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Una comparacin de las cargas de hemoparsitos entre los murcilagos (F: Phyllostomidae) de los diferentes gremios de alimentacin en San Luis, Costa Rica
A comparison of hemoparasite loads between bats (F: Phyllostomidae) of different feeding guilds in San Luis, Costa Rica
Bat hemoparasite studies have focused on bats as vectors for viruses that cause human diseases such as rabies, Ebola, and Henipavirus, while ectoparastite studies have looked at a broad range of ecological and abiotic factors that influence ectoparasite prevalence including age, sex, roosting ecology, and habitat. Yet diet had been largely ignored in both hemoparasite and ectoparasite studies. In this study, I looked at diet as a possible factor in hemoparasite load and also looked at the relationships between hemoparasites, health (with weight as a proxy), and ectoparasites. Four guilds were compared, sanguinivores, omnivores, frugivores, and nectarivores, with an overall sample size of 27 bats in San Luis, Costa Rica. I found differences in hemoparasite loads between guilds (Chi square test, p = 0.0026, x2 = 16.295, df = 4) and that bats with ectoparasites were more likely to have a small number of hemoparasites (Chi square test, p= 0.0004, x2 = 18.104, df = 3). Interesting facets to the study included the observation that frugivorous bats had the highest hemoparasite loads, including hemoparasites from all three categories studied (Plasmodium, Babesia, and unknown), and nectarivores had no hemoparasites but the highest percentage of ectoparasites. Interspecific competition between ectoparasites and hemoparasites, roosting ecology of the most common fruit bat, Artibeus toltecus, and coevolved immune signaling molecules may explain these new trends. Overall, the study introduced diet as a possible factor in hemoparasite load due to diet differences altering blood proteins that could promote hemoparasite immunity, but sample size limits the conclusiveness of the study. Future studies should further analyze these differences in the blood and include a broad sample size for each feeding guild, for the results could show the effect of diet on health, both in bats and humans.
En este estudio, mire la dieta como un posible factor para explicar la carga de hemoparsitos y su relacin con la salud y los ectoparsitos.
Text in English.
Bats--Feeds and feeding--Costa Rica--Puntarenas--San Luis
Murcilagos--Alimentos y alimentacin--Costa Rica--Puntarenas--San Luis
Tropical Ecology 2008
Hemoparasite loads on bats
Ecologa Tropical 2008
Cargas de hemoparsitos en los murcilagos
t Monteverde Institute : Tropical Ecology
A Comparison of hemoparasite loads between bats F: Phyllostomidae of different feeding guilds in San Luis, Costa Rica Rachael Zacks Department of Ecology and Evolutionary Biology, University of Colorado Boulder ABSTRACT Bat hemoparasite studies have focused on bats as vectors for viruses that cause human diseases such as rabies, Ebola, and Henipavirus, while ectoparasite studies have looked at a broad range of ecological and abiotic factors that influence ectoparasite prevalence including age, sex, r oosting ecology, and habitat. Yet diet had been largely ignored in both hemoparasite and ectoparasite studies. In this study, I looked at diet as a possible factor in hemoparasite load and also looked at the relationships between hemoparasites, health wi th weight as a proxy, and ectoparasites. Four guilds were compared, sanguinivores, omnivores, frugivores, and nectarivores, with an overall sample size of 27 bats in San Luis, Costa Rica. I found differences in hemoparasite loads between guilds Chi squar e test, p = 0.0026, x 2 = 16.295, df = 4 and that bats with ectoparasites were more likely to have a small number of hemoparasites Chi square test, p= 0.0004, x 2 = 18.104, df = 3 . Interesting facets to the study included the observation that frugivorous bats had the highest hemoparasite loads, including hemoparasites