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Atta cephalotes (Formicidae) as a reliable bioassay for anti-fungal compounds in plants of Monteverde, Costa Rica

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Atta cephalotes (Formicidae) as a reliable bioassay for anti-fungal compounds in plants of Monteverde, Costa Rica
Translated Title:
Atta cephalotes (Formicidae) como un bioensayo confiable de compuestos anti micóticas en las plantas de Monteverde, Costa Rica ( )
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Chisholm, Annie
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Ants--Behavior   ( lcsh )
Biological assay   ( lcsh )
Hormigas--Comportamiento
Ensayo biológico
Tropical Ecology 2007
Ecología Tropical 2007
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Reports   ( lcsh )
Reports

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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.
Abstract:
Estudios han demostrado que Atta cephalotes (Formicidae) es selectiva en sus plantas hospederas, evita aquellas que contienen compuestos secundarios no polares dañinos para su hongo mutualista. Debido a esta selectividad varios estudios han encontrado que estas hormigas son buenas indicadoras de las propiedades anti micóticas en las plantas.
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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 choosing host plants, avoiding those that contain nonpolar secondary comp ounds 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 19 93; Taylor 1995). This experi ment 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 antifungal 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. RESUMEN 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 propiedades anti micoticas en plantas. Este experimento prueba las propiedades anti micoticas de seis plantas. La hiptesis seria que las hormigas evitaran los extractos de estas plantas. A. cephalotes prefirio el control en lugar de los extractos anti micoticos en 6 de los 7 tratamientos, solo en el caso de Heliconia sp. esto no ocurri. Estos resultados sugieren que esta especie es una he rramienta confiable para detectar compuestos anti micoticos 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 larvae (Howard 1988). Alt hough categorized as genera lists, 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 chemistry. A. cephalotes are deterred from leaves containing non-polar secondary compounds, 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 directly harm the ant at all (Hubbell et al. 1984). Due to this selectivity, several

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studies have shown A. cephalotes to be a reliable bioassay tool for detecting antifungal properties in medicinal plan ts (Ozaki 1993; Taylor 1995). Discovering medicinal plants 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, ar e 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-f ungal 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 cr ops to fungal infection, a major issue in tropical agricu lture (Westerdijk 1915). This experiment tests the acceptability of A. cephalotes towards Costa Rican plants with known anti-fungal compounds. Ba sed 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 wet 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-fungal properties found in Monteverde, Costa Rica (T able 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 Plant 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 were 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 were then strained fr om the extract. A fungicide treatment (10% propanodiol) was prepared using a concentration of 5 l/1 L of water. Twenty oat flakes dipped in anti-fungal treatment and 20 contro l flakes dipped in methanol/hydrochloric 2

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acid solution were placed on opposit e sides of the trail, three feet from the entrance of the A. cephalotes nest. The number of oat flakes remo ved and successfully carried into the entrance of the A. cephalotes nest was recorded over a twenty-minute period. Oats were replaced upon removal, and this process was rep eated for each extract. Six trials for each anti-fungal extraction were pr eformed using one colony. The data were analyzed using a chi-squa red test to determine the preference of A. cephalotes for each anti-fungal extraction 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. (Chisquared = 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 (Chisquared=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 difference in preference between Heliconia sp. and the control treatments (Fig. 1). The fre quencies 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 signifi cant difference in preference between the six plants tested (Chi-squared = 2.653, df = 5, P = 0.753). 0 10 20 30 40 50 60 70Heli co nia s p p. Allium sati u m Ore o p an ax spp Phytolacc a rivin o ides Nectandr a me m branace a B o cco n i a f r ute sce ns C omm e r c ial f un gici d eTreatments extract control 3

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FIGURE 1. Frequency of removal of oat fl akes 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 (Chisquared = 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 comme rcial fungicide (10% propanodiol) and five of the six medicinal plant extracts: A. satium, Oreopanax sp., P. rivinoides, N. membranacea and B. frutescens These results conf irm my hypothesis an d indicate that anti-fungal compounds in plants influence diet selection in A. cephalotes The preference of these ants towards the control suggests that A. cephalotes selectively avoids plants with chemical properties toxic to their mu tualistic fungus. Although physical factors such as leaf toughness, and trichome density on l eaves 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 th e most important factor influencing plant species selection by A. cephalotes Therefore, this study, as well as, 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 show 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 saponins (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 antifungal 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 s econdary compounds can be tolerated by A. cephalotes and their fungus, but the amount removed depend s 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 a nd 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 pl ants screened produces a useful drug, costing hundreds of millions of dollars and many years of analyses (Hamilton 2004), the 4

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use of A. cephalotess may help accelerate the process of locating plants with secondary compounds useful in the medical world. Further research should ex amine diet selection of A. cephalotes across multiple colonies and test if streng th of preference among anti-f ungal treatments corresponds to effects on fungus. In addition, as A. cephalotes are a pest in tropical agriculture and an attack by A. cephalotes may significantly reduce the fitne ss 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 Tania 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 th ank Frank Joyce for the use of his A. cephalotes nest and Willow Zuchowski and Dr. Setzer for their recommendations in choosing plants with anti-fungal properties. Lastly, special thanks to my fellow CIEE cla ssmates for making everyday rock. LITERATURE CITED Bernhardt, E. 2005. Costa Rican tropical medi cinal plants and trees New Dawn Center, Costa Rica. Blanton, C. and J. Ewel. 1985. Leaf-cutting ant he rbivory in successional and agricultural tropical ecosystems. Ecology 66: 861-869. Hamilton, R. 2004. Bioprospecting, with no a pologies: Costa Rica uses home-grown scientific muscle to unlock natures treasure chest. www.iadb.org/idbamerica/index.cfm?thisid=2705 Holldobler, B. and E. Wilson. 1990. The An ts. Harvard University Press, Massachussets. Howard, J. 1987. Leaf cutting ant diet selecti on: the role of nutrients, water, and secondary chemistry. Ecology 68: 503-513. Howard, J. 1988. Leaf cutting ant diet selecti on: relative influence of leaf chemistry and physical features. Ecology 69: 250-260. Hubbell, S., Howard, J. and D. Wiemer. 1984. Ch emical 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. Melek, F., Miyase, T., Abdel-Khalik, S., He tta, M. and I. Mahmoud. 2002. Triterpenoid saponins from Oreopanax guatemalensis. Phytochemistry 2: 185-195. Nielsen, S., Anthoni, U., Christopherson, C. a nd Cornett, C. 1995. Triterpenoid saponins from Phytolacca rivinoides and Phytolacca bogotensis. Phytochemistry 39: 625630. 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 H ealing Forest. Dioscorides Press, Oregon. Setzer, W.N., M.N. Flair, K.G. Byler, J. Huang, M.A. Thompson, A.F. Setzer, D.M. 5

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6 Moriarity, R.O. Lawton and D.B. Wi ndham-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. 190191. Taylor, B. Assessment of secondary compound ch aracter of five medicinal plants 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 M onteverde, Costa Rica. CIEE 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 cloud forest. Biotropica 39: 87-93. Westerdijk, J. 1915. Phytopathol ogy 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. P hytochemical investigation of Nectandra membranacea The University of Alabama in Huntsville.


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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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Biological assay
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Ensayo biolgico
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Reports
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CIEE
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