Atta cephalotes Formicidae as a reliable bioassay for anti fungal compounds in plants of Monteverde, Costa Rica Annie Chisholm Department of Biology, Bowdoin College ABSTRACT Studies have shown Atta cephalotes Formicidae to be selective in choosi ng host plants, avoiding those that contain nonpolar secondary compounds harmful to their mutualistic fungus Howard 1988. Due to this selectivity, several studies have found A. cephalotes to be a reliable bioassay tool for detecting antifungal properties of plants Ozaki 1993; Taylor 1995. This experiment tests the acceptability of A. cephalotes towards six Costa Rican plants with known anti fungal compounds. It was hypothesized that A. cephalotes would selectively avoid extracts of these plants. The fre quency of removal of oat flakes dipped in the anti fungal and control treatments was measured. A. cephalotes preferred the control to the anti fungal extracts for six of the seven treatments: Allium satium, Oreopanax sp., Phytolacca rivinoides, Nectandra m embranacea, Bocconia frutescens, and the commercial fungicide 10% propanodiol. However, there was no difference in preference between Heliconia sp. and control treatments. These results suggest that this species is a reliable bioassay tool for detecting anti fungal compounds in medicinal plants. RESUMEN Estudios han demostrado que Atta cephalotes Formicidae es selectiva en sus plantas hospederas, evita aquellas que contienen compuestos secundarios no polares daÃ±inos para su hongo mutualista. Debido a esta selectividad varios estudios han encontrado que estas hormigas son buenas indicadoras de propiedades anti nicÃ³ticas en plantas. Este experimento prueba las propiedades anti nicÃ³ticas de seis plantas. La hipÃ³tesis seria que las hormigas evitarÃan los extractos de estas plantas. A. cephalotes prefiriÃ³ el control en lugar de los extractos anti nicÃ³ticos en 6 de los 7 tratamientos, solo en el caso de Heliconia sp. esto no ocurriÃ³. Estos resultados sugieren que esta especie es una herramienta confiable par a detectar compuestos anti nicÃ³ticos en plantas medicinales. INTRODUCTION Atta cephalotes Formicidae are herbivores that use leaves to cultivate a symbiotic fungus Basidiomycota , providing digestive enzymes to adults and the sole food source for lar vae Howard 1988. Although categorized as generalists, studies have shown A. cephalotes to be quite selective in choosing host plants Blanton and Ewel 1985. Howard 1987 found that A. cephalotes discriminate among leaves based on their secondary chemis try. A. cephalotes are deterred from leaves containing non polar secondary co mpounds, especially terpenoids that are harmful to their fungus Howard 1988. In fact, many of the plants avoided by A. cephalotes are toxic only to their fungus, and do not dir ectly harm the ant at all Hubbell et al. 1984. Due to this selectivity, several studies
2 have shown A. cephalotes to be a reliable bioassay tool for detecting antifungal properties in medicinal plants Ozaki 1993; Taylor 1995. Discovering medicinal plant s is crucial in treating current health problems. More than 50% of clinical drugs worldwide are derived from natural products Wink and Wyk 2004. Major drug companies, such as Merck, are investing in bioprospecting to target potent secondary compounds in plants to be used in creating new medicines. However, this is an expensive and timely process, which involves modified random searches Lewis 1995. Using A. cephalotes to locate bioactive anti fungal compounds in plants may be a more efficient strategy. In addition, applying the plant extracts found unpalatable to A. cephalotes may also reduce the risk of crops to fungal infection, a major issue in tropical agriculture Westerdijk 1915. This experiment tests the acceptability of A. cephalotes towards C osta Rican plants with known anti fungal compounds. Based on previous studies showing that Heliconia sp. Schultes and Raffauf 1990, Oreopanax sp. Setzer et al. 1992, Bocconia frutescens Veldman et al. 2007 , Phytolacca rivinoides Veldman et al. 2007 , Allium satium Bernhardt 2005 and Nectandra membranacea Xiujun and Setzer 2004 contain anti fungal properties , I expect A. cephalotes to selectively avoid extracts of these plants. MATERIALS AND METHODS This study was conducted in the pre montane w et secondary forest of Bajo del Tigre in Monteverde, Costa Rica around an active A. cephalotes nest. Data was collected between November 3 and November 17, 2007. Under recommendations