GLADYS V. SOBERÓN RISCO¹², CONSUELO ROJAS IDROGO^1,3, MASSUO J. KATO⁴, JORGE SAAVEDRA DÍAZ^1,5, JOSÉ ARMANDO-JR.⁶ and GUILLERMO E. DELGADO PAREDES^1,7

Key words: CH₂Cl₂:MeOH (2:1) extract. EtOH extract. In vitro propagation. Larval susceptibility. Lethal Dosis.

Resumen: Se evaluó la actividad larvicida del "matico" neotropical Piper tuberculatum. Los compuestos secundarios fueron extraídos de hojas, tallos y espigas maduras (con frutos y semillas) de plantas silvestres y de plantas in vitro de P. tuberculatum. Fue evaluada la toxicidad aguda de extractos de espigas maduras con frutos y semillas y de plantas in vitro de P. tuberculatum sobre el "cogollero" Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) mediante bioen-sayos de contacto. Solamente extractos Diclorometano:metanol (CH₂Cl₂:MeOH) (2:1) y etanólicos (EtOH) de espigas maduras y el extracto CH₂Cl₂:MeOH (2:1) de plantas in vitro mostraron niveles significativos de mortalidad. Los extractos CH₂Cl₂:MeOH (2:1) y EtOH de espigas maduras causaron 90% de mortalidad cuando dosis de 0,1850 mg/µL se aplicaron sobre S. frugiperda en 24 y 48 h de exposición, respectivamente. El extracto CH₂Cl₂:MeOH (2:1) de plantas in vitro causó 95% de mortalidad cuando dosis de 0,1850 mg/µL también se aplicaron en 48 h de exposición. Los mejores resultados para las espigas maduras fueron: CL₅₀ 0,001 mg/µL con EtOH y 0,007 mg/µL con CH₂Cl₂:MeOH (2:1) y CL₉₀ 0,027 mg/µL con EtOH y 0,103 mg/µL con CH₂Cl₂:MeOH (2:1); y en el caso de las plantas in vitro, solamente el extracto CH₂Cl₂:MeOH (2:1) fue: CL₅₀ 0,003 mg/µL y CL₉₀ 0,060 mg/µL. Se discute el valor potencial de los extractos de P. tuberculatum como un eficiente insecticida sobre S. frugiperda.

Palabras clave: Extracto CH₂Cl₂:MeOH (2:1). Extracto EtOH. Propagación in vitro. Susceptibilidad larval. Concentración letal.

Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), the fall armyworm, is a polyphagous insect of enormous agricultural importance, not only because of the damage it provokes, but also because of control difficulties (Santos et al. 2003). The species is a migratory pest endemic to the Western Hemisphere that occurs from Southern Canada to Argentina and causes considerable economic losses in several important crops such as maize, sorghum, rice, cotton, alfalfa, forage grasses, and occasionally other crops in most of the countries of its range (Clark et al. 2007). In its distribution area, two genetically distinct strains are found that differed in their plant host distribution (Pashley 1986); one strain feeds primarily on maize and sorghum (corn strain), and the other strain feeds on rice and bermuda grass (rice strain) (Pashley et al. 1985; Pashley 1986).

The indiscriminate use of synthetic insecticides has caused environmental contaminations and toxicity to living organisms (Nakata et al. 2005), indicating the need for the development of products that not hazardous to the environment, target-specific and biodegradable. Thus, the development of new insecticides from plant extracts sources can be an alternative for the control of Spodoptera bugs.

The benzene extract of the leaves of Piper futokatsura (Sieb., 1900) from Taiwan and Japan are known as a feeding deterrent to Spodoptera litura (Fabricius, 1775) larvae (Matsui and Munskata 1975), and the leaves of Piper umbellatum (Linnaeus, 1753), Piper hispidum (Sw., 1788), Piper auritum (Kunth, 1816), and others Piper spp., which are native to Central America and the Northwest Amazonian basin, are used by indigenous peoples to prevent malaria and removing head lice (Schultes 1980). Likewise, the leaf and stem petrol and dichloromethane extracts of Piper falconeri (C.DC., 1925) have shown insecticidal activity against Musca domestica (Linnaeus, 1758) (flies) and Aedes aegypti (Linnaeus, 1762) (mosquitoes); the Piper acutisleginum (DC., 1869) dichloromethane extract has also been reported to show insecticidal activity against M. domestica and A. aegypti (Parmar et al. 1997) and the dichloromethane and ethanolic extracts of spikes and in vitro plants of Piper tuberculatum (Jacq., 1795) have shown insecticidal activity against Diatraea saccharalis (Fabricius, 1794) (Soberón et al. 2006).

