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Un parasito protozoo en las poblaciones de mariposas monarcas silvestres y en cautiverio cerca de Monteverde, Costa Rica
A protozoan parasite in wild and captive monarch butterfly populations near Monteverde, Costa Rica
Although the protozoan parasite Ophyrocystis elektroscirrha was first recovered from Danaus plexippus (Nymphalidae: Danainae) and Danaus gilippus populations in Florida in 1966 and has since been found in many other monarch and queen populations worldwide, no previous studies have shown that this parasite infects butterflies in Costa Rica. This study is the first to do so, documenting the occurrence of Ophryocystis elektroscirrha in a wild and butterfly garden population of D. plexippus near Monteverde, Costa Rica. Although only a few infected individuals were found in both populations, there was a stark contrast in the number of parasites per individual between the two populations. Infected individuals in the captive population had much higher parasite spore loads, 679 and 944, compared to the spore loads of seven and three found in the two infected individuals in the wild population. This finding is significant because only monarchs with high parasite loads suffer decreased fitness and increased mortality. This study
raises the possibility that captive monarch populations can serve as a source of disease to wild monarch populations that are healthy. However, simple measures can be taken to ensure that butterfly garden monarchs are free from heavy O. elektroscirrha infections, which ensures the health of the captive population and safeguards that of the local wild population.
Aunque el parsito protozoario Ophryocystis elektroscirrha fue colectado en poblaciones de Danaus plexippus (Nymphalidae: Danainae) y de Danaus gilippus en Florida en 1966 y ha sido encontrado desde entonces en muchas otras poblaciones de mariposas monarcas y reinas en todo el mundo, no existen estudios previos que hayan muestreado que este parsito infecte mariposas en Costa Rica. Este estudio es el primero en documentar la presencia de Ophryocystis elektroscirrha en poblaciones silvestres y del mariposario de D. plexippus cerca de Monteverde, Costa Rica. Aunque solamente unos pocos individuos fueron encontrados con la infeccin en ambas poblaciones, hubo un contraste notable entre los niveles de la infeccin en las dos poblaciones. Los individuos infectados en la poblacin prisionera tenan cargas mucho ms altas (679 y 944) que las de los individuos infectados en la poblacin silvestre (7 y 3). Este resultado es importante porque solamente las mariposas monarcas con grandes cantidades del parsito sufren adaptacin disminuida y mortalidad creciente. Este estudio presenta la posibilidad de que las poblaciones prisioneras de la mariposa monarca puedan servir como fuente de enfermedad a las poblaciones silvestres de monarcas sanas. Sin embargo, se pueden tomar medidas simples para asegurar que las monarcas del mariposario estn libres de infecciones agudas del O. elektroscirrha, lo que beneficiara la salud de la poblacin prisionera y salvaguardara la de la poblacin silvestre local.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Fall 2004
Ecologa Tropical Otoo 2004
t Monteverde Institute : Tropical Ecology
1 A protozoan parasite in wild and captive monarch butterfly populations near Monteverde, Costa Rica Joanna Hsu Department of Biology, Johns Hopkins University ABSTRACT Although protozoan parasite Ophyrocystis elektroscirrha was first recovered from Dan aus plexippus (Nymphalidae: Danainae) and Danaus gilippus populations in Florida in 1966 and has since then been found in many other monarch and queen populations worldwide, no previous studies have shown that this parasite infects butterflies in Costa Ric a. This study is the first to do so, documenting the occurrence of Ophryocystis elektroscirrha in a wild and butterfly garden population of D. plexippus near Monteverde, Costa Rica. Although only a few infected individuals were found in both populations, t here was a stark contrast in the number of parasites per individual between the two populations. Infected individuals in the captive population had much higher parasite spore loads, 679 and 944, compared to the spore loads of seven and three found in the t wo infected individuals in the wild population. This finding is significant because only monarchs with high parasite loads suffer decreased fitness and increased mortality. This study raises the possibility that captive monarch populations can serve as a s ource of disease to wild monarch populations that are healthy. However, simple measures can be taken to ensure that butterfly garden monarchs are free from heavy O. elektroscirrha infections, which ensures the health of the captive population and safeguar ds that of the local wild population. RESUMEN Aunque el parsito protozoario Ophryocystis elektroscirrha fue colectado en poblaciones de Danaus plexippus (Nymphalidae: Danainae) y de Danaus gilippus en Florida en 1966 y ha sido encontrado desde entonces en muchas otras poblaciones de mariposas monarcas y reinas en todo el mundo, no existen estudios previos que hayan