of Willow Zuchowski and Dr. Setzer, I tested six plants with anti funga l properties found in Monteverde, Costa Rica Table 1. The part of the plant tested differed for each species depending on the location of anti fungal compounds Table 1. TABLE 1. Family and plant part tested for each species Family Plant Species Pla nt Part Tested Heliconiaceae Heliconia sp. leaves Liliaceae Allium satium bulb Araliaceae Oreopanax sp. leaves Phytolaccaceae Phytolacca rivinoides seeds Lauraceae Nectandra membranacea bark Papaveraceae Bocconia frutescens seeds Plant extracts w ere prepared using a 50 ml solution of 80% methanol and 1% HCl mixed in a 1:1 ratio Thurston 2007. Five grams of each plant were mashed until a somewhat viscous liquid formed, then added to the methanol/HCl solution for further mixing. Leaf particles wer e then strained from the extract. A fungicide treatment 10% propanodiol was prepared using a concentration of 5 Ã°m l/1 L of water. Twenty oat flakes dipped in anti fungal treatment and 20 control flakes dipped in methanol/hydrochloric
3 acid solution were pla ced on opposite sides of the trail, three feet from the entrance of the A. cephalotes nest. The number of oat flakes removed and successfully carried into the entrance of the A. cephalotes nest was recorded over a twenty minute period. Oats were replaced u pon removal, and this process was repeated for each extract. Six trials for each anti fungal extraction were performed using one colony. The data were analyzed using a chi squared test to determine the preference of A. cephalotes for each anti fungal ext raction and the control. RESULTS A. cephalotes preferred the control over the anti fungal extracts for six of the seven treatments: A. satium Chi squared = 6.81 , df = 1, P = 0.0091 , Oreopanax sp. Chi squared = 27.84, df = 1, P < 0.001, P. rivinoides Chi squared = 27.56, df = 1, P < 0.001 , N. membranacea Chi squared = 9.29 , df = 1, P < 0.0023 , B. frutescens Chi squared=9.6 , df=1, P=0.0019 , and the commercial fungicide Chi squared = 35.1, df = 1, P < 0.001; Fig. 1. However, there was no differe nce in preference between Heliconia sp. and the control treatments Fig. 1. The frequencies of oat removal ranged from 11 to 19 for plant extracts and 31 to 61 for control treatments. A. cephalotes preferred the control more compared to the P. rivinoides and Oreopanax spp. treatments than for the other plant extracts Fig. 1. There was no significant difference in preference between the six plants tested Chi squared = 2.653, df = 5, P = 0.753. 0 10 20 30 40 50 60 70 Heliconia spp. Allium satium Oreopanax spp. Phytolacca rivinoides Nectandra membranacea Bocconia frutescens Commercial fungicide Treatments Frequency of oats removed extract control
4 FIGURE 1. Frequency of removal of oat flakes dipped in control and antifungal treatments by A. cephalotes . A. cephalotes preferred the control over the anti fungal extracts for six of the seven treatments: A. satium Chi squared = 6.81 , df = 1, P = 0.0091, Oreopanax sp. Chi squared = 27.84, df = 1, P < 0.001, P. rivinoides Chi squared = 27.56, df = 1, P < 0.001 , N. membranacea Chi squared = 9.29 , df = 1, P < 0.0023, B. frutescens Chi squared = 9.6 , df = 1, P = 0.0019 and 10% propanodiol commercial fungicide Chi squared = 35.1 , df = 1, P < 0.001. However, there was no difference in preference between Heliconia sp. and control treatments Chi squared = 3.45, df = 1,P = 0.063. DISCUSSION A. cephalotes preferred the control over the commercial fungicide 10% propanodiol and five of the six medicinal plant extracts: A. satium, Oreopanax sp., P. rivinoides, N. membranacea and B. frutescens . These results confirm my hypothesis and indicate that anti fungal compounds in plants influence diet selection in A. cephalotes . The prefe rence of these ants towards the control suggests that A. cephalotes selectively avoids plants with chemical properties toxic to their mutualistic fungus. Although physical factors such as leaf toughness, and trichome density on leaves may also be important to consider Hubbell et al. 1984, creating plant extracts controlled for these variables. Howard 1987 found plant secondary chemistry to be the most important factor influencing plant species selection by A. cephalotes . Therefore, this study, as well a s, previous experiments Taylor 1995; Thurston 2007 indicates that A. cephalotes is a reliable bioassay tool for detecting anti fungal compounds in medicinal plants. There was no difference