The objective of this research was to investigate the insecticidal activity of extracts of leaves, stems and mature spikes, with fruits and seeds, of wild plants and in vitro plants of P. tuberculatum on third instar larval of S. frugiperda.

Plant material. Spikes with mature seeds, leaves and stems of P. tuberculatum were collected in November 2003 from 'Cumbil' river (Lambayeque, Peru). Botanical identification was performed by Doctor Guillermo E. Delgado from Universidad Nacional Pedro Ruiz Gallo (UNPRG) based on taxonomic description realized by Yuncker (1973). The botanic specimen vouchers were deposited at same herbarium of the institution (HPR).

In vitro micropropagated plants. The culture was initiated from axenic seedlings explants. A total of 50 seeds per flask were surface-sterilized by 70% ethanol (v/v) 1 min followed by 2-2.5% sodium hypochlorite (w/v) for 20 min and then washed three times with sterile water. Floating seeds were discarded; about 3-10 of them were transferred to glass test tubes containing 20 mL of MS medium (Murashige and Skoog 1962) and 2% sucrose. Shoot-tip and nodal segments, 1 cm length containing a lateral bud, taken from three-month-olds in vitro seedlings, were used as explant source. MS medium, supplemented with 0.02 mg/L indoleacetic acid (IAA), 0.02 mg/L gibberellic acid (GA₃) and 3% sucrose was used to initiate cultures, and were maintained by subculturing every six months on a fresh medium containing the same formulation. In all cultures, the same MS medium was supplemented with 100 mg/L m-inositol and 1 mg/L thiamine.HCl, adjusted to pH 5.7 ± 0.1, solidified with "Phytagel" 0.3% prior to autoclaving, dispensed into tubes (150 x 25 mm) containing 20 mL MS medium and covered with polypropylene caps. All cultures were incubated at 24-28°C in a 16-h light, 8 h dark photoperiod provided by cool white fluorescent tubes, with 5 umol m^-2 s^-1, for seed germination and 30 umol m^-2 s^-1 for clonal propagation.

Insects. Eggs of S. frugiperda were collected from a corn crop during its vegetative to early reproductive stage, in the Fundo La Peña, UNPRG - Lambayeque, Peru, and were reared in the Laboratorio de Entomología of the Facultad de Agronomía (UNPRG), under laboratory conditions. Insects were maintained in Petri dishes lined with damp filter paper (one fall armyworm per dish to avoid cannibalism) under a controlled environment (26 ± 2^oC, 80 ± 5% relative humidity, 16:8 h light:dark photoperiod). Third instar larvae of S. frugiperda were fed to repletion with fresh leaves of maize.

Topical test. Bioassays were carried out at in the Laboratorio de Entomologia of UNPRG. The stock solutions of extracts were prepared by dissolving 100 mg of dry extract in 1 mL of MeOH-water to obtain a concentration of 100 mg/mL. After 24 h, and using and Eppendorf® 0-10 µL pipette, 6.5 µL of the solution, containing an aliquot of each one of the treatments, was applied directly on the larval mesothorax of S. fru-giperda. The plant extract was tested at doses of 0.0, 0.0007, 0.0014, 0.0029, 0.0057, 0.0115, 0.0230, 0.0460, 0.920 and 0.1850 mg/µL. Twenty larvae were tested per treatment and the experiment was carried out twice. The control insects received a topic application with MeOH-water alone. Larval mortality was recorded at 24, 48 and 72 hour post-treatments, under the same conditions of temperature and humidity described above. The larvae were considered dead if they displayed no observable response to a mechanical stimulus, i.e. short-term pressure applied with a spatula.

A dose - response correlation was obtained using a linear regression model to fit the probit data to the log of the dose of each extract applied. LD₅₀ and LD₉₀ values were determined used the software US. EPA Probit Program Version 1.5 (2003).

