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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 ve ctors 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 prevale nce including age, sex, roosting ecology, and habit at. 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 g uilds were compared, sanguinivores, omnivores, frugivores, and nectarivores, with an overall sampl e 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 ha ve a small number of hemoparasites (Chi square test, p= 0.0004, x2 = 18.104, df = 3) . Interesting facets to the study included the obs ervation that frugivorous bats had the highest hemoparasite loads, including hemop arasites from all three categories studied ( Plasmodium , Babesia , and unknown), and nectarivores had no hemoparasit es but the highest percentage of ectoparasites. Interspecific competition between e ctoparasites and hemoparasites, roosting ecology of the most common fruit bat, Artibeus toltecus , and coevolved immune signaling molecules may expl ain these new trends. Overall, the study introduced diet as a possible factor in hemoparasite load due to diet differences altering blood proteins that could prom ote hemoparasite immunity, but sample size limits t he 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 h ealth, both in bats and humans. RESUMEN Los estudios de hemo-parasitos en murcielagos se ha n enfocado en estos animales como vectores de viruses, mientras que los estudios de ectoparasitos han abarcado un gran rango de factores ecologicos y abioticos que influencian la prevalencia de estos. Sin embargo, la dieta ha sido ignorada en ambos tip os de estudios. Aqui se estudio la dieta como un posible factor para explicar la carga de hemo-partasitos, y su relacion con salud y ectoparasitos. Los murcielagos con mas ectoparasitos parecen tener menos hemoparasitos. Los frugivoros parecen tener una mayor c antidad de hemo-parasitos, incluyendo los 3 tipos encontrados en el estudio ( Plasmodium, Babesia , y desconocido), los nectarivoros no tienen hemopa rasitos pero si el mayor porcentaje de ectoparasitos. Este estudio presenta la dieta como un posible factor pa ra explicar la carga de hemo-parasitos, debido a que l as diferentes dietas alteran las proteinas en la sa ngre que podrian promover la inmunidad a los hemo-parasitos, pero el tamano de muestra limitas las conclusions de este estudio. INTRODUCTION Bats (O: Chiroptera) exhibit a diverse array of fee ding strategies, consuming all types of food including fruit, insects, vertebrates, and nec tar (Kunz and Pierson 1994). With such a diversified range of feeding guilds, bats can pla y a Â“keystone roleÂ” in terrestrial community structure, pollinating flowers, dispersin g seeds, and managing insect densities (Kunz and Pierson 1994). Bats are also contributor s to the human community as bat guano provides many human amenities such as soap, f ertilizer, gasohol, and antibiotics
(Calisher et. al 2006). The prominent role of bats in community ecosystems makes the study of bat parasites 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 ecology as bats are ubiquitous in multiple habitats, migratory , and frequently come in contact with other bat species during foraging and roosting (Dic k and Patterson 2007). These combined factors could result in host switching of both ecto and endoparasites in the bat communities fostering speciation or providing vecto rs 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). O nly four insect orders have evolved true ectoparasitism, in which blood feeders spend t he majority of their lives on their hosts; Diptera, Hemiptera, Phthiraptera, and Siphon aptera (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 sp ecies of ectoparasites that can be influenced by multiple ecological and physiological factors of the host and abiotic environment (Gannon and Willig 1995). Similarly, endoparasites including trematodes, ces todes, and nematodes have been recorded on bats in North America (Jameson 1959). E ndoparasites can also include hemoparasites that are harmful to humans, such as Plasmodium falciparum , the parasite 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 ran ges (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 th ere is little doubt that new parasites will be found as technology and extent of studied s pecies increases. Yet, studies have been contradictory on the importa nce of bats as vectors for endoparasite transmission. It is acknowledged in l iterature that D. rotundus is an important vector for rabies, causing an estimated 1 00 million dollars of annual loss in domestic stock (Turner 1983) and Neotropical bats a re zoonotic vectors for other viruses such as equine encephalitis, Henipaviruses, and Ebo la (Ubico and McLean 1995, Calisher et al. 2006). Only Ubelaker et al. 1977 has shown evidence that hemoparasites such as Trypanosoma cruzi and Eimeria can be transferred from host to D. rotundus through feeding. The purpose of this study was to investigate the ef fect of feeding guild on endoparasite load, specifically hemoparasites (bloo d parasites), in four guilds of