mosterado que este parsito infecte mariposas en Costa Rica. Este studio es el primero en documentar la presencia de Ophryocystis elektrosci rrha en poblaciones silvestres y de mariposario de D. plexippus cerca de Monteverde, Costa Rica. Aunque solamente unos pocos individuos fueron encontrados con la infeccin en ambas poblaciones, hubo un contraste notable entre los niveles de la infeccin en las dos poblaciones. Los individuos infectados en la poblacin prisionera tenan cargas mucho ms altas (679 y 944) que las de los individuos infectados en la poblacin silvestre (7 y 3). Este resultado es importante porque solamente las mariposas monarca s con grandes cantidades del parsito sufren adaptacin disminuida y mortalidad creciente. Este estudio presenta la posibilidad de que las poblaciones prisioneras de la mariposa monarca puedan servir como fuente de enfermedad a las poblaciones silvestres d e monarcas sanas. Sin embargo, se pueden tomar medidas simples para asegurar que las monarcas del mariposario estn libres de infecciones agudas del O. elektroscirrha lo que beneficiara la salud de la poblacin prisionera y salvaguarda la de la poblaci n silvestre local. INTRODUCTION Parasites increase the mortality of their hosts by consuming nutrients and by creating energy costs associated with an adequate immune response. They also play an important role in regulating animal populations by influen cing host fitness, reproduction, and survival (Lanciani 1975). Insects are parasitized by a variety of microorganisms,
2 nematodes, and other insects such as small flies and wasps. Parasites can infect insects at the egg, larval, pupal, or adult stage. Ophr yocystis elektroscirrha is an obligate protozoan parasite that infects monarch butterflies ( Danaus plexippus ) and queen butterflies ( Danaus gilippus ). Like many other protozoan parasites in the phylum Aplicomplexa (Brusca & Brusca 1990), O. elektroscirrha alternates between sexual and asexual phases (Appendix A). Butterfly larvae are infected when they ingest parasite spores on contaminated eggshells or leaves of the larval foodplant (Apocynaceae). In the larval gut, the spores lyse and the emerging sporozo ites migrate to the hypoderm where they undergo two cycles of asexual reproduction. During host pupation, the parasite sexually reproduces and the butterfly emerges with external spores, primarily around the scales of its abdomen, but also on its head, win gs, and thorax. Although adults with low parasite loads appear normal, adults with heavier loads have difficulty emerging from their pupal cases and expanding their wings (McLaughlin and Myers 1970, in Altizer et al. 2000). Heavily infected adults emerge with smaller wingspans, lower body mass, and are shorter lived. High parasite loads also increase larval mortality and decrease male reproductive success, although female fecundity is unaffected by heavy loads (Altizer and Oberhauser 1999). O. elektrosci rrha is transmitted maternally when infected females scatter spores on eggs and milkweed while ovipositing (McLaughlin and Myers, 1970). Spores can be also transferred horizontally between adult butterflies during mating or other contact (Altizer 1998, in Altizer et al. 2000). Different populations of D. plexippus in North America are parasitized by O. elektroscirrha to different degrees. In one large scale study, over 70% of monarchs in one population from Florida were heavily infected, while only 30% of a western population and 8% of an eastern population were heavily infected. Parasite prevalence was correlated with host migration patterns, with non migratory populations having the highest proportion of heavily infected individuals (Altizer, et al. 2000). Since O. elektroscirrha has also been documented in monarch populations (Altizer, et al. 2000) in Australia (proportion of heavily infected individuals in three populations: 0.39, 0.27, .083), the Caribbean, and northern South America (proportion of heavi ly infected = 0.11), it is likely that D. plexippus in Costa Rica is also infected by this protozoan. In this study, the extent of O. elektroscirrha parasitism was compared between a captive and wild population for both monarch and queen butterflies. I als o intended to assess whether infection rates corresponded with butterfly sex, size, or age. Queen butterflies were included in this investigation because most studies of O. elektroscirrha have been with their monarch hosts, and it would be interesting to s ee if trends that have been shown in monarchs also apply to this closely related species. Sex of the butterfly was studied since the parasite seems to affect the sexes differentially (Altizer and Oberhauser 1999), decreasing male reproductive success but n ot that of females. Based on a previous study (Altizer and Oberhauser, 1999), I expected to find that heavily infected individuals would be smaller than other individuals. A size difference between healthy and infected monarchs could have consequences for reproductive success. For example, females with larger wingspans have longer egg laying lifespans (Oberhauser, 1997). Larger wingspans may also correlate with successful migration (Alonso Mejia et al ., 1997).