The resultant regression lines for all the extracts appeared to be very similar, showing a relatively fast intoxication process on the insects exposed to P. tuberculatum extracts. In a general way, the LD₅₀ and LD₉₀ values decreased when the time between application and evaluation increased (Table 1). The data presented confirm that mature spikes and in vitro plants extracts from P. tuberculatum presented potential insecticide activities.

The larval mortality at 90% was reached after 24 h when using 0.1850 mg/µL of CH₂Cl₂:MeOH (2:1) extract from mature spikes; and a mortality of 100% was reached with 0.1850 mg/µL EtOH extract from mature spikes in 72 h. In reference to the in vitro plants, the extract obtained with CH₂Cl₂:MeOH (2:1) alone generated a 95% larval mortality with 0.1850 mg/ µL in 48 h.The mortality of the control group was 0%.

The small variations in LD₅₀ and LD₉₀ values of both extracts with respect to time of exposure suggest a rapid toxic action. Similar to what happens with larvae of Anticarsia gemmatalis (Hubner, 1818) (Navickiene et al. 2007), almost immediately following the application of doses of each treatment, larval movement decreased and feeding practically ceased. Also, typical intoxication symptoms, as described by Marchini et al. (1992), such as spasmodic movements, regurgitation and faecal elimination, were observed, thus confirming the acute toxicity of these extracts to fall armyworm.

The results confirm that CH₂Cl₂:MeOH (2:1) and EtOH extracts of mature spikes from wild plants and QLC^MeOH (2:1) extracts from in vitro plants of P. tuberculatum showed a potent insecticidal activity on third instar larval of this Lep-idoptera species. The CH₂Cl₂:MeOH (2:1) and EtOH extracts of mature spikes showed higher toxicity than CH₂Cl₂:MeOH (2:1) extract from in vitro plants and also EtOH extract was more effective than CH₂Cl₂:MeOH (2:1) extract.

The results suggest two possibilities for the direct use: insecticidal activity of mature spike EtOH extracts from P. tuberculatum that allows rural people to freely use the extract obtained with traditional alcoholic drinks as "aguardiente, yonque or cañazo". The second possibility, even thought the in vitro plant extracts showed lower toxicity than mature spike extracts, may be an active compound biosynthesis at large scale using the establishment of cellular suspensions (Danelutte et al. 2005).

Only some species of Piper, of the flora of Peru, as Piper aduncum (Linnaeus, 1753), Piper aequale (Vahl., 1797), P. hispidum, Piper reticulatum (Linnaeus, 1753) and P. tuberculatum showed significative activity against the mosquito A. atropalpus (Bernard et al. 1995). In this paper was reported that 100 mg/L (0.1 mg/mL or 0.0001 mg/µL) hexanic crude extract of P. tuberculatum leaves presented most intense activity with 54% mortality in second instar larval of A. atropalpus, after 24 h of exposure; this mortality was attributed to isobutylamide 4,5-dihydropiperlonguminine (pure substance isolated from the active fraction) because caused 47% mortality of mosquito larvae at 0.01 mg/L in the same time of exposure. Comparing these results, in our work was showed that intermediate doses of extract, from mature spikes as 0.0115 mg/µL and 0.0230 mg/µL extract, from in vitro plants, produced a mortality exceeds 50% in S. frugiperda at 24 h of exposure.

In previous studies with seed extracts of P. tuberculatum were isolated several amides, mainly bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties (Navickiene et al. 2000; da Silva et al. 2002). The antifungal activity of each amide was determined by direct bioautography against C. sphaerospermum and C. cladosporioides with 1- 5 µg and 5 - 10 µg, respectively, as the minimum quantity of compounds, specially piperine and 5,6-dihydropiperlongu-minine, necessary to inhibit growth of the fungus (Navickie-ne et al. 2000; da Silva et al. 2002). Likewise, extracts of P. tuberculatum were used in control of dermatophyte fungi Microsporum canis (Bodin, 1902), Microsporum gypseum (Bodin, Guiart & Grigorakis, 1907) and Trichopyton rubrum (Malmsten, 1845) (Palacios et al. 2009) therefore, these amides have shown to have a potent fungicidal activity as well as a inseciticidal activity as reported in the control of the third instar larval of D. saccharalis (Soberón et al. 2006) and control of II and III instar larval and adults of A. aegypti and A. pseudopunctipennis (Bazán-Calderón et al. 2010).