in preference between the plants tested, but A. cephalotes did s how a greater preference towards the control for the P. rivinoides and Oreopanax sp. treatments. This suggests that Oreopanax sp. and P. rivinoides may contain more repellent secondary compounds. Both Oreopanax sp. and P. rivinoides contain triterpenoid sa ponins Melek et al. 2002; Nielsen et al. 1995, a compound shown in laboratory studies to inhibit growth of A. cephalotes fungus, as well as other fungi Hubbell 1984. Therefore, Oreopanax sp. and P. rivinoides may be more harmful to the symbiotic fungus of A. cephalotes than the other plants tested. There was no difference in preference between Heliconia sp. and the control. This suggests that secondary chemistry is more benign for this species and contrary to previous studies Schultes and Raffauf 1990 Heliconia sp. may not actually contain anti fungal compounds. Furthermore, Howard 1988 found that nutrients in leaves might interact with secondary chemistry to determine diet selection of A. cephalotes . This study found that plants are cut if their se condary compounds can be tolerated by A. cephalotes and their fungus, but the amount removed depends on the nutritional quality of the leaves Howard 1988. Therefore, the data on Heliconia sp. suggest that nutrient concentrations in leaves may be high in this species and may explain why there was no difference in preference for anti fungal and control treatments . These results confirm that A. cephalotes is an important bioassay for antifungal compounds in plants. As only one in 10,000 plants screened prod uces a useful drug, costing hundreds of millions of dollars and many years of analyses Hamilton 2004, the
5 use of A. cephalotes s may help accelerate the process of locating plants with secondary compounds useful in the medical world. Further research sho uld examine diet selection of A. cephalotes across multiple colonies and test if strength of preference among anti fungal treatments corresponds to effects on fungus. In addition, as A. cephalotes are a pest in tropical agriculture and an attack by A. ceph alotes may significantly reduce the fitness of individual plants Howard 1987, it would be interesting to examine if plants found unpalatable in this study are effective in reducing herbivory by A. cephalotes. ACKNOWLEDGEMENTS I would like to thank Tan ia Chavarria Pizarro for guidance and support during this project. Alan and Karen Masters for encouragement and talking through ideas. Thanks to Pablo Allen and Taegan McMan for assistance. I would also like to thank Frank Joyce for the use of his A. cepha lotes nest and Willow Zuchowski and Dr. Setzer for their recommendations in choosing plants with anti fungal properties. Lastly, special thanks to my fellow CIEE classmates for making everyday rock. LITERATURE CITED Bernhardt, E. 2005. Costa Rican tropi cal medicinal plants and trees. New Dawn Center, Costa Rica. Blanton, C. and J. Ewel. 1985. Leaf cutting ant herbivory in successional and agricultural tropical ecosystems. Ecology 66: 861 869. Hamilton, R. 2004. Bioprospecting, with no apologies: Costa Rica uses home grown scientific muscle to unlock nature s treasure chest. www.iadb.org/idbamerica/index.cfm?thisid=2705 Holldobler, B. and E. Wilson. 1990. The Ants. Harvard University Press, Massachussets. Howard, J. 1987. Leaf cutting ant diet selectio n: the role of nutrients, water, and secondary chemistry. Ecology 68: 503 513. Howard, J. 1988. Leaf cutting ant diet selection: relative influence of leaf chemistry and physical features. Ecology 69: 250 260. Hubbell, S., Howard, J. and D. Wiemer. 1984. Chemical leaf repellency to an attine: seasonal distribution among potential host plant species. Ecology 65: 1067 1076. Lewis, W., and M. Elvin Lewis. 1995. Medicinal plants as sources of new therapeutics. Annual Missouri Botanical Garden 82:16 24. Mel ek, F., Miyase, T., Abdel Khalik, S., Hetta, M. and I. Mahmoud. 2002. Triterpenoid saponins from Oreopanax guatemalensis . Phytochemistry 2: 185 195. Nielsen, S., Anthoni, U., Christopherson, C. and Cornett, C. 1995. Triterpenoid saponins from Phytolacca rivinoides and Phytolacca bogotensis. Phytochemistry 39: 625 630. Ozaki, R. Atta cephalotes as a bioassay for Fungicides in Plant leaves. UC EAP Tropical Biology Spring 1993, pp. 3 18. Schultes, R. and R. Raffauf. 1990. The Healing Forest. Dioscorides Pre ss, Oregon. Setzer, W.N., M.N. Flair, K.G. Byler, J. Huang, M.A. Thompson, A.F. Setzer, D.M.
