SHAIENE COSTA MORENO BIOACTIVITY OF NEOTROPICAL PLANT COMPOUNDS TO AGRICULTURAL AND VEGETABLE PESTS AND SELECTIVITY TO NON-TARGET INSECTS LAVRAS - MG 2011 SHAIENE COSTA MORENO BIOACTIVITY OF NEOTROPICAL PLANT COMPOUNDS TO AGRICULTURAL AND VEGETABLE PESTS AND SELECTIVITY TO NON-TARGET INSECTS Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós- Graduação em Agronomia/Entomologia, área de concentração em Entomologia Agrícola para a obtenção do título de Doutor. Orientador Dr. Geraldo Andrade Carvalho LAVRAS - MG 2011 Moreno, Shaiene Costa. Bioactivity of neotropical plant compounds to agricultural and vegetable pests and selectivity to non-target insects / Shaiene Costa Moreno. – Lavras : UFLA, 2011. 146 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2011. Orientador: Geraldo Andrade de Carvalho. Bibliografia. 1. Inseticidas botânicos. 2. Controle de pragas. 3. Metabólitos secundários de plantas. 4. Insetos-praga. 5. Pragas agrícolas. I. Universidade Federal de Lavras. II. Título. CDD – 632.7 Ficha Catalográfica Preparada pela Divisão de Processos Técnicos da Biblioteca da UFLA SHAIENE COSTA MORENO BIOACTIVITY OF NEOTROPICAL PLANT COMPOUNDS TO AGRICULTURAL AND VEGETABLE PESTS AND SELECTIVITY TO NON-TARGET INSECTS Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós- Graduação em Agronomia/Entomologia, área de concentração em Entomologia Agrícola para a obtenção do título de Doutor. APROVADA em 04 de abril de 2011. Dr. Marcelo Coutinho Picanço UFV Dr. Celso Omoto ESALQ Dr. Martin Francisco Pareja Piaggio UFLA Dr. Ronald Zanetti Bonetti Filho UFLA Dr. Geraldo Andrade Carvalho Orientador LAVRAS - MG 2011 A Deus, a quem devo a vida, pelo amor e bênçãos concedidas. Agradeço Aos meus pais, Wagner e Maria Amélia, pelo apoio em todos os momentos; Aos meus irmãos, Shenia, Sarah e Wagner, pela amizade e alegria; A minha amiga e irmã Clarissa, que mesmo longe está sempre presente; Ao meu esposo, Diogo, pelo amor e companheirismo; Aos meus avós, pelo amor e carinho. Dedico Ao povo brasileiro e aos cientistas. Ofereço AGRADECIMENTOS A DEUS, pela vida, saúde, amor e por me acompanhar todos os dias da minha vida. À Universidade Federal de Lavras e ao Programa de Pós-Graduação em Agronomia/ Entomologia, pela oportunidade de realização deste curso. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela concessão da bolsa de estudo. Ao orientador e querido amigo, professor Geraldo Andrade de Carvalho, pela amizade, pelos ensinamentos e pelo estímulo ao longo desses anos. À sua esposa Ana Paula e ao seu filho Vinícius, pelo agradável convívio. Ao querido co-orientador, professor Marcelo Coutinho Picanço, pela amizade, apoio e ensinamentos, que tanto contribuíram para minha formação. Agradeço pelos exemplos diários de dedicação e amor à pesquisa. À sua esposa Kátia e aos seus filhos Mayara, Luíza e Marcelo Filho, pelo agradável convívio. Aos professores Celso Omoto, Martin Francisco Pareja Piaggio e Ronald Zanetti Bonetti Filho, componentes da banca, pela cordialidade em aceitar o convite e pela forma como participaram. Ao professor Raul Narciso Carvalho Guedes pela valiosa contribuição no planejamento dos experimentos. Ao Dr. Márcio Dionízio Moreira, pela grande ajuda na seleção e coleta de plantas, sem o qual seria impossível a realização desse trabalho. Ao professor Lúcio Antônio de Oliveira Campos, pelo fornecimento de adultos da abelha jataí e ao Sr. Geraldo, funcionário do apiário da UFV, pela ajuda nas coletas. Aos todos os professores que me acompanharam e me incentivaram e que foram os responsáveis pela minha formação. Aos amigos do laboratório de Seletividade, Andrea, Dejane, Rodrigo, Letícia, Olinto, Stephan, Jader, Jander e Valéria pela amizade, companheirismo e convivência. Aos amigos do Laboratório de Manejo Integrado de Pragas pela grande amizade, convívio e companheirismo ao longo da minha vida acadêmica em Viçosa. Em especial, gostaria de expressar minha gratidão ao Eliseu, Elisângela, Jorgiane, Rogério, Silvério e Vânia pela ajuda na condução e avaliação dos bioensáios. Ao meu esposo, Diogo Carvalho de Gouvêa, pelo amor, amizade, companheirismo, apoio, confiança e paciência demonstrada ao longo desses anos de convivência. A todos os meus familiares, que diretamente ou indiretamente ofereceram condições para que eu progredisse na minha caminhada. Em especial, aos meus pais Wagner da Silva Moreno e Maria Amélia de Silva Moreno, que me deram a vida e souberam me conduzir para que tivesse uma boa educação. A todos os colegas dos cursos de Entomologia e Agronomia pelo agradável convívio durante as disciplinas cursadas e pela relação de amizade, entretenimento e divergência de idéias que fazem da