This fact has a profound ecological significance since it presupposes an advantage to using plant extracts as a source of complex molecules that exhibit various bioactivities, raising the levels of toxicity in relation to chemically pure individual compounds, in addition to the risk of induce resistance (Bobadilla et al. 2005). It is knew, chemical studies on Piperaceae species have revelled the occurrence of various compounds as alkaloids, amides, propenylphenols, lignans, neolignans, terpenes, steroids, kawapyrones, piperolides, chalcones, dihydrochalcones, flavones, flavanones and miscellaneous compounds (Parmar et al. 1997).

In this context Scott et al. (2002) demonstrated that the amides presents in P. tuberculatum plants has higher toxicity when combined in binary, tertiary and quaternary mixtures compared to single compounds or binary mixtures; one of the four amide compounds, 4,5-dihydropiperlonguminine, was the most toxic in mosquito larvae bioassays. Navick-iene et al. (2007) reported that seed extracts of P. tuberculatum may be more powerful than the pellitorine isolated, therefore, would be advisable the preferential use of crude extracts. There are no simple explanations for the observed differences in the efficacy of the whole extract from different parts of the plant and the isolated piperamides. Variations in the concentration of the insecticide compounds among the plant tissues suggest that varied selective pressures operate in the plants, and a great number of combinations of compositions can arise inside individuals in certain species (Jones and Firn 1991), which can provide a higher protection level to the plant against herbivores (Berenbaum and Zangerl 1996). Our results make it possible to conclude that Piper extracts may be good candidates for use in crop protection.

The action mechanism of the pellitorine, 4,5-dihidrop-iperlonguminina and other related compounds (piperamides) found in Piper species on third instar larval of S. frugiperda is not well known, however could be attributed the toxicity at the presence of methylenedioxyphenyl ring (MDP) in the molecular structure (Bernard et al. 1995; Scott et al. 2003), like just as reported to other compounds of similar structure at pipercida, guineensinamida, guineensina, pellitorine and kalecida isolated from P. guineense and very actives in the control of adults of M. domestica (Gbewonyo et al. 1993).

The first three amides were the most active against adults of M. domestica and each of these contain a MDP structural moiety. The 5,6-dihydropiperlonguminine, isolated from P. tuberculatum, also has an MDP ring and is the main component of the active fraction of the spikes extract (Navickiene et al. 2000). Greger (1988) pointed out that these types of amides, including olefinic and alkyl isobutylamides, are common to a restricted number of related plant families, namely the Piperaceae, Asteraceae, and Rutaceae. In all these families are abundant in the tropics particularly in the humid tropics in the case of the Piperaceae, where herbivory is a selective potent force.

In conclusion, P. tuberculatum is an abundant species and is semidomesticated in Peru where is used as a hedge plant. It is for the reasons that the potential use of this species as a source of botanical insect control material looks promising.

The authors are grateful to the Prof. Jorge Chanamé-Céspedes for the statistical analyses.

ALLAN, E. J.; STUCHBURY, T; MORDUE (LUNTZ), A. J. 1999. Azadirachta indica A. Juss. (Neem Tree): In vitro culture, micropropagation, and the production of azadirachtin and other secondary metabolites. pp. 11-41. In: Bajaj, Y. P. S. (Ed.). Biotechnology in Agriculture and Forestry 43. Medicinal and Aromatic Plants XI. Springer-Verlag, Berlin.         [ Links ]

BALANDRIN, M. F.; KLOCKE, J. A.; WURTELE, E. S.; BOLLINGER, W. H. 1985. Natural plant chemicals: Source of industrial and medicinal materials. Science 228: 1154-1160.         [ Links ]

BALDOQUI, D. C.; KATO, M. J.; CAVALHEIRO, A. J.; BOLZANI, V. DA S.; YOUNG, M. C. M.; FURLAN, M. 1999. A chromene and prenylated benzoic acid from Piper aduncum. Phytochemistry 51 (7): 899-902.         [ Links ]

