Piper spp. s cent as an i ndicat or of larvicidal a ctivity Erica Jansen Department of Biology, Hope College ABSTRACT Cebus capucinus monkeys search for licorice scented Pipers to use as an insect repellent (Brown 1996). Bioassays measuring mosquito larvicidal activity of si x Piper plants were completed in Monteverde, Costa R ica to see if scent is a reliable cue . Overall, there was no relationship between strength of scent and larvicidal activity ( Linear Regression F=0.064, P<0.05 ) . The species in the study that exhibited the highest larvicidal activity was Piper auritum , a species that con tains the essential oil safrole and has a strong licorice odor. Piper auritum and Piper marginatum (the only species C. capucinus is reported to use) both had a licorice like scent and together had significantly higher larvicidal activity than the four species tested that lacked the licorice like scent (Independent Samples T test t=2.74, DF=15, P<0.05 ). Altogether these data show that the qual ity of Piper scent can indicate insect repellent ability but strength cannot. Thus, it makes sense that C. capucinus use scent cues to find effective plant repelle nts as specific scents can indicate larvicidal essential oils. RESUMEN Los monos de la especie Cebus capucinus buscan plantas del gÃ©nero Piper con esencia a regaliz y lo utilizan como repelente para insectos. Bioensayos midiendo el efecto larvicida en seis plantas de Piper se realizÃ³ en Monteverde, Costa Rica para ver si el olor es una pista confiable. Sobre todo, no hubo relaciÃ³n alguna entre lo fuerte del olor y el efecto larvicida (F= 0.064, p <0.05). La especie en este estudio que exhibenun mayor efecto larvicida fue Piper auritum , esta especie contiene el aceite esencia l safrol y un fuerte olor a regaliz. Piper auritum y P. marginatum (la Ãºnica especie reportada como usada por los monos) tienen un fuerte olor a regaliz y juntas tienen significativamente un mayor efecto larvicida que las otras cuatro especies que carecen de olor a regaliz (t=2.74, DF=15, P<0.05). Estos datos sugieren que la calidad de la esencia de Piper puede indicar habilidades como repelente de insectos pero no la fuerza. AsÃ, tiene sentido que C. capucinus utilice la esencia como una pista para enc ontrar repelentes efectivos y que estas esencias pueden indicar aceites esenciales con efectos larvicidas. INTRODUCTION Many p lants of the tropical rainfore st have medicinal v alue due to their vast array of secondary metabolites which function in herbivore defense (Balandrin e t al. 1985) . S of all cancer drugs have been derived fr om natural plant products (Cragg and Newman 2007) . Some n on human a nimals also use plants for medicinal purposes. Zoopharmacognosy is a term that r efers to non human species selecting and using plants for disease treatment or parasite protection. This activity has been documented in many different animals including dusky footed wood rats ( Neotoma fuscipes ) , coatis ( Na sua narica ) , ants , and several primates (Raman & Kandula 2008) . The Piper plant genus (family Piperaceae) is distributed pantropically and has charac teristic attributes, one of these being a peppery 1993 ), given by essential oils of the plant . Cebus capuchinus monkeys look for Piper marginatum by smell in order to rub the leaves on t heir fur as insect repelle nt (Baker 1996) . It is hypothesized that C.
