
monarchs are less likely to remigrate long distances to summer breeding sites. However,
I found no differences in the frequency of heavily infected adults among eastern
or western North American monarchs throughout the overwintering period, suggesting
that this parasite does not affect overwintering mortality. Changes in the prevalence
of monarchs with low parasite loads demonstrate that spore transfer occurs during
migration and overwintering, possibly when adult butterflies contact each other
as a result of their clustering behavior. This study of geographical and temporal
variation in O. elektroscirrha among populations of D. plexippus demonstrates
the potential role of seasonal migration in mediating interactions between hosts
and parasites, and suggests several mechanisms through which migratory behavior
may influence parasite prevalence.
The prevalence and spread of infectious diseases can be influenced by a variety
of factors, including host density, parasite transmission mode, and the spatial
structure of host populations (Getz & Pickering, 1983; Anderson & May, 1991;
Antonovics & Thrall, 1995; Lockhart et al., 1996). In animal systems,
seasonal host migration will also likely to affect pathogen prevalence. For example,
seasonal migration has been implicated in the reduction of parasite prevalence in
reindeer and baboons (Hausfater & Meade, 1982; Folstad et al., 1991;
Shaw 1994). In this study, I examined large-scale temporal and geographic variation
in the prevalence of an obligate protozoan parasite, Ophryocystis elektroscirrha,
in monarch butterflies, Danaus plexippus. Monarchs form both migratory and
non-migratory populations, and within the migratory populations not all generations
migrate similar distances. Thus, this system provides the opportunity to examine
how both inter- and intra-population variation in parasite prevalence relate to
host movement.
Within North America, three monarch populations show varying degrees of migratory
behavior. Eastern North American monarchs migrate up to 5200 km to coniferous forests
in the trans-volcanic mountains of central Mexico (Urquhart & Urquhart, 1978;
Brower & Malcolm, 1991; Calvert & Lawton, 1993; Fig. 1), arriving from late
October to November, and overwinter in densely populated roosting sites that harbor
tens of millions of individuals (Brower & Malcolm, 1991; Calvert & Lawton,
1993). In February and March, these same individuals break diapause and mate before
flying north to recolonize their breeding range (e.g. Brower & Malcolm, 1991;
Van Hook, 1993). Three to four summer reproductive generations breed each year between
phases of migration and overwintering. Western North American monarchs migrate a
shorter distance to the coast of California in September and October (Nagano et
al., 1993; Brower, 1995; Fig. 1). Monarchs in a non-migratory population in
southern Florida appear to move very little during the year, but recent evidence
indicates that this population has a significant influx of autumn migrants from
the larger eastern population (Knight, 1997). Monarchs also populate parts of Australia,
Central and South America, and many Pacific and Caribbean Islands (Ackery &
Vane-Wright, 1984; James, 1993), with varying degrees of seasonal movement.
Fig. 1. Summer breeding ranges and major migratory routes for three North American
monarch butterfly populations: (1) eastern migratory population, (2) western migratory
population, and (3) southern Florida population (modified from Brower, 1995).
Click for more information on monarch
migration and geographical distribution
The protozoan parasite, O. elektroscirrha, has been reported in both eastern
and western populations of North American monarchs and in D. gilippus, the
Florida queen butterfly (McLaughlin & Myers, 1970; Leong et al., 1992,
1997a), although recent work suggests that the same parasite strains do not infect
both monarchs and queens (Leong et al., 1997a; S. M. Altizer, unpublished).
The life cycle of O. elektroscirrha is correlated closely with monarch development.
Asexual vegetative replication occurs within larvae and pupae, and sexually produced
spores are found in the developing adult integument. Parasites are transmitted vertically
from infected females to their offspring when females scatter spores on milkweed
plants during oviposition. Spores can also be transferred horizontally during mating
or other contact between adults, although larval ingestion is required to generate
new infections.
Click for more information on parasite development and
life cycle
The objective of this study was to quantify variation in parasite prevalence among
monarch butterfly populations with different migratory patterns. I examined samples
from monarchs collected over the past three decades to determine historical patterns
of parasite prevalence. Short-term changes in parasite prevalence within migratory
populations were examined to test the hypothesis that heavily infected hosts suffer
higher mortality during migration or overwintering. Finally, I studied changes in
the frequency of monarchs with low parasite loads to investigate whether horizontal
spore transfer occurs among adults during migratory and overwintering periods, when
monarchs cluster in dense aggregations.