bats; sanguinivore, frugivore, omnivore, and nectarivore. A side note to the study recorded both ectoparasite presence and weight to see if hem oparasite loads had direct associations with these paralleled factors. I hypothesized that difference in diet would result in different hemoparasite loads for five species of ba ts in San Luis, Costa Rica. I expected to find an overall higher hemoparasite mean in sang uinivores because they are constantly exposed to blood through feeding on both domestic a nd wild animals, which provide a diverse community of hemoparasites which could be t ransmitted by direct blood contact and ingestion. I also expected to find that ectopar asites were more common on bats with hemoparasites because arthropod vectors are known t o transmit hemoparasites (Kudo 1966). The results of the study are relevant to pa rasitism ecology in general to elucidate
diet as an important indicator for blood parasite c omposition and concentration in mammals, and for adding to the knowledge of bat hem oparasite ecology, which is vital both for the survival and health of the bat populat ion 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 geoffroyi (nectar), Carollia perspicillata (fruit) Artibeus toltecus (fruit), and Phyllostomus discolor (omnivore) are known to have similar social roostin g sites and social behaviors (pers comm. LaVal), with the exception of A. toltecus in which much of its roosting 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 nectar 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 stu dy 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 anim als, although it also feeds on wild mammals as well (Greenhall et al. 1983). Specifical ly D. rotundus bites around the neck, anus, vulva, legs and snout of the animal (Greenhal l et al. 1983), using modified incisors to make a painless incision, and uses an anticoagul ant to allow constant blood flow (Greenhall et al. 1983). As many as five 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 Three study sites in the Holdrige tropical moist fo rest of San Luis, Costa Rica at approximately 1000 m in altitude provided the neces sary environments, banana patches and cow pastures, to catch the frugivores, nectariv ores, omnivores and sanguinivores. The Brenes Salazar family farm in La Finca Bella an d 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 positio ned near a chicken coop 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 behi nd the lodge in the large banana patch. The sanguinivores were caught at MachoÂ’s residence, a nearby dairy farm outside 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 October 25th and November 12th 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 characteristics (e.g. fac ial 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 ectoparasites 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 cl oth bag and on the weight. To collect blood samples, I used a lancet to prick the promine nt vein running along the uppermost part of the plagiopatagium and used a capillary tub e to remove less than 10 microliters. The blood was immediately blown onto a clean slide and a blood smear was made on sight. 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 m ade the slides were stained with Geimsa (Appendix 1). Analysis of the blood and iden tification of hemoparasites was conducted with the help of Dr. Carlos Alfonso, a mi crobiologist at Emergencias Monteverde. A drop of oil immersion was added to ea ch slide and the slides were analyzed under 100x magnification. The number of pa rasites per specimen was recorded in mm3 (e.g. 8 parasites per mm3 of blood) and due to the difficulty of hemoparasit e identification to the species level, hemoparasites were categorized into a generalized types consisting of Plasmodium , Babesia , or unknown (Figure 1). Photos of all parasites found were taken using Dr. AlfonsoÂ’s camera, and tw o blood samples were thrown out because it was not an efficient sample or it was di fficult to tell if the parasites were stain debris or actual organisms. Figure 1: Simplified drawings of red blood cells co ntaining parasites, similar to the real cells observed under the microscope. From left to r ight, 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 th e 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 there was a significant difference between the numb er 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 hem oparasite load in the frugivorous 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 caugh t including two D. rotundus , two P. discolor , one C. perspicillata , 15 A. toltecus , and seven A. geoffroyi . 