3 I also anticipated that heavily infected indivi duals would be younger because heavy parasite loads increase mortality, decreasing the likelihood that hosts will survive to old age. Wing wear, which is indicative of butterfly age as well as behavior, was correlated to infection levels in the 1999 Altize r and Oberhauser study. Captured individuals with low or intermediate spore loads had greater degrees of scale loss and wing tatter than their non infected counterparts. This could be because older or more active individuals are more likely to acquire spor es through contact with infected adults, which would not support my hypothesis that heavily infected individuals tend to be younger. An alternative explanation for the 1999 study results proposed by the authors is that infected monarchs may acquire more wi ng damage while foraging for nectar or water because they are physiologically stressed. Finally, higher incidence and virulence of O. elektroscirrha was expected in captive populations than in wild populations because a depressed gene pool in inbred capti ve populations would make individuals more susceptible to disease. Furthermore, a variety of factors facilitate parasite transmission in captive populations, including non sterile rearing techniques and accumulation of spores on a limited quantity of milkw eed plants. MATERIALS AND METHODS This study took place over a three week period during the transition from wet to dry season in October and November 2004. Wild D. plexippus and D. gilippus thersippus were captured in open field areas around 1200 m in e levation where Asclepias curassavica (Apocynaceae) was abundant in San Luis Valley, Costa Rica, a Premontane Tropical Moist Forest. Captive butterflies were obtained from Selvatura Butterfly Garden in Monteverde, Costa Rica. All individuals caught were sex ed, and based on wing wear (fresh, intermediate, or worn), each butterfly was placed into one of three age categories: young, intermediate, or old. The forewing length from the thorax to the edge of the wing was measured for each butterfly using a caliper. Individuals were sampled for O. elektroscirrha spores according to the method outlined by Davis, et al. (2004). A cotton swab was used to swipe one side of the butterfly abdomen forward from the posterior end. This was repeated four times on the same sid e of the abdomen using the same cotton swab. The scales and spores on the swab were transferred onto a standard microscope slide by tapping the swab on the slide several times. The slides were covered with transparent Scotch tape and examined under a comp ound microscope at 100x for spores. Spores were easily identifiable as brown football shaped objects approximately 1/50 th the size of a butterfly scale. Two samples from each butterfly, one from each side of the abdomen, were taken and the final spore coun t was the average of the two counts. In between butterfly captures, Scotch tape was used to clean fingers and equipment to limit accidental spore transfer. Butterflies were marked on the back of their hind wings before they were released to ensure that the same butterfly was not re sampled.
