1 Hemoparasite load in bats with varying roost size in San Luis, Costa Rica Robin Miller Department of Biology, The George Washington University ABSTRACT Bats, as well as most other mammals, are host to many endopara sites that can damage or even cause death in their host (Baron 1996). I surveyed blood parasites from 28 bats in San Luis, Costa Rica. This study focused on six different species of bats with varying roosting behaviors to determine the amount and type of b lood parasites differs between bat species. I identified four different types of blood parasites as well as a gram negative bacterium. Using a proportion between the total number of parasites and the number of individuals caught per species, I found Myotis keaysi, which roosts in groups of over 500 individuals, to have more total average parasites (x 2 = 28.47, p = 0.00424, df = 4, p < 0.0001). I also found significance difference between each individual species of parasite and the different species of bats (x 2 = 42.70091393, p = 7.88447E 70, df = 8, p < 0.0001). Although data indicates that bats roosting in extremely large groups have more average blood parasites, more data is needed, in terms of sample size, in order to make further conclusions. RESUMEN Los murciÂŽlagos, asÂ’ como la mayorÂ’a de los otros mamÂ’feros, son anfitriones de muchos endoparasitos que puedan daÂ–ar o aÂœn causar muerte en su anfitriÂ—n (Baron 1996). ExaminÂŽ parÂ‡sitos de la sangre a partir de 28 murciÂŽlagos en San Luis, Costa Rica. Este estudio se centrÂ— en seis especies de murciÂŽlagos con comportamientos diferentes para pasar la noche se determinÂ— la cantidad y el tipo de parÂ‡sitos de la sangre entre las especies del murciÂŽlagos. IdentifiquÂŽ cuatro diversos tipos de parÂ‡sitos de la sangr e asÂ’ como una bacteria gram negative. UsÂŽ una proporciÂ—n entre el nÂœmero total de parÂ‡sitos y el nÂœmero de individuos por especie, encontrÂŽ ese Myotis keaysi que los para pasar la noche son grupos sobre de 500 individuos, tuvieron mÂ‡s parÂ‡sitos (x 2 = 28 .47, p = 0.00424, df = 4, p < 0.0001). TambiÂŽn encontrÂŽ diferencia de la significaciÂ—n entre cada tipo del parÂ‡sito y las diversas especies de murciÂŽlagos (x 2 = 42.70091393, p = 7.88447E 70, df = 8, p < 0.0001). Aunque los datos indican que los murciÂŽlagos que tienen grupos para pasar la noche extremadamente grandes tienen mÂ‡s parÂ‡sitos mÂ‡s medios de la sangre, mÂ‡s datos son necesarios, en tÂŽrminos del tamaÂ–o de muestra, para hacer otras conclusiones. INTRODUCTION Parasites harm their host organism in or der to gain food, nutrients, and habitat or to gain an overall advantage in their environment. Although not always necessary for the parasite to survive, this relationship always harms the host or brings it to some disadvantage during a parasitic relations hip. Parasitic diseases in humans are becoming increasingly important due to various epidemics, such as AIDS, that make immunological compromised people more susceptible to a plethora of different pathogenic parasites. In addition, increasing travel to and from areas with parasites is increasing the rate of infection all over the world (Baron 1996).
2 Bats have not been studied extensively for blood parasites, or hemoparasites, because they are not necessarily of medical importance to humans. However, since bats have a complex and understudied life style and social behaviors, it would be interesting to see how these behaviors influence parasite ecology (Coggins 1998). Bats are host to various medically relevant hemoparasites, such as Leishmania mexicana and T rypanosoma cruzi both of which cause disease in humans (Eberly 1997). Some cave dwelling bats are thought to be a possible blood source for Lutzomyia longipalpis, which is a vector for American visceral leishmaniasis in the New World (Lampo et al. 2000). A better understanding of bat pathogens is necessary in light of recent pollinator declines, including bats, in North America (Gordon et al. 1998). Previous studies have shown bats that roost in lower altitudes has a higher rate of infection with hemop arasites than bats that live in higher altitudes in Costa Rica (Eberly 1997). Studies have also shown insectivorous bats have a greater variety and number of hemoparasites than frugivorous or nectivorous bats. This is because insectivorous bats are more su sceptible to insect bites (Coggins 1988). Although numerous studies examined variation of ectoparasite and endoparasite loads based on altitude and diet, very little information is available on endoparastic load of bat species that vary in their roost size (Coggins 1998 and Eberly 1997). Since bat species that roost in very large communities tend to have more ectoparasites, it would be interesting to determine whether or not they are more susceptible to insects carrying blood parasites as well (Krutzsch 195 5). Bats that roost in extremely large groups should be infected with more hemoparasites than bats who roost single or in smaller groups. The purpose of this experiment was to see if species of bats with varying roosting behaviors are host to different blo od parasite loads and species. METHODS I mist netted bats in the tropical moist forest at the University of Georgia's Ecolodge in San Luis, Costa Rica. I used three; 12 m long mist nests and set them up at different sites along the trails at the Ecolod ge for six nights between April 18 th and April 25 th between 5:00pm and 10:30pm. I determined the species and then used a needle from a 250 ml syringe to prick a vein on the bat's tibia. I used a capillary tube or simply allowed the blood to drip into a 500 l Microtainer Brand tube containing heparin. I obtained between 50 l and 250 l of blood from each bat. After each night of blood collection, I stored the blood in a refrigerator at approximately 20Â¡C. I analyzed the blood samples with the guidance of Dr. Alfonso Artavia at the Laboratorio de Monteverde in Santa Elena. I began by dropping approximately 100 l of the blood sample onto a clean glass slide and using another slide to spread the blood evenly across to the slide. To fix and stain the samples I used the Geimsa Stain Protocol from the Department of Microbiology, University of Costa Rica, to make thick slides for each bat blood sample (Appendix 1). Then, I used a 1000x oil immersion microscope to analyze each slide and recorded the type and num ber of parasites found in each sample (Weikel Magden 2000 and Botero 1984).