BAZÁN-CALDERÓN, J.; VENTURA-FLORES, R.; KATO, M. J.; ROJAS-IDROGO, C.; DELGADO-PAREDES, G. E. 2011. Actividad insecticida de Piper tuberculatum Jacq. sobre Aedes aegypti L. (Diptera: Culicidae) y Anophelespseudopunctipennis Tehobal (Diptera: Culicidae). Anales de Biología 33: 135-147.         [ Links ]

BERENBAUM, M .R.; ZANGERL, A. R. 1996. Phytochemical diversity. Adaptation or random variation?. pp. 1-24. In: Beren-baum, M.R. (Ed.). Phytochemical Diversity and Redundancy in Ecological Interaction; Recent Advances in Phytochemistry. Plenum Press, New York.         [ Links ]

BERNARD, C. B.; KRISHNAMURTY, H. G.; CHAURET, D.;DURST, T.; PHILOGÉNE, B. J. R.; SÁNCHEZ-VINDAS, I.; HASBURN, C.; POVEDA, L.; SAN ROMÁN, L.; ARNASON. J. T. 1995. Insecticidal defenses of Piperaceae from the neotropics. Journal of Chemical Ecology 21: 801-814.         [ Links ]

BOBADILLA, M.; ZAVALA, F.; SISNIEGAS, M.; ZAVALETA,G.; MOSTACERO, J.; TARAMONA, L. 2005. Evaluación larvicida de suspensiones acuosas de Annona muricata Linnaeus "guanabana" sobre Aedes aegypti Linnaeus (Diptera, Culicidae). Revista Peruana de Biología 12: 145-152.         [ Links ]

BULLANGPOTI, V.; WAJNBERG, E.; AUDANT, P.; FEYEREI-SEN, R. 2012. Antifeedant activity of Jatropha gossipyfolia and Melia azedarach senescent leaf extracts on Spodoptera frugiperda (Lepidoptera: Noctuidae) and their potential use as synergists. Pest Management Science, in press.         [ Links ]

CLARK, P. L.; MOLINA-OCHOA, J.; MARTINELLI, S.; SKODA, S. R.; ISENHOUR, D. J.; LEE, D. J.; KRUMM, J. T.; FOSTER, J. E. 2007. Population variation of the fall armyworm, Spodoptera frugiperda, in the Western Hemisphere. Journal of Insect Science 7: 1-10.         [ Links ]

DANELUTTE, A. P.; COSTANTIN, M. B.; DELGADO, G. E.; BRAZ-FILHO, R.; KATO, M. J. 2005. Divergence of secondary metabolism in cell suspension cultures and differentiated plants of Piper cernuum and P. crassinervium. Journal of the Brazilian Chemical Society 16 (6B): 1425-1430.         [ Links ]

DA SILVA, R. V.; NAVICKIENE, H. M. D.; KATO, M. J.; BOLZANI, V. DA S.; MÉDA, C. I.; YOUNG, M. C. M.; FURLÁN, M. 2002. Antifungal amides from Piper arboreum and Piper tuberculatum. Phytochemistry 59: 521-527.         [ Links ]

DE SOUZA TAVARES, W.; CRUZ, I.; PETACCI, F.; DE ASSIS Jr., S. L.; DE SOUZA FREITAS, S.; ZANUNCIO, S. C.; SERRAO, J. E. 2009. Potential use of Asteraceae extracts to control Spodoptera frugiperda (Lepidoptera: Noctuida) and selectivity to their parasitoids Trichogramma pretiosum (Hymenoptera: Trichogrammidae) and Telenomus remus (Hymenoptera: Scelionidae). Industrial Crops and Products 30: 384-388.         [ Links ]

EDAHIRO, J-I.; SEKI, M. 2006. Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture. Journal of Bioscience and Bioengineering 102: 8-13.         [ Links ]

GBEWONYO, W. S. K.; CANDY, D. J.; ANDERSON, M. 1993. Structure-activity relationships of insecticidal amides from Piper guineense root. Pesticide Science 37: 57-66.         [ Links ]