4 RESULTS A total of 50 monarch and queen butterflies were included in this study. Two out of 16 (13%) wild monarchs and two out of 20 (10%) captive monarchs were infected with O. elektroscirrha The infected monarchs i n the wild population were both male and had spore counts of seven and three, while the infected monarchs from the captive population were both female and had spore counts of 679 and 944 (Fig. 2). Unfortunately, sample sizes and number of infected indivi duals were too small to determine whether parasite infections correlated significantly with monarch age, sex, or size. Several observations unrelated to parasite infection were also made between the wild and captive D. plexippus populations. The two popu lations had a significantly different sex ratio ( 2 = 5.61, df = 1, p < .025). The wild population was dominated by males, while the captive population had a 50 50 sex ratio (Table 1). The age structure also differed significantly between the two D. plexi ppus populations ( 2 = 11.16, df = 2, p < .005), with the captive population having no old individuals and a much higher frequency of young individuals (Table 2). Wing size did not differ significantly between the wild and captive populations (t = .156, df = 26, P = .88). No O. elektroscirrha spores were found in wild or captive D. gilippus thersippus populations, although only one individual could be found in the butterfly garden to be sampled. Unlike the sex ratio of the wild monarch population, the sex r atio of the wild queen butterflies was female biased. Of the 13 butterflies sampled, nine were females. But like the wild D. plexippus population, the majority of the D. gilippus sampled in the wild were of intermediate age (Table 3). ____________________ _______________________________ TABLE 1. Sex ratios of monarch butterflies in a wild population from San Luis Valley, Costa Rica and a captive population from the butterfly garden at the Selvatura Butterfly Garden in Monteverde, Costa Rica. The wild pop ulation has a clearly male biased sex ratio. ________________________________________________________ Population Sample size males females ________________________________________________________ Wild 16 14 2 Captive 20 10 10 TABLE 2. Age structures of monarch butterflies in a wild population from San Luis Valley, Costa Rica and a captive population from the butterfly garden at Selvatura Butterfly Garden in Montev erde, Costa Rica. Wing wear (fresh, intermediate, worn) was used an indicator of age. Population sample size young intermediate old Wild 16 1 10 5 Captive 20 9 10 0
5 ___________________ ___________________________________________________________ FIGURE 2. Image of Ophryocystis elektroscirrha spores from a heavily infected D. plexippus individual in the Selvatura Butterfly Garden in Monteverde, Costa Rica. The larger object is an abdomen s cale from the butterfly. ______________________________________________________________________ TABLE 3. Age structures of queen butterflies in a wild population from San Luis Valley, Costa Rica and a captive individual from the butterfly garden at Selvatura Butterfly Garden in Monteverde, Costa Rica. Wing wear (fresh, intermediate, worn) was used an indicator of age. Population sample size young intermediate old Wild 13 3 9 1 Captive 1 0 1 0 DISCUSSION Sex Ratio and Age Structure of Wild and Captive Danainae The sex ratio of the wild population differed significantly from that of the captive local population. The captive population of monarchs had the expected 50 50 sex ratio, but
6 wild monarchs exhibited a heavily male biased sex ratio that may be related to local monarch migratory patterns. The San Luis site is a mid elevation moist forest where A. curassavica still survives during the onset of the local dry season, when this study too k place. It has been proposed (Haber 1993) that D. plexippus and D. gilippus migrate from the Pacific lowlands to these mid elevation forests at the beginning of the dry season. Unlike other monarch populations that migrate further distances, these queen and monarch populations do not enter reproductive diapause while they migrate (Haber 1993). The Danaines breed in these moist forests and do not continue their migration over the continental divide to the wet Atlantic slope until the dry season overcomes t he last milkweeds in February or March. I hypothesize that male monarchs migrate to the moist forests earlier than females, competing with each other to mate with the first females that arrive. This would explain the male bias early in the dry season. Thi s explanation is supported by the fact that the only two female monarchs included in the study were caught during the last day of sampling. Further support of this hypothesis would be gained if a survey of the population later during the dry season reveale d sex