3 RESULTS I caught 28 bats in San Luis and observed blood parasites in 22 of the individuals (Appendix 2). During this time, I captured six Anoura geoffroyi six Artibeus toltecu s, 11 Carollia brevicauda, three Myotis keaysi, one Glossophaga commissarisi and one Sturnira ludovici. The roosting behavior for each species is listed in Table 1. The blood parasites I found included Plasmodium species, Paragonium species, Nematoda, Bart onella/Bordetella bacteria species as well as an unknown blood parasite. My results showed significant difference between the proportion of the average number of each species of blood parasites and the different species of bats as shown in Figure 1 (x 2 = 4 2.70091393, p = 7.88447E 70, df = 8, p < 0.0001). This indicates that the five different species of bats all had different species and numbers of hemoparasites. As shown in Figure 2, I used a proportion between the total number of average parasites and the total number of individuals caught per bat species and found that Myotis keaysi has more average parasites than the other bat species (x 2 = 28.47, p = 0.00424, df = 4, p < 0.0001). I was unable to identify the bacteria species found, but I believe it to b e either a Bartonella species or a Bordetella species. Bartonella is a facultative, intracellular, gram negative bacterium (Foucault et al. 2006). Only four individuals were infected with the bacteria Bartonella/Bordetella. Furthermore, only one species of bat, Myotis keaysi, contained a nematode parasite. As shown in Figure 1, Plasmodium was prevalent in five out of the six species of bats. Table 1: Family, subfamily and species name for all six different species of bats captured at the Ecolodge in San Lu is, Costa Rica. I recorded both the social and behavioral characteristics. I captured five species of Phyllostomidae, one species of the family Vespertilionidae. Family and Subfamily Species Social Behavior & Characteristics Phyllostomidae Stenodermat inae Artibeus toltecus Frugivore. Constructs leaf tents in Costa Rica. They are very social and roost in fairly large groups (LaVal 2002) Phyllostomidae Stenodermatinae Sturnira ludovici Frugivore. Believed to roost in very small groups or solitary (La Val 2002) Phyllostomidae Carollinae Carollia brevicauda Frugivore. Roosts in large groups (LaVal 2002) Phyllostomidae Glossophaginae Anoura geoffroyi Nectarivore. Roost singly or in groups of up to 20 (Nowak 1994) Phyllostomidae Glossopha ginae Glossophaga commissarisi Nectarivore. Roosts in caves, tunnels and tree hollows (LaVal 2002) and in small to large colonies (Nowak 1991) Vespertilionidae Myotis keaysi Insectivore. Roosts are extremely large, over 500 bats (LaVal 2002)
4 0 20 40 60 80 100 120 Artibeus toltecus 6 Carollia brevicauda 11 Anura geoffroyi 6 Sturnia ludovici 1 Myotis keaysi 3 species frequency parasites Plasmodium Paragonium unknown Figure 1: The average frequency of each of the four blood parasites found for each bat species tested. Glossophaga commissarsi showed no parasites while Sturnira ludovici was only infected with Plasmodium. Only Myotis keaysi, showed infecti on with a nematode. Figure 2: The average frequency of total blood parasites found for each bat species tested as a proportion of the total number of bats caught per species. Myotis keaysi, which roosts in groups of over 500 individuals, shows signific antly more average total parasites than those who roost in smaller groups.