Universidade um ambiente propício à formação profissional e intelectual. E finalmente, a todos aqueles que, de alguma forma, contribuíram para a execução deste trabalho, os meus sinceros agradecimentos. RESUMO A demanda por novos produtos para o manejo de pragas é crescente. Os riscos ambientais do uso indiscriminado de pesticidas sintéticos para controle de pragas agrícolas são evidentes e vêm sendo amplamente discutidos. Consequentemente, os pesticidas naturais, especialmente os de origem vegetal, são considerados alternativas promissoras. Este trabalho teve como objetivo avaliar o efeito de plantas sobre importantes pragas agrícolas e alguns insetos não-alvo. Inicialmente realizou-se uma seleção de plantas bioativas, avaliando- se extratos hexânicos e etanólicos de 23 plantas. O extrato hexânico da planta Acmella oleracea (L.) R.K. Jansen foi o que apresentou maior atividade inseticida, sendo selecionado para isolamento e identificação de compostos bioativos. Foram isolados três alkamidas do extrato hêxanico de A. oleracea: spilanthol, (E)-N-isobutylundeca-2-en-8,10-diynamide and (R,E)-N-(2- methylbutyl)undeca-2-en-8,10-diynamide. Avaliou-se então a atividade inseticida desses compostos sobre Tuta absoluta Meyr. (Lepidoptera: Gelechiidae), Ascia monuste Latr. (Lepidoptera: Pieridae), Diaphania hyalinata L. (Lepidoptera: Crambidae), e Plutella xylostella L. (Lepidoptera: Plutellidae), e a seletividade sobre o predador Solenopsis saevissima Smith (Hymenoptera: Formicidae) e sobre o polinizador Tetragonisca angustula Latr. (Hymenoptera: Apidae: Meliponinae). Os efeitos de extratos e compostos presentes em A. oleracea também foram avaliados sobre os pulgões Myzus persicae Sulz. e Lipaphis erysimi Kalt. (Hemiptera: Aphididae), e sobre o parasitóide Diaeretiella rapae McIntosh (Hymenoptera: Braconidae) e o predador Orius insidiosus Say (Hemiptera: Anthocoridae). O extrato de A. oleracea e as alkamidas avaliadas aprentaram elevada atividade inseticida sobre os insetos pragas e foram seletivos aos insetos não-alvo. Também foi objetivo deste trabalho avaliar o efeito letal e comportamental de extratos de plantas sobre operários das formigas cortadeiras Atta sexdens rubropilosa Forel, Atta laevigata Smith e Acromyrmex subterraneus molestans Santschi (Hymenoptera: Formicidae). Todos os extratos testados apresentaram efeito inseticida sobre as formigas e o extrato de A. oleracea foi o mais tóxico para todas as espécies, além de não apresentar efeito no comportamento de caminhamento das formigas. Palavras-chave: Inseticidas botânicos. Metabólitos secundários de plantas. Controle de pragas. ABSTRACT The demand for new products to control pests is growing. The number of environmental issues stemming from the use of synthetic pesticides to control agricultural pests is also increasing. Accordingly, natural pesticides, particularly those of plant origin, are now considered to be promising alternatives. This study aimed to evaluate the effects of plants on important agricultural pests and several non-target insects. An initial bioassay screening with hexane and ethanol extracts from 23 plants was performed. The hexane extract from Acmella oleracea (L.) R.K. Jansen exhibited the highest activity of all extracts, and the structure of its bioactive compounds was identified. The following three alkamides were isolated from the hexane extract of the aerial parts of A. oleracea: spilanthol, (E)-N-isobutylundeca-2-en-8,10-diynamide and (R,E)-N- (2-methylbutyl)undeca-2-en-8,10-diynamide. We analyzed the insecticidal activity of these compounds on Tuta absoluta Meyr. (Lepidoptera: Gelechiidae), Ascia monuste Latr. (Lepidoptera: Pieridae), Diaphania hyalinata L. (Lepidoptera: Crambidae), Plutella xylostella L. (Lepidoptera: Plutellidae), the predator Solenopsis saevissima Smith (Hymenoptera: Formicidae) and the pollinator Tetragonisca angustula Latr. (Hymenoptera: Apidae: Meliponinae). We also evaluated the effects of extracts and compounds present in A. oleracea on the aphids Myzus persicae (Sulz.) and Lipaphis erysimi (Kalt.) (Hemiptera: Aphididae), the aphid parasitoid Diaeretiella rapae McIntosh (Hymenoptera: Braconidae), and the predator Orius insidiosus (Say) (Hemiptera: Anthocoridae). The extracts and alkamides of A. oleracea showed high insecticidal activity against pest insects and were selective to non-target insects. Another objective of this research was to assess the lethal and behavioral effects of plant extracts on the leaf-cutting ants Atta sexdens rubropilosa Forel, Atta laevigata Smith and Acromyrmex subterraneus molestans Santschi (Hymenoptera: Formicidae). All extracts showed some insecticidal effect on the ants, and the A. oleracea extract was the most toxic to all ant species studied. No extract affected the walking behavior of the ants. Keywords: Botanical insecticides. Plant secondary metabolites. Pest control. SUMARY PART ONE .............................................................................................. 