ratios to be more equal. However, if this hypothesis is true, it does not apply to queen butterflies, which were female biased in their sex ratios in the wild. D. gilippus also do not continue their elevational migration over to the Atlantic slope, b ut remain abundant in the Pacific slopes during the entire dry season (Haber 1993). The age structure between the wild and captive populations of monarchs also differed significantly. The percentage of young, intermediate, and old individuals in the wild population most likely represents a normal age structure for a healthy population of monarchs, with the majority of monarchs being neither very young nor very old. The lack of old individuals in the captive garden can most likely be attributed to the decre ased lifespan of butterflies in a butterfly garden. The coexistence of so many butterflies in such a small space leads to increased competition for resources and possibly even density dependent mortality. Limited genetic diversity due to high levels of inb reeding also decreases the opportunity for adaptive traits that increase butterfly fitness. O. elektroscirrha Parasitism in D. plexippus It was impossible to predict the proportion of adults highly infected with O. elektroscirrha in Costa Rican populatio ns since this figure ranges from near zero to almost 100% in populations worldwide and even fluctuates seasonally within populations (Altizer, et al. 2000). This study shows that the parasite O. elektroscirrha occurs in Costa Rican populations of D. plexip pus though in low frequencies compared to other studied populations of monarchs. Variation in parasite prevalence between populations can be attributed to many factors that can differ between populations such as genetic differences in the host or the pa rasite. More resistant monarchs or less virulent O. elektroscirrha could explain the low infection rates in both the wild and captive population of monarchs. Environmental factors such as temperature or humidity (Benz, 1987 in Altizer et al. 2000) could al so play a role. For example, O. elektroscirrha is naturally destroyed by a deep freeze (Groth 2000) and some speculate that this could explain why populations of monarchs in the northern United States have lower levels of infection than populations in area s that do not
7 experience freezes. Although deep freezes clearly do not explain the low prevalence of infection in Costa Rican monarchs, other non biotic aspects of the environment here may be less favorable for parasite growth and reproduction. Infection r ates might also be low if the parasite is newly established in this region. Size of the host population could also explain low parasite prevalence. Parasites require a threshold number of hosts in order to establish and increases in host population size us ually are followed by an increase in parasite prevalence (Dobson & Carter 1992). It is possible that the population size in San Luis is not large enough to sustain high frequencies of infection. This is even more likely in the butterfly garden. Furthermore the prevalence of younger butterflies in the garden suggests that mortality is high and early in the garden, perhaps keeping the parasite incidence down. Infection rates could also be correlated with host migratory patterns as demonstrated in North Amer ican monarch populations (Altizer et al. 2000). Populations in North America that migrate the furthest have the lowest parasite prevalence. The authors of the study propose that if infected butterflies suffer disproportionate mortality during migration, pa rasite prevalence should decrease as migratory distances increase since increasing host mortality should theoretically be accompanied by declines in parasite prevalence. The populations of D. plexippus and D. gilippus studied in San Luis were likely elevat ional migrants from the Pacific lowlands and it is possible that their migration plays a role in keeping infection rates low. Even if parasite incidence was similar in both populations in this study, there was a striking difference in parasite load for eac h infected individual. Individuals with O. elektroscirrha in the butterfly garden population were heavily infected, while infected individuals in the wild had very low spore loads. This difference in level of infection is very important because only heavy spore loads negatively affect monarch fitness. Low levels of infection such as those found in the wild population appear to be harmless to the host. A A s s e e p p t t i i c c r r e e a a r r i i n n g g Most of the butterflies in the butterfly garden are purchased from butterfly rearing faci lities when they are pupae. Because of this, it is likely that levels of infection in the garden can be traced back to practices used in the rearing facilities. One possible explanation for the very high