5 DISCUSSION I found that Myotis keaysi, which roosts in groups of over 500 individuals, has more average blood parasites as a proportion to the number of total species sampled. Th is indicates that there is some relationship between roost size and blood parasite prevalence. I was, however, lacking a sample size with enough individuals from varying roosting behaviors. A. toltecus, C. brevicauda, and G. commissarisi, which lives in m edium to large groups, did have fewer average blood parasites than Myotis keaysi which roosts in extremely large groups. Furthermore, A. geoffroyi and S. ludovici, which roost in small groups, did not show fewer blood parasites than other species. This ma y be because all of these bats are somewhat social and therefore would not have significantly different types or amount of parasites (LaVal 2002 and Nowak 1994). The sample size of the extremely large roosting bats, Myotis keaysi, was quite small as was th e sample size for the very small or even solitary roosting bats, S. ludovici. A larger sample size with more individuals from varying social groups would possibly prove to expand on the results found here. Based on these results, roosting size may influe nce prevalence of blood parasites but other factors are involved. For instance, bats that roost in caves with water may be more likely to come in contact with arthropod vectors of hemoparasites. Also, bats that travel longer distances, especially to places where some of these parasitic diseases are prevalent are may also be more likely to have parasites. The blood parasites found suggest that mosquitoes, specifically those in the genus Anopheles, have Plasmodium, the protozoan parasite that causes malaria Although there are extremely few cases of malaria in San Luis, Costa Rica, it is possible that these bats are traveling long distances and have been bitten by infected mosquitoes from other locations in Costa Rica where malaria is more common (CDC Yellow Book). Bats, in addition, may have contracted Paragonium from ingesting arthropods (CDC Paragonimiasis). Since approximately 78% of the bats captured had parasites, further studies should be implemented determine where the bats are contacting the paras ites in order to protect against declines in bat population and decreases in bat pollinated and dispersed plant species as well as to understand how the prevalence of these parasites can affect human health. ACKNOWLEDGMENTS Thank you to Arturo Cruz for al l his help and knowledge of catching and identifying bats and to the University of Georgia's Ecolodge for allowing us to use their trails to set up nets. Another thanks to Dr. Alfonso Artavla, who helped me identify the blood parasites and share his lab sp ace with him. Thank you to Richard LaVal for getting me interested in bats and formulating a project idea. I would also like to thank Alan, Karen, Tania, Cam and Tom for their patience. Finally, to my wonderful host family, Rita, Catalina and Gabriel Varga s, who were so kind and understanding about my late night hours spent catching bats.
6 LITERATURE CITED Baron, S. 1996. Medical Microbiology. University of Texas Medical Branch at Galveston. Galveston, Texas. Botero, D., Restrepo, M. 1984. ParÂ‡sitos Hu manas. Corporacion para Investigaciones Biologicas (CIB). Medellin, Colombia. Center for Diease Control. Paragonimiasis. http://dpd.cdc.gov/dpdx/html/Paragonimiasis.htm. Cited 8 May 2007. Center for Disease Control. 2005 2006. Travelers' Health: Yellow Book. Yellow Fever Vaccination Requirements and Information on Malaria Risk and Prophylaxis. http://www2.ncid.cdc.gov/travel/yb/utils/ybGet.asp?section=YBOne&obj=CR&c ssNav=browseoyb. Cited 8 May 2007. Coggins, J.R. 1988. Methods for the Ecological Stud y of Bat Endoparasites. In: Ecological and Behavioral Methods for the Study of Bats, T.H Kunz (ed). Smithsonian Institution Press, Washington DC. pp. 475 89. Eberly, J. 1997. Altitude and Parasites in Bats. In: Tropical Ecology and Conservation; Counci l on International Educational Exchange. New York. Foucault C, Brouqui P, Raoult D. 2006. Bartonella quintana Characteristics and Clinical Management. Emergering and Infectious Diseases http://www.cdc.gov/ncidod/EID/vol12no02/05 0874.htm Cited 8 May 2007 Gordon A.H., P. Bernhard, R. Bitner, A. Burquez, S. Buchmann, J. Cane, P.A Cox, V. Dalton, P. Feinsinger, M. Ingram, D. Inouye, C.E. Jones, K. Kennedy, P. Kevan, H. Koopowitz, R. Medellin, S. Medellin Morales, G.P. Nabhan, B. Pavlik, V. Tepedino, P. T orchio, S. Walker. 1998. The Potential Consequences of Pollinator Declines on the Conservation of Biodiversity and Stability of Food C Crop Yields. Conservation Biology 12: 8 17. Krutzsch, P.H. 1955. Ectoparasites from Some Species of Bats from Western N orth America. Journal of Mammalogy. 36(3): 457 459. Lampo, M., M.D. Feliciangeli, L.M Marquez, C. Bastidas, P. Lau. 2000. A Possible Role of Bats as a Blood Source for the Leishmania vector Lutzomyia longipalpis (Diptera: Psycodidae). The American Socie ty of Tropical Medicine and Hygiene. 62(6): 718 719. LaVal, R.K and H.B. Rodriguez. 2002. MurciÂŽlagos de Costa Rica. Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica. pp. 86 272. Nowak, R.M. 1991. Walker's Bats of the World. The Johns Hopkins University Press. Baltimore, Maryland. pp. 144 145. Weikel Magden, O. 2000. A Comparison of Abundance and Diversity of Blood Parasites in Common Vampire Bats ( Desmondus rotundus) and Cattle. In: University of California Education Abroad Program