capuchinus preferentially choose P. marginatum over other Pipers because of the licorice like odor (Baker 1996) that comes from the essential oil safrole found in its leaves (Andrade et al . 2008). Humans also use the essential oils of Piper spp. in traditional medicine as an insect repelle nt and to relieve skin problems, sore muscles, and swelling (Bernhardt 2008). Bioassays measuring mosquito larvicidal activity of Piper spp. elucidate which species have adult mosquito repellent capabilities. M osquito larvicidal activity varies across the Piper genus with a t least 611 active ingredients from ap proximately 110 species identified as having larvicidal activit y (Dyer et al . 2004), though not every species has the same larvicidal effectiveness . For instance, P. nigrum and P. retrofractum have been isolated from groups of Piper spp. as being more toxic to mosquito larvae than other species of Piper (Park et al . 2002 & Chansang et al . 2005 respectively) . The fact that C. capuc inus prefers a licorice scented Piper species (Baker 1996) could suggest that Pipers with this particular scent have high larvicidal activity . Additionally, it is possible that the strength of P. marginatum could be a cue to the monkeys, as the strength could reflect essential oil concentration in the leaf. This study looks at six different species of Piper in order to elucidate the correlation between the scent of the leaves and the larvicidal activity. The type of scent and the strength of the scent should give an indication of the essential oi l compositio n of the leaf and c ould provide information about the potential repellent uses of different Piper species. METHODS Plant Collection Six Piper species were collecte d and identified from Monteverde , Costa Rica in the P re montane Moist F orest Holdridge life zone at 800 1450 m . Mean annual rainfall is 2000 4000 mm with a mean annual temperature between 17 24Â°C ( Haber 2000). Young fully opened leaves w ere collected (between 5 7 leaves) to ensure the highest possible secondary metabolite concentration . Scent Quantification Piper leaves from each species (approximately five ) were washed with water to remove dust and then ground with a mortar a nd pestle. The leaves were allowed to so ak in water for 24 hours in a ratio of 50 g wet leaf weight : 1 liter water (me thods adapted from Chansang et al . 2005 ). The fi ltrate from each species was diluted in 2 fold serial dilution s from 50 g/L to 0.75 g/L with water and stored in capped vials. A panel of 10 human subjects quantified the strength of the scent by smelling the vials from the weakest to the strongest co ncentration (smelling water in between each concentration) and indicatin g when the scent was first detectable. The subjects were also asked to describe the odors of each speci es in order to group the species into licorice scents and non licorice scents. Species described by over half of the participants as having a licorice like scent were recorded as possessing a licorice scent. Mosquito larvae bioassay Mosquito larva e (Culicidae) were obtained from bromeliad tanks and small, shallow pools of water in Monteverde and kept for up to two weeks in Tupperware containers of the water and
leaf matter from which they were obtained. Ten 3 rd and 4 th instar mosquito larvae were added to 15 mL of water in a 50 mL plastic cup along with 0.5 g of ground up Piper leaf picked from the plant on the same day as the test (a concentration of 0.03 g crude Piper extract/mL water ). Twenty four hours later, t he number of dead larvae was coun ted; a larva whose appendages did not move upon touching with a toothpick constitute d a dead organism. A mosquito larva e cup of 10 individuals with no treatment served as the contr ol for each species . Five tests were completed for each species of Piper . RESULTS Scent Classification and Quantification Of the six Piper species, two species were classified as licorice scented while the other four TABLE 1. A description of six Piper species collected from Premontane Moist Forest in Monteverde, Costa Rica. Scent descriptions were determined by a concensus from a 10 person panel. Number Species Description of Leaf Description of Scent 1 P. friedrichsthalii Small, elongated, drip tip Peppery, ranalean 2 P. methysticum Large, rounded, rough texture Sweet, ranalean 3 P. auritum Very large and long, asymmetrical petiole, hairy texture Licorice 4 P. amalago Small, very thin, smooth, drip tip Sweet, ranalean 5 P. aequale Small, drip tip, tough leathery text ure Sweet, ranalean 6 P. marginatum Very large heart shaped, drip tip Licorice The scent strengths varied among the different Piper species (Kruskal Wallace Test, H=30.9, DF=5, P<0.05 ) . Piper auritum had the lowest mean detectable scent concentration (indicating a strong scent strength) of 3.025% +/ 1.9% and was significantly lower than all other species except for P. amalago (Post hoc multiple comparisons, P<0.05) which had a mean detectable scent concentration of 6.4% +/ 3.9%, although this scent was described as sweet and not licorice like. However, the mean detectable concentration of P. amalago was only si gnificantly lower than P. aequale (Post hoc multiple comparisons, P<0.05) , the Piper with the largest mean detectable scent of 33.75% +/ 27.0 (in dicating a weak scent strength) . This detectable concentration is about 5 times greater than P . amalago and over 10 times greater than P . auritum . Piper friedrichstalii had a mean detectable scent of 21.35% +/ 16.5%, P . methysticum had a mean detectable scent of 13.75% +/ 6.5%, and P . marginatum had a mean det ectable scent of 16.9% +/ 14.7%, none of which differ from one another. All of the means had large standard deviations, a measure of the variability in the smell capabilities of human test subjects.
