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1 Atta cephalotes as a bioassay tool to identify the presence of polar secondary compounds in medicinal plants of Monteverde, Costa Rica. John Thurston Department of Biology, University of Minnesota Abstract: Atta cephalotes has shown potential as a bio assay tool for antifungal characteristics of medicinal plants (Hubbel, et al. 1983). This experiment tests a variety of plants with differing secondary compounds to explore further uses of A. cephalotes as a bioassay tool. A polar solvent was used to ma ke crude extractions of twelve different medicinal plants to test the preference of Atta for the extract compared to a control. It was predicted that A. cephalotes would show a negative preference towards plants with antifungal compounds. However, negati ve preference for Brugmansia suaveolens, a plant with no known antifungal compounds, deterred A. cephalotes the most. Resumen: Atta cephalotes ha mustrado ser un potential bio catador para plantas medicinales con propiedades fungicidas. Este proyecto es un prueba para plantas con componentes secundarios para explorar el uso de A. cephalotes como una herramiento bio catadora. Hice un extraction de doce plantas medicinales para probar preferencia de A. cephalotes. Se predijo que A. cephalotes tendria una negativa preferencia a plantas con fungicidas. Sinembargo, tuvo preferencia negativa para Brugmansia suaveolens, una planta sin fungicidas. Introduction: Analyzing plants and secondary compounds is a costly and time consuming endeavor. Bioprospectors must choose where to start, typically performing "modified random searches" (Lewis 1995). Leaf cutter ants, Atta cephalotes are pan tropical and use leaves to grow their only food, fungus (Stevens, 1983). This makes leaf cutter ants a potential bioassa y tool, a crude first step in narrowing the search for potential medicinal plants. Studies have shown that leaf cutters will avoid non polar terpenoids more than polar compounds such as phenolics, alkaloids and glycosides (Howard 1987). Plants contain m any combinations of secondary compounds and many have yet to be extensively studied (Hubert 1985), especially the synergistic effects of polar compounds. Natural products and their derivatives, including antibiotics, represent more than 50% of all drugs in clinical use in the world (Wink and Wyk, 2004). Botanical medicine has been used by traditional cultures for centuries. Uncovering the mechanism by which phytochemical's act on the physiology of the human body has added scientific rational to many na tural remedies. However, many traditionally used medicinal plants have yet to be extensively studied.
2 This study examines traditionally used medicinal plants of Costa Rica by observing the preference of A. cephalotes for polar secondary compounds. It is expected that A. cephalotes will show a negative preference for plants with known antifungal compounds. However, preference shown by leaf cutter ants for plants high in alkaloids could represent plants worth researching further. Methods: Twelve plants w ere chosen to study based upon their medicinal qualities, growth in the Monteverde area (VanZandt 2002) and the recommendation of Lucas Villalobos, a medicinal plant expert at the Ecolodge in San Luis, Costa Rica. Recommendations were made about which pla nts were the most toxic and thus contained a wide variety and concentration of strong secondary compounds. Plant species used in this experiment were the following: Family Genus and Specie Asclepiadaceae Asclepius curassavica Acanthaceae Justicia pec toralis Asteraceae Neurolaena lobata Asteraceae Chaptalia nutans Rutaceae Ruta chalepensis Solanaceae Brugmansia suaveolens Passifloraceae Passiflora biflora Simaroubaceae Quassia amara Solanaceae Solanum mammosum Euphorbiaceae Jatropha gossypifoli a Apocynaceae Plumeria rubra Euphorbiaceae Chamaesyce hyssopifolia Crude secondary extractions were performed with a polar solution of 80% methanol and 1% hydrochloric acid mixed in a 1:1 ratio (Coley et al. 1989). 10 grams of plant leaves and 40 ml of solution were grinded for twenty minutes using the backside of a steel spoon upon a plastic plate. The plant extraction was then strained into a small vial to be carried to the leaf cutter colony. Leaf picking, plant extraction and data collection wer e always performed within one day, ensuring a fresh extraction. The leaf cutter colony observed was located in San Luis, Costa Rica on the farm of Zaida Villalobos from April 15 to May 1 of 2007. The study site was at approximately 1100 meters in pre mon tane wet secondary forest. Using forceps, oat flakes were dipped into the control, the polar solution, or the plant extraction and placed six inches from the entrance to the leaf cutter nest. Two "control flakes" and two "extract flakes" were placed alte rnately on a large leaf cutter trail, so that the lines of flakes were perpendicular to the trail. Respective oat flakes were replaced as soon as they were removed and recorded. Oat flakes were observed until they entered the leaf cutter nest. Flakes we re dropped or carried the wrong way very few times these instances were recorded as well. Each plant extract was observed and recorded for one hour, generally in the afternoon. A chi square test was then run for each plant to determine the preference of A. cephalotes for each specific extraction.