6 Moriarity, R.O. Lawton and D.B. Windham Carswell. 1992. Antimicrobial and cytotoxic activity of crude extracts of Araliaceae from Monteverde, Costa Rica. Brenesia 38: 123 130. Stevens, G.C. 1983. Atta cephalotes Leaf cutting Ants. In: Costa Rican Natural History, D.H. Janzen ed. The University of Chicago Press, Chicago, IL, pp. 190 191. Taylor, B. Assessment of secondary compound character of five medicinal p lants using Atta cephalotes as a biological indicator. UC EAP Tropical Biology Fall 1995, pp. 205 217. Thurston, J. Atta cephalotes as a bioassay tool to identify the presence of polar secondary compounds in medicinal plants of Monteverde, Costa Rica. CI EE Tropical Ecology and Conservation Spring 2007, pp. 271 276. Veldman, K., Murray, G., Hull, G., Garcia C, J., Mungall, W. Rotman, G., Plosz, M. and L. McNamara. 2007. Chemical defense and persistence of pioneer plant seeds in the soil of a tropical clou d forest. Biotropica 39: 87 93. Westerdijk, J. 1915. Phytopathology in the tropics. Annals of the Missouri Botanical Garden 2: 307 313. Wyk, B. and M. Wink. 2004. Medicinal Plants of the World. Tien Wah Press, Singapore. Xiujun, W. and W. Setzer. 2004. Phytochemical investigation of Nectandra membranacea . The University of Alabama in Huntsville.
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Atta cephalotes (Formicidae) como un bioensayo confiable de compuestos anti micticas en las plantas de Monteverde, Costa Rica
Atta cephalotes (Formicidae) as a reliable bioassay for anti-fungal compounds in plants of Monteverde, Costa Rica
Studies have shown Atta cephalotes (Formicidae) to be selective in choosing host plants, avoiding those that contain nonpolar secondary compounds harmful to their mutualistic fungus (Howard 1988). Due to this selectivity, several studies have found A. cephalotes to be a reliable bioassay tool for detecting antifungal properties of plants (Ozaki 1993; Taylor 1995). This experiment tests the acceptability of A. cephalotes towards six Costa Rican plants with known anti-fungal compounds. It was hypothesized that A. cephalotes would selectively avoid extracts of these plants. The frequency of removal of oat flakes dipped in the anti-fungal and control treatments was measured. A. cephalotes preferred the control to the anti-fungal extracts for six of the seven treatments: Allium satium, Oreopanax sp., Phytolacca rivinoides, Nectandra membranacea, Bocconia frutescens, and the commercial fungicide (10% propanodiol). However, there was no difference in preference between Heliconia sp. and control treatments. These results suggest that this species is a reliable bioassay tool for detecting anti-fungal compounds in medicinal plants.
Estudios han demostrado que Atta cephalotes (Formicidae) es selectiva en sus plantas hospederas, evita aquellas que contienen compuestos secundarios no polares dainos para su hongo mutualista. Debido a esta selectividad varios estudios han encontrado que estas hormigas son buenas indicadoras de las propiedades anti micticas en las plantas.
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Tropical Ecology 2007
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