from all three categories studied Plasmodium , Babesia , and unknown, and nectarivores had no hemoparasites but the highest percentage of ectoparasites. Interspecific competition between ec toparasites and hemoparasites, roosting ecology of the most common fruit bat, Artibeus toltecus , and coevolved immune signaling molecules may explain these new trends. Overall, the study introduced diet as a possible factor in hemoparasite load due to diet differences altering blood proteins that could promote hemoparasite immunity, but sample size limits the conclusiveness of the study. Future studies should further analyze these differences in the blood and include a broad sample size for each feeding gu ild, for the results could show the effect of diet on health, both in bats and humans. RESUMEN Los estudios de hemo parÃ¡ sitos en murciÃ©lagos se han enfocado en estos animales como vectores de viruses, mientras que los estudios de ectoparÃ¡sitos han abarcad o un gran rango de factores ecolÃ³gicos y abiÃ³ticos que influencian la prevalencia de estos. Sin embargo, la dieta ha sido ignorada en ambos tipos de estudios. AquÃ se estudiÃ³ la dieta como un posible factor par a explicar la carga de hemo par Ã¡ sitos, y su relaciÃ³n con salud y ectoparÃ¡sitos . Los murciÃ©lagos con mÃ¡s ectoparÃ¡sitos parecen tener menos hemo parÃ¡ sitos. Los frugÃvoros parecen tener una mayor cantidad de hemo parÃ¡ sitos, incluyendo los 3 tipos encontrados en el estudio Plasmodium, Babesia , y descono cido, los nectarÃvoros no tienen hemo parÃ¡ sitos pero si el mayor porcentaje de ectoparÃ¡sitos . Este estudio presenta la dieta como un posible factor par a explicar la ca rga de hemo parÃ¡ sitos, debido a que las diferentes dietas alteran las proteÃnas en la san gre que podrÃan promo ver la inmunidad a los hemo parÃ¡ sitos, pero el tamaÃ±o de muestra s limitan las conclusiones de este estudio. INTRODUCTION Bats O: Chiroptera exhibit a diverse array of feeding strategies, consuming all types of food including fruit, i nsects, vertebrates, and nectar Kunz and Pierson 1994. With such a diversified range of feeding guilds, bats can play a Â€keystone roleÂ in terrestrial community structure, pollinating flowers, dispersing seeds, and managing insect densities Kunz and Pie rson 1994. Bats are also contributors to the human community as bat guano provides many human amenities such as soap, fertilizer, gasohol, and antibiotics
Calisher et. al 2006. The prominent role of bats in community ecosystems makes the study of bat p arasites of equal importance because parasites can alter both the fitness and densities of bat hosts causing a cascade of effects in the nearby ecosystem Plowright et. al 2008. Similarly, the study of parasites in bats is fundamental for parasitism ecolo gy as bats are ubiquitous in multiple habitats, migratory, and frequently come in contact with other bat species during foraging and roosting Dick and Patterson 2007. These combined factors could result in host switching of both ecto and endoparasites i n the bat communities fostering speciation or providing vectors for transmission to other zoonotic hosts Dick and Patterson 2007. Studies have provided data on the presence of both ecto and endoparasites in bat communities Jameson 1959, Ubelaker et al . 1977. Only four insect orders have evolved true ectoparasitism, in which blood feeders spend the majority of their lives on their hosts; Diptera, Hemiptera, Phthiraptera, and Siphonaptera Dick and Patterson 2007. All but one order, Phthiraptera, have groups restricted to bat hosts Dick and Patterson 2007. Phyllostomidae bats are known to harbor over 250 species of ectoparasites that can be influenced by multiple ecological and physiological factors of the host and abiotic environment Gannon and Wi llig 1995. Similarly, endoparasites including trematodes, cestodes, and nematodes have been recorded on bats in North America Jameson 1959. Endoparasites can also include hemoparasites that are harmful to humans, such