GREGER, H. 1988. Comparative phytochemistry of the alkamides. pp. 159-178. In: Lam, J.; Breteler, H.; Arnason H.; Hansen, I. (Eds.). Chemistry and Biology of Naturally Occurring Acetylenes and Related Compounds, Bioactive Molecules. Vol. 7. Elsevier, Amsterdam.         [ Links ]

IVBIJARO, M. F.; BOLAJI, O. O. 1990. Effects of cypermethrin + dimethoate and extracts of P. guineense and Azadirachta indica on the pests and yield of cowpea, Vigna unguiculata. Journal Agriculture Science 115: 227-231.         [ Links ]

LEATEMIA, J.; ISMAN, B. 2004b. Toxicity and antifeedant activity of crude seed extracts of Annona squamosa (Annonaceae) against lepidopteran pests and natural enemies. International Journal Tropical Insect Science 24: 150-158.         [ Links ]

MARCHINI, L. C.; ALVES, S. B.; NAKANO, O. 1992. Sericultura. Curso de Entomología Aplicada à Agricultura. FEALQ, Piracicaba, Brazil, pp. 705-736.         [ Links ]

MARQUIS, R. J. 1991. Herbivore fauna of Piper (Piperaceae) in a Costa Rican wet forest: Diversity, specificity, and impact. pp. 179-208. In: Price, P. W.; Lewinsohn, T. M.; Fernandes, G. W.; Benson, W. W. (Eds.). Plant-Animal interactions: Evolutionary ecology in tropical and temperate regions. John Wiley and Sons, New York.         [ Links ]

MATSUI, K.; MUNSKATA, K. 1975. The structure of piperenone, a new insect antifeeding substance from Piperfutokadzura. Tetrahedron Letters 10: 1905-1908.         [ Links ]

MIYAKADO, M.; NAKAYAMA, I.; OHNO, N. 1989. Insecticidal unsaturated isobutylamides: From natural products to agro-chemical leads. pp. 183-187. In: Arnason, J. T.; Philogène, B. J. R.; Morand, P. (Eds.). Insecticides of plant origin. ACS Symposium Series 387, American Chemical Society, New York.         [ Links ]

MURASHIGE, T.; SKOOG, F. 1962. A revised medium for rapid growth an bioassays with tissue culture. Physiology Plantarum 15: 473-497.         [ Links ]

NAKATA, H.; HIRAKAWA, Y.; KAWAZO, M.; NAKABO, T.;ARIZONO, K.; ABE, S. I.; KITANO, T.; SHIMADA, H.; WATANABE, I.; LI, W.; DING, X. 2005. Concentrations and compositions of organochlorine contaminants in sediments, soils, crustaceans, fishes and birds collected from Lake Tai, Hangzhou Bay and Shangai city region. China Environmental Poly 133: 415-429.         [ Links ]

NAVICKIENE, H. M. D.; ALÉCIO, A. C.; KATO, M. J.; BOL-ZANI, V. DA S.; YOUNG, M. C. M.; CAVALHEIRO, A. J.; FURLAN, M. 2000. Antifungal amides from Piper hispidum and Piper tuberculatum. Phytochemistry 55: 621-626.         [ Links ]

NAVICKIENE, H. M. D.; MIRANDA, J. E.; BORTOLI, S. A.; KATO, M. J.; BOLZANI, V. DA S.; FURLAN, M. 2007. Toxicity of extracts and isobutyl amides from Piper tuberculatum: potent compounds with potential for the control of the velvetbean caterpillar, Anticarsia gemmatalis. Pest Management Science 63: 399-403.         [ Links ]

PALACIOS, Z. G. F.; DELGADO, G. E.; MORENO, M. C.; KATO, M. J.; ROJAS, C. 2009. Actividad antifúngica in vitro de extractos crudos de Piper tuberculatum. Revista Peruana de Biología 16: 209-214.         [ Links ]

PARMAR, B. S.; TOMAR, S. S. 1983. Review of research on insecticide synergists in India - retrospect and prospect. International Journal of Tropical Agricultural 1: 7-17.         [ Links ]

PARMAR, V. S.; JAIN, S. C.; BISHT, K. S.; JAIN, R.; TANEJA, P.; JHA, A.; TYAGI, O. D.; PRASAD, A. K.; WENGEL, J.; OL-SENl, C. E.; BOLL, P. M. 1997. Phytochemistry of the genus Piper. Phytochemistry 46: 597-673.         [ Links ]