spore loads in the captive population is that asept ic techniques in butterfly rearing facilities create conditions where individuals are not exposed to spores more frequently than in the wild, but when they are (dirty rearing containers, etc.), individuals are exposed to huge numbers. But once infected ind ividuals are introduced to the enclosed butterfly garden, the artificial environment there can be perfect for the cultivation of disease. In an enclosed butterfly garden, a small population size but high population density results in elevated levels of in breeding, which decrease already small gene pools. This limited genetic base for parasite resistance makes the population more susceptible to disease. A captive population of monarchs also shares many of the characteristics with nonmigratory monarch pop ulations that Altizer (2001) suggests may contribute to high O. elektroscirrha frequency and virulence in these populations. In these populations, spores can accumulate on milkweed and create high rates of horizontal transmission. This effect is exacerba ted in butterfly gardens, which have reduced area and limited quantities of milkweed. The parasite also has more vertical transmission opportunities in nonmigratory populations, which have more breeding generations per year than populations that migrate lo ng distances. In a butterfly garden, adult butterflies are constantly reproducing,
8 even though new individuals are also regularly being released into the garden. Due to increased densities, reproduction rates are high, facilitating maternal and paternal tr ansmission of the parasite. Like livestock operations and zoos, a butterfly garden confines animals for human purposes. And just as human maintained populations transmitted disease to wild populations in the cases of Rinderpest virus (Dobson 1997) and avi an cholera (Christensen et al. 1998), a butterfly garden can also serve as a source of disease to nearby populations in the unlikely but not impossible event of an escape. This possibility is especially worrisome when butterfly garden individuals are heavi ly infected, but wild individuals are healthy as was the case in this study. Even though the parasite is already a the captive population are not typical in he San Lu is wild population. Because they can be reservoirs of disease, captive populations pose a risk to wild populations of the same or related species. For this reason, it is important that captive populations remain healthy. In a paper presented at the 2000 An Association, Jacob Groth offers several suggestions to breeders for preventing the incidence and spread of O. elektroscirrha in butterfly rearing facilities: 1.) Check that breeding stocks are parasi te free. 2.) Sterilize all plant material and feeding material used until pupation using 10% bleach solution and rinse at least three times. 3.) Sterilize butterfly eggs using a commercially available decontamination method. 4.) aterpillars separate and isolated from adults butterflies. 5.) Sterilize rearing containers, tools, etc. with a 10% bleach solution daily. These sterile practices are especially important in the early stages of larval rearing because the protozoan multipli es in the caterpillar and in the chrysalis; individuals that are infected earlier usually end up more heavily infected (Altizer & Oberhauser 1999). Similar steps can also be taken to ensure that monarchs in butterfly garden populations are healthy. Purchas ed chrysalises and emerging monarch and queen butterflies should be checked for parasite spores using the cotton swab method described here, and only butterflies with less than 10 spores should be released into the garden. All milkweeds should be regularly sterilized with 10% bleach. Periodic checks of monarchs in the garden should also be conducted to ensure that high spore loads are not accumulating on any individuals. Minimizing levels of infection and parasite prevalence promotes the health of not only captive butterflies, but also that of wild butterflies nearby. This study was the first to document the occurrence of O. elektroscirrha in Costa Rican D. plexippus populations. But sample sizes in this study were small and many more studies need to be und ertaken before this host parasite system in this population of monarchs is well understood. Future studies should determine whether infection corresponds with butterfly size, age, and sex and whether local migratory patterns play a role in regulating paras ite prevalence. Parasite prevalence should be monitored throughout the entire year so that any seasonal variation can be assessed. D. gilippus populations should be further surveyed to confirm that they are indeed free from O. elektroscirrha infection. It would also be interesting to see if the parasite infects other Danainae butterflies such as Lycorea spp.