3 Results: 0 10 20 30 40 50 60 70 80 Asclepius curassavica Justicia pectoralis Neurolaena lobata Chaptalia nutans Ruta chalepensis Brugmansia suaveolens Passiflora biflora Quassia amara Solanum mammosum Jatropha gossypifolia Plumeria rubra Chamaesyce hyssopifolia Frecuency Extract Control Figure 1. Frequency of selection for oats dipped in a polar extraction for selected plants compared to selection of the control (pure solvent) by A. cephalotes Atta showed the greatest preferen ce in the case of Brugmansia suaveolens (X 2 = 4.955, p = 0.026, df = 1). In some cases Atta picked up more extract flakes than control flakes, as in Asclepius and Solanum
4 TABLE 1: Chi squared test's with calculated p values for each plant. Plant Species p value chi square df Asclepius curassavica 0.24504217 1.351351351 1 Justicia pectoralis 0.66252058 0.19047619 1 Neurolaena lobata 0.66252058 0.19047619 1 Chaptalia nutans 0.30500721 1.052173913 1 Ruta chalepensis 0.66981536 0.181818182 1 Br ugmansia suaveolens 0.02601448 4.95505618 1 Passiflora biflora 0.5929801 0.285714286 1 Quassia amara 0.11914146 2.428571429 1 Solanum mammosum 0.18978349 1.719298246 1 Jatropha gossypifolia 0.8585862 0.031746032 1 Plumeria rubra 0.7815113 0.076923077 1 Chamaesyce hyssopifolia 0.8596838 0.03125 1 Brugmansia suaveolens showed the greatest preference between extract and control and represents the only trial statistically significant (p = .026, X 2 = 4.955). TABLE 2: Additional observations: dropped or c arried the wrong way. Plant species trials that never exhibited dropped or carried the wrong way behavior by A. cephalotes were omitted from this table. In some rare cases Atta would drop the oat flake prior to bringing it into its nest. The o at flake was only recorded as dropped if Atta walked away from it after dropping it. However, every dropped flake was eventually picked up by another leaf cutter and carried into the nest. In some cases Atta dropped the flake to get a better grip, this w as not recorded. Another oddity observed was Atta carrying the oat flake away from the nest. This was only recorded if Atta walked against the traffic, away from the nest, for a distance greater than 10 cm. Brugmansia extract seemed to provoke this reac tion more than other plants (Table 2). Discussion: Plant Species Dropped (Extract) Wrong Way (Ex tract) Dropped (Control) Wrong Way (Control) Neurolaena lobata 0 1 0 0 Ruta chalepensis 0 0 0 1 Brugmansia suaveolens 1 8 0 1 Quassia amara 0 2 0 0 Solanum mammosum 0 1 1 1 Jatropha gossypifolia 0 1 0 0
5 These results were surprising. It was expected that Atta would show negative preference towards plants with known antifungal properties, such as Plumeria rubra (Hubbel, et al. 1983). Some of the plant s are also listed as containing compounds known to be insecticides, as in Quassia and Solanum (Duke, 1994). It is interesting that the greatest negative preference was shown towards Brugmansia. Brugmansia contains an abundance of tropane alkaloids, such as scopolamine and atropine. These alkaloids are in the same class as cocaine and are strong antagonists at muscarinic acetylcholine receptors, inhibiting smooth muscle contraction. These compounds are also able to cross the blood brain barrier, creating hallucinations (Wyk and Wink, 2004). Brugmansia also experienced a much higher proportion of cases where A. cephalotes would carry the flake the "wrong way" (Table 2). Perhaps these compounds are also able to upset the behavior of A. cephalotes It is possible that the strength of tropane alkaloids and there high concentration in Brugmansia are capable of deterring A. cephalotes. This is the first study to show that non antifungal secondary compounds can deter A. cephalotes. It has been well documente d that terpenoids contain the greatest antifungal activity (Hubert, 1985). In this experiment a highly polar solvent was used that is not effective in extracting the non polar terpenoids. Perhaps there is more to the selectivity of substrate for fungal g rowth by Atta than is known. It would be interesting to isolate tropane alkaloids to see is they are the deterring factor or if it is a synergistic effect of many classes of compounds that determine the preference of A. cephalotes Minima were observed t o be present in different concentrations on different