Larvae Bioassays There is di fferential larvicidal activity across the six Piper species (Kruskal Wallace Test, H=23.9, DF=5, P<0.05). However, the only significant difference in mean larvae mortality is between P . auritum and the plants which exhibited no larvicidal activity ( P. methysticum and P. amalago , Post hoc multiple comparisons, P<0.05) . P . auritum sh owed a mean mortality percentage of 88 +/ 11 % (Figure 1) . Piper friedrichstalii had the second highest mean mortality proportion of 64 +/ 18 %. P i per aequale and P. marginatum had mean mortality proportions of 28 +/ 16 % and 32 +/ 33 % respectively which is between one half and one thirds the mean mortality proportions of P . friedrichstalii and P. auritum . An interesting observation is that P . marginatum has an extreme ly high standard deviation. In contrast to the other tests, the testing of P. marginatum was complete d with a leaf that was 3 days old . It is also interesting to note that P. friedrichstalii had the second highest larvicidal activity even though it did n ot possess a licorice scent or a strong scent. None of the control tests yielded larvae mortality. 0 10 20 30 40 50 60 70 Mean Scent Detectable Concentraion Piper Species 0 10 20 30 40 50 60 70 80 90 100 Larvae Mean Percent Mortality Piper Species FIGURE 2. Larvae mean proportion mortality differed among six Piper species in Monteverde, Costa Rica(Kruskal Wallace Test, H=23.9, DF=5, P< 0.05). Post hoc tests show the differences are found between Piper auritum and the species with no larvicidal activity ( P. methysticum and P. amalago , Post hoc multiple comparison tests, P<0.05). FIGURE 1. The scent detectable concentration as a percentage of pure solution (50 g crude Piper extract/L) for each Piper species differed among six Piper species in Monteverde, Costa Rica (Kruskal Wallace, H=30.9, DF=5, P< 0.05). Low detectable concentrations indicate strong scents. Differences are found between P. auritum and all species except for P. amalago , and between P. amalago and P. aequale (Multiple Comparison Post Hoc Test, P<0.05).
There is no correlation between strength of Piper scen t and mean proportion larva e mortality (Linear Regression, F=0.064, P>0.05). As expected, the Piper with the strongest scent ( Piper 3) also had the highest mean proportion larvae mortality (Figure 3 ) ; however, the Pipers with the second and third strongest scents showed virtually no larvicidal activity. A comparison of mean larvae mortality proportions in comparing Pipers based on type of smell show that the means vary between Pipers with a licorice scent and Pipers without a licorice scent (Independent samples T test, t=2.74, DF=15, P< 0.05). The licorice scented Piper plants 3 and 6 had a mean larvae mortality proportion of 0.6 +/ 0.37 whereas the other Pipers had a combined mean larvae mortality proportion of 0.23 +/ 0.29 , almost a three fold difference (Figure 4) . y = 0.0422x + 17.332 RÂ² = 0.0177 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 Mean Lowest Detectable Scent Concentration Proportion of Larvae Mortality 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Licorice Scent Other Mean Larvae Mortality Proportion Types of Scents FIGURE 3. Strength of Piper scent does not correspond to larvicidal activity among six Piper species in Monteverde, Costa Rica (Linear Regression, F=0.064, P<0.05). FIGURE 4. The mean larvae mortality proportions differ significantly between Pipers possessing a licorice scent (2 species) and those lacking a licorice scent (4 species) in Monteverde, Costa Rica (Independent samples T test, t=2.74, DF=15, P<0.05) .
DISCUSSION Th e six Piper species differ in their larvicidal bioas say activity, which is a well established finding in the Piper genus (Dyer et al . 2004 ). Piper auritum showed the highest larvicidal activity, which confirms previous findings of Piper auritum having high insecticidal activity (Leyva et al . 2009). The three other species that showed larvicidal activity als o have been previously documented as larvicides ( P. aequale, Marquis 1991 ; P. marginatum , Autran et al . 2008; and m onoterpenoids found in P. friedrichsthalii , Lee 1997) , though no studies have compared these specific species. It is interesting that in this study P. marginatum (the species used by C. capucinus ) did not show significantly greater larvicidal activity tha n the control. The high variability in mean percent larvae mortality could be explained by possible leaf degradation in certain parts of the leaf since the tests were not completed the same day as leaf collection. This is congruent with a study showing l oss of larvicidal activity as a function of Piper fruit age (Chansang et al . 