9 ACKNOWLEDGMENTS My first thanks goes to my mom and dad, who have always supported me and finally let me go study abroad this year. I would like to th ank Alan Masters, who advised me throughout this project and whose enthusiasm constantly motivated me. Thank you to Sonia Altizer for her practical advice on sampling monarchs and to Bill Haber for giving me information and ideas about local monarch migrat ions. Thank you to Cristian and Mario Andr s Sol r zano at Selvatura Park for allowing me to sample butterfies in their butterfly garden. Thanks to Javier Mendez, Ollie Hyman, and Matt Gasner for assisting me in this project in all sorts of ways and for pr oviding me with Picaronas while I worked. I am also deeply indebted to everyone who took mercy on my lack of butterfly catching skills and came out with me into the field to catch monarchs: Alan, Matt, Tom, Chai, Jenier, Jaqueline, Waldir, and little Josue I appreciate Matt, Javier, and Kathleen for reading a draft of this paper and Alicia for her making the parasite life cycle diagram. My love and thanks also goes to Yenny Cruz, the best mam tica ever and the rest of the Cruz family, who treated me like one of their own and made San Luis a wonderful and indelible memory in my mind. Finally, thank you Alan, Karen, the rest of the CIEE staff, and my new friends for showing me amazing things and for teaching me so much this semester. ________________________ ________________________________________ LITERATURE CITED Alonso Mejia, A., Rendon Salinas, E., Monetsinos Patino, E., and Brower, L.P. 1997. Use of lipid reserves by monarch butterflies overwintering in Mexico: Implications for conservation. Ecol. Apply. 7: 934 947. Altizer, S.M., K.S. Oberhauser, and L.P. Brower. 2000. Associations between host migration and the prevalence of a protozoan parasite in natural populations of adult monarch butterflies. Ecological Entomology 25: 124 139. Altizer, S.M. Migra tory behaviour and host parasite co evolution in natural butterflies infected with a protozoan parasite. 2001. Evolutionary Ecology Research 3: 611 632. Altizer, S.M. and Oberhauser, K.S. 1999. Effects of the protozoan parasite, Ophryocystis elektroscirrh a, on the fitness of monarch butterflies ( Danaus plexippus ). J. Invert. Path. 74: 76 88. Benz, G. 1987. Environment. Epizootiology of insect diseases, J.R. Fuxa and Y. Tanada eds. John Wiley and Sons, New York, pp. 177 214. Brusca, G. and Brusca, R. 1990 Invertebrates. Sinauer Associates, Inc., Sunderland, Massachusetts. Christensen, J.P. & Bisgaard, M. 1999 Phenotypic and genotypic characters of isolates of Pasteurella multocida obtained from back yard poultry and two outbreaks of avian cholera in t he avifauna in Denmark. Avian Pathology 27: 373 381. Davis, A.K., S.A. Altizer, and E. Friedle. 2004. A non destructive, automated method of counting spores of Ophryocystis elektroscirrha (Neogregarinorida: Ophyrocystidae) in infected monarch butterflies (Lepidoptera: Nymphalidae). Florida entomologist 87 (2): 231 234. Dobson, A. 1997. Infectious disease and the conservation of biodiversity. In: Principles of conservation biology, G.K Meffe and C.R Carroll. Sinauer Associates, Inc., Sunderland, Massachus etts, pp.256 257. Dobson, A., and R. Carper. 1992. Global warming and potential changes in host parasite and disease vector relationships. In Global warming and biodiversity ed. R. L. Peters and T. E. Lovejoy. New Haven, CT: Yale University Press. Groth J. 2000. Sterilization for Disease Control. 2000 IBBA Annual Convention. Available from http://www.butterflybreeders.com/pages/sterilizationfordiseasecontrol jg .html
10 Haber, W.A. and Stevenson, R.D. 2004. Diversity, migration, and conservation of butterflies in northern Costa Rica. In: Biodiversity conservation in Costa Rica, Frankie, G.W., A. Mata, and S.B. Vinson, eds. University of California Press, USA, pp. 99 114. Haber, W.A. 1993. Seasonal migration of monarchs and other butterflies in Costa Rica. In: Biology and conservation of the monarch butterfly, ed. S.B. Malcolm and M. Zalucki. Los Angeles County Museum of Natural History, Los Angeles. Lanciani, C.A 1975. Parasite induced alterations in host reproduction and survival. Ecology 56: 689 695. McLaughlin, R.E. and Myers, J. 1970. Ophryocystis elektroscirrha sp. n. a neogregarine pathogen of the monarch butterfly Danaus plexippus(L.) and the Florida quee n butterfly Danaus gilippus berenice Cramer. Journal of Protozoology 17: 300 305. Monarchs in the Classroom. Monarch Lab: Parasites and Natural Enemies: Life cycle of O. elektroscirrha. [Updated March 2003]. Available from http://www.monarchlab.umn.edu/research/PNE/life.html Oberhauser, K.S. 1997. Fecundity, lifespan and egg mass in butterflies: Effects of male derived nutrients and female size. Funct. Ecol. 11: 166 175.