as Plasmodium falciparum , the par asite responsible for virulent malaria Duval et al. 2007. The extant of described hemoparasites in bats is probably only a sliver of the actual number, and species distribution may be different across geographic ranges Calisher et al. 2006. For example , a new species of nematode, Capillaria sp ., was recently found in D. rotundus bats in Costa Rica Rojas and Guerrero 2007 and there is little doubt that new parasites will be found as technology and extent of studied species increases. Yet, studies h ave been contradictory on the importance of bats as vectors for endoparasite transmission. It is acknowledged in literature that D. rotundus is an important vector for rabies, causing an estimated 100 million dollars of annual loss in domestic stock Turn er 1983 and Neotropical bats are zoonotic vectors for other viruses such as equine encephalitis, Henipaviruses, and Ebola Ubico and McLean 1995, Calisher et al. 2006. Only Ubelaker et al. 1977 has shown evidence that hemoparasites such as Trypanosoma c ruzi and Eimeria can be transferred from host to D. rotundus through feeding. The purpose of this study was to investigate the effect of feeding guild on endoparasite load, specifically hemoparasites blood parasites, in four guilds of bats; sanguinivor e, frugivore, omnivore, and nectarivore. A side note to the study recorded both ectoparasite presence and weight to see if hemoparasite loads had direct associations with these paralleled factors. I hypothesized that difference in diet would result in diff erent hemoparasite loads for five species of bats in San Luis, Costa Rica. I expected to find an overall higher hemoparasite mean in sanguinivores because they are constantly exposed to blood through feeding on both domestic and wild animals, which provid e a diverse community of hemoparasites which could be transmitted by direct blood contact and ingestion. I also expected to find that ectoparasites were more common on bats with hemoparasites because arthropod vectors are known to transmit hemoparasites K udo 1966. The results of the study are relevant to parasitism ecology in general to elucidate
diet as an important indicator for blood parasite composition and concentration in mammals, and for adding to the knowledge of bat hemoparasite ecology, which i s vital both for the survival and health of the bat population as well as the plants they pollinate, animals they eat, and seeds they disperse. MATERIALS AND METHODS Study Species The primary bats for this study, Desmodus rotundus vampire, Anoura geoffr oyi nectar, Carollia perspicillata fruit Artibeus toltecus fruit, and Phyllostomus discolor omnivore are known to have similar social roosting sites and social behaviors pers comm. LaVal, with the exception of A. toltecus in which much of its roo sting behavior is unknown. C. perspicillata eats fruit, specializing on the genus Piper , and A. toltecus eats mainly successional shrubs Solanum , figs, and guava from the family Myrtaceae LaVal and Rodriguez 2002. A. geoffroyi is the largest of the ne ctar bats, and is commonly seen visiting banana flowers and hummingbird feeders in Monteverde LaVal and Rodriguez 2002. P. discolor is an omnivorous bat which eats pollen, fruit and insects, and is an important pollinator in Costa Rica where in one study 82% of the bats examined carried pollen LaVal and Rodriguez 2002. D. rotundus is very different in its diet in that it consists entirely of blood, mainly of domestic animals, although it also feeds on wild mammals as well Greenhall et al. 1983. Specif ically D. rotundus bites around the neck, anus, vulva, legs and snout of the animal Greenhall et al. 1983, using modified incisors to make a painless incision, and uses an anticoagulant to allow constant blood flow Greenhall et al. 1983. As many as fi ve D. rotundus individuals have been seen feeding from one wound site Greenhall et al. 1971. D. rotundus generally feeds for 9 40 minutes Greenhall et al. 1971 and