2005). S trength of the scent did not indicate larvicidal activity, but instead type of scent was much more important in determining larvicidal activity. This supports the hypothesis that certain types of odors ( licorice like odors ) can confer repellent ability (Brown 1996). These data point to the difference in chemical functions of the essential oils in the Piper genus; though the plants are phylogenetically related, their secondary metabolite defenses are not equal. Chemical structures of essential oils found in Piper include alkaloids/amides, lignans, terpenes, propenylphenols, steroids, kavapyrones, chalcones, flavones, flavanones, and pi perolides (Dyer et al . 2004) , indicating the vast array of chemical defense mechanisms found in Piper spp . The scent types of Piper species can indicate the chemical composition of the essential oils; t hus , it makes sense that scent type rather than scent strength is a better indicator of larvicidal activity. For instance, the second and third strongest scented plants did not show larvicidal activity, indicating that though their essential oil content is high, the ir chemical defense ab ilities do not impact mosquito larvae. The licorice sce nt present in P. auritum and P. marginatum is due to safrol e , the main essential oil present in both species (Andrade et al . 2008 and Hansel et al . 1975). Not surprisingly, saf rol e is already used commercially a s a precursor to piperonal butoxide, an insecticide (Tozzi 1998) . In regards to the efficacy of safrole as a potential natural insecticide, there are mixed reports. Safrole has also been implicated as a weak hepatocarcinogen in rats (Liu et al . 1999) and is banned as a food additive by the FDA (FDA 2010) ; however, one study showed that while carcinogenic metabolites were found in the urine of rats, no carcinogenic metabolites were found in the urine of humans, bringing to question the actual car cinogenicity (Benedetti and Broillet 1977). Perhaps concentration or chemical modification of safrole could provide a new mosquito repellent product. R egardless, it is interesting to find that the licorice scent that Cebus capucinus search for as insect repellent is indeed indicative of larvicidal essential oils. Cebus capucinus monkeys have not been previously recorded as using P. auritum even though it has a strong licorice odor and high larvicidal activity. However, it is likely that this has to do with limited research; the fur rubbing behavior was observed in monkeys at the CurÃº Refugio de Vida Silvestre in the Nicoya Peninsula of Costa Rica (Brown 1996), a location n ot recorded in the distribution of P. auritum (INBio 1997). Thus, it is probable that in cases where the two co exist, C. capucinus use P. auritum as insect repellent. Further observations of fur rubbing behavior could confirm this conjecture and could also give further insight into other possible larvicidal plants.
Altogether this st udy provides a good example of nonhuman animals locating and using plants for medicinal purposes, and it points to the value of allowing animals of the rainforest to guide our learning a bout plants of the rainforest. ACKNOWLEDGEMENTS I would like to tha nk Alan Masters for his guidance on this project. I would a lso like to thank Moncho CalderÃ³ n for his help with finding mosquito larvae, and Willow and Bill Zuchowski for identifying Piper species and for collecting P. marginatum . Finally, I thank CIEE for providing supplies to complete the project. LITERATURE CITED Balandrin, M. F., J. A. Klocke, E. S. Wurtele, and W. H. Bollinger. 1985. Natural plant chemicals: Sources of industrial and medicinal materials. Science 228, 1154 60. Baker, M. 1996. Fur rubbing: Use of medicinal plants by capuchin monkeys (Cebus capucinus). American Journal of Primatology 38: 263 270. Benedetti, M., A. MalnoÃ«, and A. Broillet. 1977. Absorption, metabolism and excretion of safrole in the rat and man. Toxicology 7, 69 83. Bernhardt, E. Medicinal Plants of Costa Rica. pp. 85. Zona Tropical Publication. China. Chansang, U., N. Zahiri, J. Bansiddhi, T. Boonruad, P. Thongsrirak, J. Mingmuang, N. Benjapong, M. Mulla. 2005. Mosquito larvicidal activity of aqueous extracts of lon g pepper ( Piper retrofractum ) from Thailand. Journal of Vector Ecology 30: 195 201. Cragg GM and Newman DJ. 2007. Natural Products as Sources of New Drugs over the Last 25 Years. Journal of Natural Products 70, 461 477. Dyer, L.A., J. Richard, and C.D. Dodson. 