7 APPENDIX 1: Geimsa Stain Protocol From the Department of Microbiology, University of Costa Rica (From Roelands and Taft 1999) 1. Prepare buffer solution first. The buffer solution must be at a pH 7.2. In order to get this mix 39 ml of solutio n A and 61 ml of Solution B in 900 ml of distilled water a. Solution A. (KH2PO4) 9.5 g/L b. Solution B. (Na2HPO4) 9.07 g/L 2. Place thick smear samples upright and not overlapping in a large container of distilled water until smear has become clear white, not red. About 10 15 minutes. You may gently move the sample to remove the last bit of color. 3. Place slides on a rack to dry. Please thin smears adjacent to thick. 4. After thick is dry, wash both the thick and thin smears with methanol and allow methanol to sit on s lides for five minutes 5. Allow methanol to dry off or shake off excess and wipe 6. Take 9 ml of the pre made buffer solution and 1 ml of the filtered Geimsa stain in a syringe and distribute over slides. Blow on slides to spread stain over slide evenly. Does no t need to be a thick layer. Allow stain to sit for 30 minutes. 7. Wash slides with distilled water and allow to dry before viewing.
8 APPENDIX 2: Complete list of all speices of bats captured in San Luis, Costa Rica. Includes roosting behavior of each species as well as the number and type of blood parasite or bacteria identified from each individual. Species Roosting Behavior Bartonella/ Bordella Nematoda Plasmodium Paragonium Unknown Anoura geoffroyi Singly or up to 20 15 Anoura geoff royi Singly or up to 20 10 Anoura geoffroyi Singly or up to 20 Anoura geoffroyi Singly or up to 20 yes 12 Anoura geoffroyi Singly or up to 20 13 Anoura geoffroyi Singly or up to 20 11 Sturnira ludovici Small 10 Artibeus t oltecus Large 10 4 Artibeus toltecus Large 10 Artibeus toltecus Large 5 Artibeus toltecus Large 5 Artibeus toltecus Large 7 Artibeus toltecus Large 10 7 Carollia brevicada Large yes 21 Carollia brevicada Large Carollia brevicada Large 11 Carollia brevicada Large Carollia brevicada Large yes 15 1 Carollia brevicada Large Carollia brevicada Large 10 Carollia brevicada Large Carollia brevicada Large 15 Carollia brevicada Large 20 Ca rollia brevicada Large yes 17 Glossophaga commissarisi Small to large Myotis keaysi Very large 7 Myotis keaysi Very large 10 Myotis keaysi Very large 20 10
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Carga de hemoparsitos en murcilagos con diferentes tamaos de percha en San Luis, Costa Rica
Hemoparasite load in bats with varying roost size in San Luis, Costa Rica
Bats, as well as most other mammals, are host to many endoparasites that can damage or even cause death in their host (Baron 1996). I surveyed blood parasites from 28 bats in San Luis, Costa Rica. This study
focused on six different species of bats with varying roosting behaviors to determine the amount and type of blood parasites differs between bat species. I identified four different types of blood parasites as well as a gram-negative bacterium. Using a proportion between the total number of parasites and the number of individuals caught per species, I found Myotis keaysi, which roosts in groups of over 500 individuals, to have more total average parasites (x2 = 28.47, p = 0.00424, df = 4, p < 0.0001). I also found significance
difference between each individual species of parasite and the different species of bats (x2 = 42.70091393, p = 7.88447E-70, df = 8, p < 0.0001). Although data indicates that bats roosting in extremely large groups
have more average blood parasites, more data is needed, in terms of sample size, in order to make further conclusions.
Los murcilagos, as como la mayoria de los otros mamferos, son hospederos de muchos endoparsitos que pueden daar o an causar la muerte en su hospedero (Baron 1996). Examin los parsitos de sangre de 28 murcilagos en San Luis, Costa Rica. Este estudio se centro en seis especies de murcilagos con diferentes comportamientos de percha para determinar la cantidad y el tipo de parsitos de sangre entre las especies de murcilagos.
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Tropical Ecology 2007
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