can consume up to 20 ml of blood per day Greenhall et al. 1983. Study Sites Th ree study sites in the Holdridge tropical moist forest of San Luis, Costa Rica at approximately 1000 m in altitude provided the necessary environments, banana patches and cow pastures, to catch the frugivores, nectarivores, omnivores and sanguinivores. The Brenes Salazar family farm in La Finca Bella and the University of GeorgiaÂ‚s Ecolodge were primary sites for the capture of A . toltecus, A. geoffroyi, C. perspicillata and P. discolor . The nets at the Brenes Salazar farm were positioned near a chicken co op where fruit was stored and bats were known to roost, as well as near banana flowers. The nets at the Ecolodge were positioned on trails behind the lodge in the large banana patch. The sanguinivores were caught at MachoÂ‚s residence, a nearby dairy farm o utside of La Finca Bella with approximately 20 cattle which were being continually bitten by D. rotundus . Collection and Field Experiments Two mist nets, 12 m and eight m respectively, were set up from 5:00 p.m. to 9:30 p.m. for 10 nights between Octob er 25 th and November 12 th 2008 totaling approximately 45 mist netting hours. Upon capture, the species were removed from the net and identified with A Field Key to the Bats of Costa Rica by Robert M. Timm and Richard K. LaVal
unpublished, using body char acteristics e.g. facial stripes, nose shape and forearm measurements as primary IDs. Next, the presence or absence of ectoparasites was noted through direct visualization of flies and mites in the batsÂ‚ fur or on the wing membrane. If no initial ectopar asites were found, I blew on the bats fur to try and disrupt ectoparasites that may have been hiding in order to confirm that no ectoparasites were present. The weight was then measured by placing the bat in a cloth bag and on the weight. To collect blood samples, I used a lancet to prick the prominent vein running along the uppermost part of the plagiopatagium and used a capillary tube to remove less than 10 microliters. The blood was immediately blown onto a clean slide and a blood smear was made on sig ht. Following the removal of a blood sample, a small patch of hair was removed from the shoulder of the bat to avoid recapture. Blood Analysis Twenty four to 72 hours after the blood smear was made the slides were stained with Geimsa Appendix 1. Anal ysis of the blood and identification of hemoparasites was conducted with the help of Dr. Carlos Alfonso, a microbiologist at Emergencias Monteverde. A drop of oil immersion was added to each slide and the slides were analyzed under 100x magnification. The number of parasites per specimen was recorded in mm 3 e.g. 8 parasites per mm 3 of blood and due to the difficulty of hemoparasite identification to the species level, hemoparasites were categorized into a generalized types consisting of Plasmodium , Babesi a , or unknown Figure 1. Photos of all parasites found were taken using Dr. AlfonsoÂ‚s camera, and two blood samples were thrown out because it was not an efficient sample or it was difficult to tell if the parasites were stain debris or actual organisms. Figure 1: Simplified drawings of red blood cells containing parasites, similar to the real cells observed under the microscope. From left to right, the first drawing is a Babesia parasite, followed by a Plasmodium parasite, a Plasmodium malaria parasite, and finally an unknown parasite with parasitic nuclei around the edge of the cell. The highest concentrations of parasites found were unknown. Statistical Analysis After completion of the blood sample analysis, two Chi square tests were done to see if th ere was a significant difference between the number of hemoparasites for each feeding guild and the number of hemoparasites in bats with or without ectoparasites. Similarly, a correlation was done between weight and average hemoparasite load in the frugiv orous bats to determine if the presence of hemoparasites had a direct affect on bat health.