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 f eeding guilds was different (p = 0.0026, x2 = 16.295, df = 4). Specifically, frugivores had a n overall higher number of parasites, and were the only feeding guild to have Babesia hemoparasites (Figure 2). Both sanguinivores and omnivores had parasites in a ll of the species present, yet nectarivores had no hemoparasites in any of the sam ples (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 that none of the nectarivores had any blood parasites. The sample sizes were as f ollows: frugivores (n = 15), nectarivores (n = 6), sanguinivores (n = 2), and om nivores (n = 2), for a total of 25 samples. Also, there was a difference between the number of hemoparasites in bats with and without ectoparasites (p = 0.0004, x2 = 18.104, df = 3). Bats with ectoparasites had a smaller number of hemoparasites, and bats with a la rger number of hemoparasites had ectoparasites present (Figure 3). This applied spec ifically to the nectarivores in which all but one nectarivore had ectoparasites (Figure 4), a nd there were no hemoparasites recorded in nectarivores. Hemoparasite Abundances in Different Feeding Guilds for Phyllostomidae Bats0 10 20 30 40 50 60 70 FrugivoresNectarivoresSanguinivoresOmnivoresFeeding Guilds Parasite Abundance (mm3) Plasmodium Babesia Unknown
Relationship between Ectoparsite and Hemoparasite Load in Phyllostidae Bats-20 -10 0 10 20 30 40 50 60 70 80 PBUHemoparasite Type Number of hemoparasites in (mm3) Ectoparasites None Figure 3: The Relationship between Ectoparasite and Hemoparasite Load. There is a difference between the number of hemoparasites in b ats 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 p er feeding guild that have ectoparasites. Nectarivores had the most ectoparasi tes; only one individual in the sample size of seven did not have ectoparasites. Similarly , no sanguinivores had ectoparasites. There was no correlation between weight and hemopar asite number in frugivores (p = .473, n = 14, Rho = -.209) indicating that althoug h there might be a negative trend for approximately 20% of the data, weight and hemoparas ite load do not seem to be related. DISCUSSION Differences in hemoparasite loads between bat feedi ng guilds could elucidate diet as another ecological factor contributing to parasitis m, yet the unequal sample sizes of this Ectoparasite Distributrion across Feeding Guilds0 1 2 3 4 5 6 7 NectarivoresFrugivoresOmnivoresSanguinivoresFeeding GuildNumber of individuals Nectarivores Frugivores Omnivores Sanguinivores
study make the exact role of diet inconclusive. My original prediction that sanguinivores would have the highest abundance of hemoparasites w as not correct. Paradoxically, the frugivores, made up of mostly A. toltecus individuals, had the most hemoparasites per mm3 of blood (Figure 1). The result is unexpected becau se frugivorous bats pass food so rapidly through their digestive tract (15-20) minut es that it has been hypothesized there is hardly any bacterial action (Nowak 1994). This wou ld make it very unlikely that ingestion is the route in which frugivores contract hemoparasites. In order to find out why frugivores had the highes t abundance of hemoparasites in this study, it is necessary to look at other parasi te factors such as roosting ecology and ectoparasite presence, which may have played a more prominent role than diet. Originally, I had predicted that the presence of ec toparasites would be associated with an increase in hemoparasite load, yet the results reve aled that individuals were more likely to have hemoparasites if there were no ectoparasite s present (Figure 2). This translated to nectarivores having the most ectoparasites and no h emoparasites, while frugivores had the most hemoparasites with fewer ectoparasites (Fi gure 4). The latter trend could possibly be explained by differences in immunology in the blood of individuals with and without ectoparasites. It has been shown in past st udies, that ectoparasites use immune signaling molecules that are similar to their hosts , thereby avoiding aggressive immuniosurvelance responses and detection by the ho st (Salazet et. al 2000). Perhaps, the specific molecules shared between ectoparasites and their hosts are so tightly coevolved that any blood parasites are quickly reco gnized by the host immune system and destroyed. This difference in immune proteins between the blo od of frugivores and nectarivores could also be due to their diet. Obse rvations by Dr. Alfonso and I while analyzing samples concluded that the Geimsa stain r eacted differently with nectarivore blood compared to frugivore blood. Nectarivore bloo d was significantly cloudier, and the frugivore blood was clearer showing individual bloo d 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 possibl y protecting nectarivoreÂ’s from hemoparasite infection. This would be similar to th e 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 exp lain the high level of hemoparasites found in frugivores. All the study sp ecies, 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. toltecus has been seen making leaf tents in Costa Rica (LaVal and Rodriguez 2002) whic h could put them into greater contact with arthropod vectors which transmit hemop arasites (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 hig her parasite loads and harbor more species, this study was only done with ectoparasiti c flies. Babesia and Plasmodium are transmitted by ticks and mosquitoes (Kudo 1966) res pectively 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 ass ess due to the fact that the vectors are unknown.