PASHLEY, D. P. 1986. Host associated genetic differentiation in fall armyworm (Lepidoptera: Noctuidae): a sibling species complex?. Annals of the Entomological Society of America 79: 898904.         [ Links ]

PASHLEY, D. P.; JOHNSON, S. J.; SPARKS, A. N. 1985. Genetic population structure of migratory moths: the fall armyworm (Lepidoptera: Noctuidae). Annals of the Entomological Society of America 78: 756-762.         [ Links ]

PÉREZ-GUTIÉRREZ, S.; ZAVALA-SÁNCHEZ, M. A.; GONZÁLEZ-CHÁVEZ, M. M.; CÁRDENAS-ORTEGA, N. C.; RAMOS-LÓPEZ, M. A. 2011. Bioactivity of Carica papaya (Caricaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae). Molecules 16: 7502-7509.         [ Links ]

RAMOS-LÓPEZ, M. A.; PÉREZ, S.; RODRÍGUEZ-HERNÁNDEZ, C.; GUEVARA-FEFER, P.; ZAVALA-SÁNCHEZ, M. A. 2010. Activity of Ricinus communis (Euphorbiaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae). African Journal of Biotechnology 9: 1359-1365.         [ Links ]

RUBERTO, G.; RENDA, A.; TRINGALI, C.; NAPOLI, E. M.; SIMMONDS, M. S. J. 2002. Citrus limonoides and their semi-synthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study. Journal of Agricultural and Food Chemistry 50: 6766-6774.         [ Links ]

SCHULTES, R. F. 1980. De plantis toxicariis e mundo novo com-mentationes XXVI. Ethnopharmacological notes on the flora of northwestern South America. Botanical Museum Leaflet of Harvard University 28: 1-45.         [ Links ]

SCHULTES, R. F.; RAFFAUF, R. F. 1990. Medicinal and toxic plants of the Indians of northwest Amazonia, Piperaceae. pp. 362-368. In: Schultes, R. E.; Raffauf, R. F. (Eds.). The Healing forest: medicinal and toxic plants of the northwest Amazonia. Historical, Ethno- & Economic Botany Series. Vol. 2. Di-oscoride Press, Portland, Oregon.         [ Links ]

SCOTT, I. M.; JENSEN, H.; SCOTT, J. G.; ISMAN, M. B.; ARNASON, J. T.; PHILOGÉNE, B. J. R. 2003. Botanical insecticides for controlling agricultural pests: piperamides and the Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Archives of Insect Biochemistry and Physiology 54: 212-225.         [ Links ]

SOBERÓN, G. V.; ROJAS, C.; SAAVEDRA, J.; KATO, M. J.; DELGADO, G. E. 2006. Acción biocida de plantas de Piper tuberculatum Jacq. sobre Diatraea saccharalis (Lepidóptera, Pyralidae). Revista Peruana de Biologia 13: 107-112.         [ Links ]

SUKAMAR, K.; PERICH, M. J.; BOOBAR, L. R. 1991. Botanical derivatives in mosquito control: a review. The Journal of the American Mosquito Control Association 7: 210-237.         [ Links ]

TAGLIARI, M. S.; KNAAK, N; FIUZA, L. M. 2010. Efeito de extractos de plantas na mortalidade de lagartas de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae). Arquivos do Instituto Biológico de Säo Paulo. 77: 259-264.         [ Links ]

US Environmental Protection Agency (EPA). Probit program version 1.5. 2003. Ecological Monitoring Research Division. Environmental Monitoring Systems Laboratory. U.S. Environmental Protection Agency Cincinnati, OH. USA.         [ Links ]

VANISREE, M.; LEE, C. Y.; NALAWADE, S. M.; LIN, C. Y.; TSAY, H. S. 2004. Studies on the production of some important secondary metabolites from medicinal plants by tissue culture. Botanical Bulletin of Academia Sinica 45: 1-22.         [ Links ]

YUNCKER, T. G. 1973. The Piperaceae of Brazil II: Piper - Group V; Ottonia, Pothomorphe; Sarcorhachis. Hoehnea 3: 29-284.         [ Links ]