RESULTS Over the ten nights of trapping, 27 bats were caught including two D. rotundus , two P. discolor , one C. perspicillata , 15 A. toltecus , and seven A. geoffro yi . Two samples were disregarded during blood analysis because the blood samples were not good enough to make clear observations of the presence or absence of hemoparasites. This brought the total available samples for statistical analysis to 25. Ten out of the 25 bats were infected with at least one hemoparasite 40% and 11 out of 27 bats had ectoparasites present 41%. The abundance of hemoparasites between different feeding guilds was different p = 0.0026, x 2 = 16.295, df = 4. Specifically, fru givores had an overall higher number of parasites, and were the only feeding guild to have Babesia hemoparasites Figure 2. Both sanguinivores and omnivores had parasites in all of the species present, yet nectarivores had no hemoparasites in any of the s amples Figure 2. Figure 2: A graph showing the relative abundance of hemoparasites by feeding guild. Overall, frugivores had the highest concentrations of parasites and were the only feeding guild to have Babesia parasites. Another strange result was tha t none of the nectarivores had any blood parasites. The sample sizes were as follows: frugivores n = 15, nectarivores n = 6, sanguinivores n = 2, and omnivores n = 2, for a total of 25 samples. Also, there was a difference between the number of he moparasites in bats with and without ectoparasites p = 0.0004, x 2 = 18.104, df = 3. Bats with ectoparasites had a smaller number of hemoparasites, and bats with a larger number of hemoparasites had ectoparasites present Figure 3. This applied specifica lly to the nectarivores in which all but one nectarivore had ectoparasites Figure 4, and there were no hemoparasites recorded in nectarivores. Hemoparasite Abundances in Different Feeding Guilds for Phyllostomidae Bats 0 10 20 30 40 50 60 70 Frugivores Nectarivores Sanguinivores Omnivores Feeding Guilds Parasite Abundance mm 3 Plasmodium Babesia Unknown
Relationship between Ectoparsite and Hemoparasite Load in Phyllostidae Bats -20 -10 0 10 20 30 40 50 60 70 80 P B U Hemoparasite Type Number of hemoparasites in mm 3 Ectoparasites None Figure 3: The Relationship between Ectoparasite and Hemoparasite Load. There is a differe nce between the number of hemoparasites in bats with ectoparasites and those without. In general, bats without ectoparasites had more hemoparasites than bats with ectoparasites in all three hemoparasite categories. Figure 4: Graph showing the number of individuals per feeding guild that have ectoparasites. Nectarivores had the most ectoparasites; only one individual in the sample size of seven did not have ectoparasites. Similarly, no sanguinivores had ectoparasites. There was no correlation between wei ght and hemoparasite number in frugivores p = .473, n = 14, Rho = .209 indicating that although there might be a negative trend for approximately 20% of the data, weight and hemoparasite load do not seem to be related. Ectoparasite Distributrion across Feeding Guilds 0 1 2 3 4 5 6 7 Nectarivores Frugivores Omnivores Sanguinivores Feeding Guild Number of individuals Nectarivores Frugivores Omnivores Sanguinivores
DISCUSSION Differences in hemopa rasite loads between bat feeding guilds could elucidate diet as another ecological factor contributing to parasitism, yet the unequal sample sizes of this study make the exact role of diet inconclusive. My original prediction that sanguinivores would have the highest abundance of hemoparasites was not correct. Paradoxically, the frugivores, made up of mostly A. toltecus individuals, had the most hemoparasites per mm 3 of blood Figure 1. The result is unexpected because frugivorous bats pass food so rapidl y through their digestive tract 15 20 minutes that it has been hypothesized there is hardly any bacterial action Nowak 1994. This would make it very unlikely that ingestion is the route in which frugivores contract hemoparasites. In order to find out why frugivores had the highest abundance of hemoparasites in this study, it is necessary to look at other parasite factors such as roosting ecology and ectoparasite presence, which may have played a more prominent role than diet. Originally, I had predic ted that the presence of ectoparasites would be associated with an increase in hemoparasite load, yet the results revealed that individuals were more likely to have hemoparasites if there were no ectoparasites present Figure 2. This translated