2004. Isolation,synthesis, and evolutionary ecology of Piper amides. In: L.A. Dyer and A.D.N. Palmer (eds.) A model genus for studies of phytochemistry, ecology, and evolution. pp.117 139. Kluwer/Plenum. New York. F DA. 2010. Safrole: CFR Code of Federal Regulations Title 21. Retrieved from http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=189.180. Gentry, A. 1993. Piperaceae. In: Gentry, A. A Field Guide to the Families and Genera of Woody Pla nts of Northwest South America (Colombia, Ecuador, Peru ) . pp. 683 684. University of Chicago Press . Chicago. Habe r, W. 2000. Plants and Vegetation . In Nadkarni, N. and N. Wheelwright. Monteverde, pp. 90. Oxford University Press, New York. Hansel, R., A. Leuschke, and A. Gomez Pompa. 1975. Aporthine type alkaloids from Piper auritum . Lloydia 38, 529 530. INBio. 1997. List of Specimens of Piper auritum. Retrieved from http://www.inbio.ac.cr/bims/k03/p13/c045/o0234/f01525/g008580/s027005.htm . Lee, S., R. Tsao, C. Peterson, and J. Coats. 1997. Insecticidal activity of monoterpenoids to western corn rootworm (Coleoptera: Chrysomelidae), twospotted spider mite (Acari: Tetranychidae), and house fly (Diptera: Muscidae). Journal of Economic Entomolog y 90: 883 892. Leyva, M., M. Marquetti, J. Tacoronte, R. Scull, O. Tiomno, A. Mesa, and D. Montada . 2009. Actividad larvicida de aceites e senciales de plantas contra de Aedes aegypti . Rev Biomed 20, 5 13. Marquis, R. 1991. Herbivore fauna of Piper (Piperaceae) in a Costa Rican wet forest: Diversity, specificity, and impact. In: P. Price, Lewinsohn T., FernÃ¡ndez G.,and Benson W. (eds.). Plant Animal Interactions: Evolutionary Ecology in Tropical and Temperate Regions. pp. 177 199. J. Wiley and Sons. United States. Park, I., S. Lee, S. Shin, J. Park, and Y. Ahn. 2002. Larvicidal Activity of Isobutlyamindes Identified in Piper nigrum Fruits against Three Mosquito Species. Journal of Agricultural Food Chemistry 50: 1866 1870. Raman, R. and S. Kandula. Zo opharmacognosy: Self Medication in Wild Animals. Resonance March 2008: 245 253. Tozzi, A. 1998. A Brief History of the Development of Piperonyl Butoxide as an Insecticide Synergist. In : D. Jones (Ed.) Piperonyl Butoxide : The Insecticide Synergist . Pp. 1 5. San Diego .
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Esencia de Piper spp. un indicador de la actividad larvicida
Piper spp. scent as an indicator of larvicidal activity
Cebus capucinus monkeys search for licorice-scented Pipers to use as an insect repellent (Brown 1996). Bioassays measuring mosquito larvicidal activity of six Piper plants were completed in Monteverde, Costa Rica to see if scent is a reliable cue. Overall, there was no relationship between strength of scent and larvicidal activity (Linear
Regression F=0.064, P<0.05). The species in the study that exhibited the highest larvicidal activity was Piper auritum, a species that contains the essential oil safrole and has a strong licorice odor. Piper auritum and Piper marginatum (the only species C. capucinus is reported to use) both had a licorice-like scent and together had significantly higher larvicidal activity than the four species tested that lacked the licorice-like scent (Independent Samples T-test t=2.74, DF=15, P<0.05). Altogether these data show that the quality of Piper scent can indicate insect repellent ability but strength cannot. Thus, it makes sense that C. capucinus use scent cues to find effective plant repellents as specific scents can indicate larvicidal essential oils.
Los monos de la especie Cebus capucinus buscan plantas del gnero Piper con esencia a regaliz y lo utilizan como repelente para insectos. Los bioensayos de medicin de la actividad de los mosquitos larvicida de seis plantas de Piper se realizaron en Monteverde, Costa Rica para ver si el olor es una seal confiable. En general, no hubo relacin alguna entre la fuerza del olor y el efecto larvicida (F= 0.064, p <0.05). La especie en este estudio que presento un mayor efecto larvicida fue Piper auritum, esta especie contiene el aceite esencial de safrol y tiene un olor fuerte a regaliz. Piper auritum y P. marginatum (reportada como la nica especie usada por los monos) tienen un fuerte olor a regaliz y juntas tienen significativamente un mayor efecto larvicida que las otras cuatro especies que carecen de olor a regaliz (t=2.74, DF=15, P<0.05). En total, estos datos muestran que la calidad del aroma de Piper puede indicar las habilidades como repelente de insectos pero no la fuerza. Por lo tanto, tiene sentido que C. capucinus utilicen el olor como una seal para encontrar los repelentes efectivos y que estas esencias puedan indicar los aceites esenciales con efectos larvicidas.
Text in English.
Insect baits and repellents
Costa Rica--Puntarenas--Monteverde Zone
Cebos y repelentes de insectos
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Fall 2010
Ecologa Tropical Otoo 2010
t Monteverde Institute : Tropical Ecology