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  |  Background  |  Methods   |  Results  |  Discussion  |  Acknowledgments  |   References  |  Appendices  |  Sonia's Research Questions

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.

BACKGROUND

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.

METHODS

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 Appendix 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 Scotch^TM 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).

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