Also, some anomalies were found in this project tha t could have a dramatic influence on the study. Frugivorous bats have the h ighest proportion in mm3 of unknown parasites. Yet, 60 of the unknown parasites were fo und in one frugivorous bat indicating that parasite abundance may just be affected 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 i n other studies (Eberly 1997). Still, the study has elucidated specifics ab out 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 and frui t 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 elucidate facet s of bat parasite ecology. In conclusion, diet could be an important factor in ba t 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 hem oparasites with larger sample sizes of all the feeding guilds to be able to make more conc lusive 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 directions for my project. To Dr. Carlos Alfonso, thank you for helpi ng me to identify blood parasites and magnifying my virology directions for the future. Thank you to Pa blo for helping me translate my abstract and gettin g supplies. I also could not have survived without m y bat catching partners, Moncho and Adam. Thank you Moncho, for handling the feisty vampires and keepin g the nights exciting with stories of other blood sucking parasites from the past. Adam, thank you fo r keeping my Spanish skills sharp and pointing me t o the right farms for vampires, frugivores, and necta rivores. Acknowledgements also need to go to the University of GeorgiaÂ’s Ecolodge and MachoÂ’s farm f or their permission to trap bats. Finally, thank yo u to my host family at the Brenes Salazar farm for letti ng 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. Holme s and T. Schountz. 2006. Bats: important reservoir hosts of emerging 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. Was hington, D.C. pp. 475-485. Dick, C.W. and B. D. Patterson. 2007. Against all o dds: Explaining high host specificity in dispersal Â– prone parasites. International Journal for Parasi tology. 37: 871-876. Duval L., V. Robert, G. Csorba, A. Hassanin, M. Ran drianarivelojosia, J. Walston, T. Nhim, S. M. Goodman, and F. Ariey. 2007. Multiple host-switch ing 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 International 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 ecto parasites from Tropical bats. Environm. Entomol. 24: 1495-1503. Greenhall, A.M., G. Joermann, U. Schmidt and M.R. S eidel. 1983. Desmodus rotundus . In: Mammalian Species . American Society of Mammalogists. pp. 1-Greenhall , A.M., U. Schmidt and W. LopezForment. 1971. Attacking behavior of the vampire b at, 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: 6 1-65. Kudo, R.R. 1966. Protozoology 5th edition. Thomas Books. Springfield, IL. pp 718-732, 745-749 . Kunz, T.H. and E.D. Pierson. 1994. Bats of the Worl d: An Introduction. In: WalkerÂ’s Bats of the World , R. M. Nowak, ed Johns Hopkins University Press, Balt imore, MD, pp 1-46. LaVal, R.K and H.B. Rodriguez. 2002. MurciÃ©lagos d e 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. Ro osting habits of bats affect their parasitism by ba t flies (Diptera: Streblidae). Journal of Tropical Ecolog y 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. 2007. Capillaria sp ., isolated from Desmodus rotundus (Chiroptera: Phyllostomidae) in Costa Rica. MastozoologÃa Neot ropical. 14: 101-102. Salazet, M., A. Capron and G.B. Stefano. 2000. Mole cular crosstalk in host Â– parasite relationships: Schistosome Â– and leech Â– host interactions. Para sitology Today. 16: 536-540. Turner, D.C. 1983. Desmodus rotundus (Vampiro, Vampire Bat). In: Costa Rican Natural Hi story , D.H. Janzen, ed The University of Chicago Press, Chica go, IL, pp. 467-468. Ubelaker, J.E., R.D. Specian and D.W. Duszynski. 19 77. 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 N aturalist. 4: 61-65. Ubico, S.R. and R. G. McLean. 1995. Serologic surve y of Neotropical bats in Guatemala for virus antibodies. Journal of Wildlife Diseases. 31: 1-9 . Weikel-Magden, O. 2000. A Comparison of abundance a nd 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 Departme nt 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 solution A and 61 ml of Solut ion 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 overlappi ng 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 b it of color. 3. Place slides on a rack to dry. Please thin smears a djacent to thick. 4. After thick is dry, wash both the thick and thin sm ears with methanol and allow methanol to sit on slides for five minutes 5. Allow methanol to dry off or shake off excess and w ipe 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 slide s to spread stain over slide evenly. Does not need to be a thick layer. Allow st ain to sit for 30 minutes. 7. Wash slides with distilled water and allow to dry b efore viewing.