days. On May 1, 2007 an extremely high abundance of minima were present. This day was different in that there was extremely dense cloud cover and the smell of ozone in the air. Perhaps Atta can sense biotic conditions such as air pressure and light intensity and change their behavior accordingly. Some days there was more foraging behavior by Atta (Fig. 1). On day 1 they collected a total of 74 oat flakes and on day 12 they collected 128 oat flakes. Data collection commenced in mid April and ended in the first week of May. Perhaps Atta can sense the onset of the rainy season and increases foraging behavior before the rains hit. Plants produce varying amounts of secondary compounds at different times of the year, month and even day (Wyk and Wink 2004). The method of extraction used in this experiment was also very crude. For these reasons was difficult to tell whether a sufficient concentration of secondary compounds was obtained in each extraction. Future studies should compare polar and non polar secondary compounds by utilizing different extraction techniques and Atta 's preference towards them. There are also many more plants traditionally used as medicine that future studies could unveil usin g the same techniques described here. Acknowledgements: Thanks Alan Masters for helping to formulate this study. Tania for helping with statistical analysis and her time and energy in reviewing my paper. Lucas Villalobos for the use of his medicinal ga rden and his guidance on medicinal plants. Zaida for being a wonderful momma tica and the use of her farm. And finally my fellow peers for listening to my ideas and contributing there own.
6 WORKS CITED: Duke, R., and R. Vasquez. 1994. Amazonian Et hno botanical Dictionary. CRC Press, Boca Raton, Florida. Dyer, L., D. Dodson and G. Gentry. 2003. A bioassay for insect deterrent compounds found in plant and animal tissues. Phytochem. Anal. 14, 381 388. Howard, J. 1987. Leaf cutting ant diet se lection: the role of nutrients, water and secondary chemistry. Ecology 68: 503. Hubert, T., and D. Wiemer 1985. Ant repellent terpenoids from Melampodium divaricatum. Phytochemistry 24: 1197 1198. Lewis, W. 1995. Medicinal plants as sources of new therapeutics. Annual Missouri Botanical Garden 82: 16 24. Mabberley, D.J. 1987. The Plant Book: A Portable Dictionary of the Higher Plants. Cambridge University Press, Great Britain, Avon. Stevens, G. C. 1983. Atta cephalotes, (Leaf cutting An ts, Zompopas). In: Costa Rican Natural History D. H. Janzen, ed. The University of Chicago Press, IL, pp. 190 191. Vanzandt, M. Fall 2002. Medicinal Plants of Monteverde CIEE Tropical Ecology and Conservation 243 278. Wyk, B. and M. Wink. 2004. Medicinal Plants of the World. Tien Wah Press, Singapore. Zuchowski, W. 2005. A Guide to Tropical Plants of Costa Rica. Zona Tropical, Miami, Florida.
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Atta cephalotes como una herramienta de bioensayo para identificar la presencia de compuestos polares secundarios en plantas medicinales de Monteverde, Costa Rica
Atta cephalotes as a bioassay tool to identify the presence of polar secondary compounds in medicinal plants of Monteverde, Costa Rica
Atta cephalotes has shown potential as a bioassay tool for antifungal characteristics of medicinal plants (Hubbel, et al. 1983). This experiment tests a variety of plants with differing secondary compounds to
explore further uses of A. cephalotes as a bioassay tool. A polar solvent was used to make crude extractions of twelve different medicinal plants to test the preference of Atta for the extract compared to a control. It was predicted that A. cephalotes would show a negative preference towards plants with antifungal compounds. However, negative preference for Brugmansia suaveolens, a plant with no known antifungal compounds, deterred A. cephalotes the most.
Atta cephalotes ha mostrado potencial como una herramienta de bioensayo para las caractersticas anti-hongos de las plantas medicinales (Hubbel, et al. 1983). Este experimento prueba una variedad de plantas con diferentes compuestos secundarios para explorar los usos adicionales de A. cephalotes como herramientas de bioensayo.
Text in English.
Tropical Ecology 2007
Ecologa Tropical 2007
Herramienta de bioensayo
t Monteverde Institute : Tropical Ecology