to nectar ivores having the most ectoparasites and no hemoparasites, while frugivores had the most hemoparasites with fewer ectoparasites Figure 4. The latter trend could possibly be explained by differences in immunology in the blood of individuals with and witho ut ectoparasites. It has been shown in past studies, that ectoparasites use immune signaling molecules that are similar to their hosts, thereby avoiding aggressive immuniosurvelance responses and detection by the host Salazet et. al 2000. Perhaps, the s pecific molecules shared between ectoparasites and their hosts are so tightly coevolved that any blood parasites are quickly recognized by the host immune system and destroyed. This difference in immune proteins between the blood of frugivores and nectar ivores could also be due to their diet. Observations by Dr. Alfonso and I while analyzing samples concluded that the Geimsa stain reacted differently with nectarivore blood compared to frugivore blood. Nectarivore blood was significantly cloudier, and the frugivore blood was clearer showing individual blood cells. The nectarivoreÂ‚s diet may provide a different complement of proteins than the frugivoreÂ‚s blood which reacts differently to the Geimsa stain, as well as possibly protecting nectarivoreÂ‚s from he moparasite infection. This would be similar to the results in little red flying foxes from Australia in which nutrition had a direct affect on seroprevalence of the Hendra virus Plowright et al. 2008. Roost ecology is another possibility that could expla in the high level of hemoparasites found in frugivores. All the study species, except for A. toltecus have similar roosting behavior. They are all generalist roosters in caves and hollow trees with ranging colony sizes from 25 to 400 individuals. A. tolte cus has been seen making leaf tents in Costa Rica LaVal and Rodriguez 2002 which could put them into greater contact with arthropod vectors which transmit hemoparasites Patterson et. at 2007. Although Paterson et al. 2007 found that bats that roost in more permanent enclosed structures are more likely to be infested, have higher parasite loads and harbor more species, this study was only done with ectoparasitic flies. Babesia and Plasmodium are transmitted by ticks and mosquitoes Kudo 1966 respective ly which may have more
access to bats which roost out in the open in leaf tents. The reason for the high number of unknown parasites in frugivores is difficult to assess due to the fact that the vectors are unknown. Also, some anomalies were found in thi s project that could have a dramatic influence on the study. Frugivorous bats have the highest proportion in mm 3 of unknown parasites. Yet, 60 of the unknown parasites were found in one frugivorous bat indicating that parasite abundance may just be affecte d on an individual basis. If there had been more equal sampling sizes of all the feeding guilds I may have found that indeed D. rotundus has the highest average of parasites as was shown in other studies Eberly 1997. Still, the study has elucidated spec ifics about the difference in blood composition of bats of different feeding guilds. Future studies should explore the possibility of interspecific competition between hemoparasites and ectoparasites as well as investigating if the differences in nectar an d fruit blood are indeed due to diet. If certain proteins have immunologic properties from consuming different diets this could be used in human disease studies as well as elucidates facets of bat parasite ecology. In conclusion, diet could be an importan t factor in bat hemoparasite ecology, but seems to be overshadowed by roosting ecology and immunology in this study. Future studies need to investigate the true association of diet and hemoparasites with larger sample sizes of all the feeding guilds to be able to make more conclusive results. ACKNOWLEDGEMENTS I would like to thank Tania Chavez for her guidance and sympathy as I battled parasites of my own, and Richard LaVal for his huge pool of bat knowledge in the form of species, locations, and directi ons for my project. To Dr. Carlos Alfonso, thank you for helping me to identify blood parasites and magnifying my virology directions for the future. Thank you to Pablo for helping me translate my abstract and getting supplies. I also could not have survi ved without my bat catching partners, Moncho and Adam. Thank you Moncho, for handling the feisty vampires and keeping the nights exciting with stories of other blood sucking parasites from the past. Adam, thank you for keeping my Spanish skills sharp and p ointing me to the right farms for vampires, frugivores, and nectarivores. Acknowledgements also need to go to the University of GeorgiaÂ‚s Ecolodge and MachoÂ‚s farm for their permission to trap bats. Finally, thank you to my host family at the Brenes Salaz ar farm for letting me spend the nights on their farm catching bats and allowing me to become part of the family. LITERATURE CITED Calisher, C.H., J.E. Childs, H.E. Field, K.V. Holmes and T. Schountz. 