Parasite prevalence in natural populations
I assessed parasite prevalence among adult D. plexippus captured from three
populations in North America (Fig. 1) between September
1968 and September 1997, several locations in Australia in 1996, and in South America
and Cuba in 1995 and 1996 (Appendix A). Adults
were placed in individual glassine envelopes and either held in a freezer for long-term
storage, or released immediately following data collection. Butterflies sampled
before 1994 were collected by Lincoln Brower and his associates for other purposes
and stored until sampled; more recent collections were made explicitly for the purposes
of parasite assessment. Parasite loads were evaluated for 12,000 adults from the
eastern North American migratory population, 2,141 adults from the western North
American migratory population, 446 adults from southern Florida, 105 adults from
Central and South America, and 108 adults from Australia.
Many samples listed in Appendix A represent collections from multiple dates or locations.
For example, monarchs were collected from the eastern and western North American
migratory populations at monthly or semi-monthly intervals during both reproductive
and overwintering periods to assess short-term changes in parasite prevalence. In
summer 1997, breeding adults were collected at 10 locations throughout western North
America to determine whether parasite loads declined with increasing distance from
overwintering sites. Dates and locations of samples used to evaluate short-term
temporal and geographic variation in prevalence are shown in
Appendices B and C.
Sampling techniques
To examine butterflies for parasite loads, I cut transparent Scotch™ tape into
approximately 1 cm2 units. This tape was held with fine forceps and pressed
against the ventral abdomen to remove a sample of abdominal scales. Each tape sample
was placed on a clear glass microscope slide or white index card, and viewed under
a microscope at 400x. Spores appear as dark brown, oval-shaped bodies approximately
1/50 the size of a butterfly scale (Leong et al., 1992).
All spores on the tape were counted, and butterflies were assigned to parasite load
classes according to the following scale: 0 = no spores, 1 = 1 spore, 2 = 2 to 20
spores, 3 = 21 to 100 spores, 4 = 101-1 000 spores, and 5 = > 1,000 spores. To
limit accidental spore transfer during handling of the butterflies, latex gloves
were worn and all objects contacting the monarchs were rinsed periodically with
a solution of 95% ethanol or 15% chlorine bleach.
Because two less extensive surveys of disease prevalence used a wash-and-count method
for parasite assessment (Leong et al., 1992, 1997a), I examined he correspondence
between the ScotchTM tape technique and the previously published method.
In July 1995, approximately 200 adults were reared in each of 3 calibrated inoculation
treatments: 0, 10, 100, or 1,000 spores administered per larva. I prepared inoculum
by vortexing the abdomens of infected adults in deionized water. I used a hemacytometer
to estimate the number of spores per volume of inoculum. Inoculum was passed through
a dilution series to obtain the desired number of spores per 10 m l, and this volume
of suspension was dropped onto a 1cm2 piece of milkweed leaf. Individual
first-instar larvae were placed in sterile Petri dishes with a contaminated leaf,
and after consuming the leaf tissue, they were transferred to plastic containers
and reared to adulthood.
Emerging adults were placed into glassine envelopes and frozen for later disease
assessment. After obtaining a tape sample from the ventral side of each individual,
I removed the abdomen and placed it in a glass vial containing 10 ml deionized water.
Vials were vortexed for 1 min, allowed to stand for 5 min, and vortexed for an additional
1 min to dislodge spores from the abdomen. The hemacytometer method outlined in
Leong et al. (1992) was then used to estimate the total number of spores
per vial.
Combining parasite load categories
To simplify many of the figures in the results that follow, I assigned infected
monarchs to one of two categories: heavily infected monarchs (parasite load categories
4 and 5) and those with low parasite loads (classes 1, 2, and 3). These categories
were selected because previous work indicated that high and low parasite loads result
from qualitatively different transmission modes and have different consequences
for host fitness. For example, laboratory experiments indicated that uninfected
female monarchs acquire low numbers of spores by mating with heavily infected males
(samples from females mated to infected males contained from 1 to 258 spores, mean
= 85 spores, n = 12 matings; Altizer 1998). However, monarchs that consume spores
as larvae almost always emerge heavily infected (Altizer & Oberhauser, in press).
Therefore, monarchs with low parasite loads are likely to have acquired spores as
adults, whereas monarchs with high parasite loads are most likely to have become
infected as larvae. Laboratory experiments have also demonstrated that heavily infected
monarchs (classes 4 and 5) experience measurable fitness declines in comparison
with uninfected monarchs or those with low parasite loads (Altizer & Oberhauser,
in press).