2006. Bats: important reservoir hosts of eme rging viruses. Clinical Microbiology Reviews. 19: 531 545. Coggins, J.R. 1988. Methods for ecological study of bat endoparasites. In: Ecological and Behavioral Methods for the Study of Bats . T.H. Kunz ed Smithsonian Institution Press. Washington, D.C. pp. 475 485. Dick, C.W. and B. D. Patterson. 2007. Against all odds: Explaining high host specificity in dispersal Âƒ prone parasites. International Journal for Parasitology. 37: 871 876. Duval L., V. Robert, G. Csorba, A. Hassanin, M. Randrianariveloj osia, J. Walston, T. Nhim, S. M. Goodman, and F. Ariey. 2007. Multiple host switching of Haemosporidia parasites in bats. Malaria Journal. 6: 1 8. Eberly, J. 1997. Altitude and parasites in bats. In: Tropical Ecology and Conservation; Council on In ternational Educational Exchange. New York. Ellorin, N.R. 1996. Hemoparasites of insectivorous, frugivorous and nectarivorous bats. In: University of California Education Abroad Program. Gannon, M.R. and M.R. Willig. 1995. Ecology of ectoparasites from Tropical bats. Environm. Entomol. 24: 1495 1503. Greenhall, A.M., G. Joermann, U. Schmidt and M.R. Seidel. 1983. Desmodus rotundus . In: Mammalian
Species . American Society of Mammalogists. pp. 1 Greenhall, A.M., U. Schmidt and W. Lopez Forment. 1971. Attacking behavior of the vampire bat, Desmodus rotundus , under field conditions in Mexico. Biotropica. 3: 136 141. Hoare, C.A. 1965. Vampire bats as vectors and hosts of equine and bovine trypanosomes. Acta Trop. 22: 204 216. Jameson, D. K. 1959. A survey of the parasites of five species of bats. Southwestern Naturalist. 4: 61 65. Kudo, R.R. 1966. Protozoology 5 th edition. Thomas Books. Springfield, IL. pp 718 732, 745 749. Kunz, T.H. and E.D. Pierson. 1994. Bats of the World: An Introduction. In: Wa lkerÂ‚s Bats of the World , R. M. Nowak, ed Johns Hopkins University Press, Baltimore, MD, pp 1 46. LaVal, R.K and H.B. Rodriguez. 2002. MurciÃ©lagos de Costa Rica. Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica. pp. 134 140, 158 161, 208, 222 225. Nowak, R.M. 1994. WalkerÂ‚s Bats of the World . The Johns Hopkins University Press. Baltimore, MD. pp. 135 137, 144 145, 150 152, 161 163. Patterson, B.D, C. W. Dick and K. Dittmar. 2007. Roosting habits of bats affect their paras itism by bat flies Diptera: Streblidae. Journal of Tropical Ecology 23: 177 189. Plowright, R.K., H.E. Field, C. Smith, A. Divljan, C. Palmer, G. Tabor, P. Daszak and J.E. Foley. 2008. The Royal Society. 275: 861 869. Rojas, A. and R. Guerrero. 200 7. Capillaria sp ., isolated from Desmodus rotundus Chiroptera: Phyllostomidae in Costa Rica. MastozoologÃa Neotropical. 14: 101 102. Salazet, M., A. Capron and G.B. Stefano. 2000. Molecular crosstalk in host Âƒ parasite relationships: Schistosome Âƒ and leech Âƒ host interactions. Parasitology Today. 16: 536 540. Turner, D.C. 1983. Desmodus rotundus Vampiro, Vampire Bat. In: Costa Rican Natural History , D.H. Janzen, ed The University of Chicago Press, Chicago, IL, pp. 467 468. Ubelaker, J.E., R.D. Specian and D.W. Duszynski. 1977. Endoparasites. In: Biology of Bats of the New World Family Phyllostomidae. Part II, R.J. Baker, J.K. Jones, Fr. And E.C. Carter eds. Spec. Publ. Museum Texas Tech. University, TX. As cited in: Jameson, D. K. 1959 . A survey of the parasites of five species of bats. Southwestern Naturalist. 4: 61 65. Ubico, S.R. and R. G. McLean. 1995. Serologic survey of Neotropical bats in Guatemala for virus antibodies. Journal of Wildlife Diseases. 31: 1 9. 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 .
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 3 9 ml of solution 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 w hite, 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 metha nol to sit on slides 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 not need to be a thick layer. Allow stain to sit for 30 minutes. 7. Wash slides with distilled water and allow drying before viewing.