10 1 GENERAL INTRODUCTION .............................................................. 10 2 THEORETICAL BACKGROUND....................................................... 14 2.1 Plant secondary metabolism: a source of bioactive compounds ......... 14 2.2 The classes of secondary metabolites..................................................... 16 2.3 Chemical ecology..................................................................................... 17 2.4 Botanical insecticides .............................................................................. 19 3 CONCLUSION ....................................................................................... 30 REFERENCES........................................................................................ 31 PART TWO – ARTICLES..................................................................... 39 ARTICLE 1 Bioactivity of compounds from from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species....................................................... 39 ARTICLE 2 Efficacy of compounds from Acmella oleracea against lepidopteran vegetable pests and selectivity to beneficial species ....... 69 ARTICLE 3 Lethal and behavioral effects of amazon plant extracts on leaf-cutting ant workers .................................................................. 100 ARTICLE 4 Effects of Acmella oleracea extracts on the aphids Myzus persicae and Lipaphis erysimi and on two natural enemies ............... 119 10 PART ONE 1 GENERAL INTRODUCTION A variety of factors affect crop production. Insect pests account for approximately 30% of agricultural production losses. To avoid such losses, producers use control methods including behavioral, biological, genetic, cultural and chemical approaches. Among these methods, the application of insecticides is the most common, and it offers distinct advantages, such as high efficiency, low costs and ease of use (SODERLUND, 1995). Botanical insecticides are one type of insecticide used in pest control. The use of botanical pesticides is not new and dates back to ancient times when it was one of the main methods of pest control. However, after the Second World War, the advent of synthetic organic pesticides and the emergence of molecules such as HCH, DDT, aldrin, dieldrin and chlordane (LAGUNES; RODRÍGUEZ, 1992; VIEGAS JR., 2003) quickly led to the replacement of botanical compounds (CASIDA; QUISTAD, 1998; FLINT; VAN DEN BOSCH, 1981; THACKER, 2002). Now, the excessive use of these synthetic products is seriously affecting the environment. In addition to occupational, food and public health hazards, the indiscriminate use of insecticides can reduce the population of beneficial insects, contribute to the resurgence and outbreak of pests and decrease the effectiveness of insecticides through the selection of resistant populations (CAMPANHOLA, 1990; GUEDES, 1999; KAY; COLLINS, 1987; MALTBY, 1999; SIQUEIRA, 2000). Although synthetic insecticides (e.g., chlorinated hydrocarbons, organophosphates and pyrethroids) have been an important part of pest management strategies for years, their disadvantages and risks are now apparent. As a result, there is a continuous search for less hazardous alternatives to 11 conventional synthetic insecticides (MARICONI, 1981; VIEIRA et al., 2001). Ideally, insecticides should reduce pest populations, be target-specific (i.e., kill pests but not other organisms), break down quickly and have a low level of toxicity for humans and other mammals. Studying natural products of plant origin can help to discover more specific and less persistent pesticides. Plants, as organisms that co-evolved with insects, are natural sources of insecticidal substances. These compounds are products of secondary metabolism and are likely related to defense mechanisms (CATEHOUSE, 2002; MANN, 1995). Plant secondary metabolites play an important role in plant–insect interactions, and therefore, such compounds may have insecticidal, hormonal or antifeedant effects on insects. Natural pest control products are advantageous because they are not persistent in the environment, rapidly control pests and have a low level of toxicity for natural enemies and humans. However, the biological activity of a compound cannot be associated only with the mortality that it causes. Sublethal effects, which are not usually considered, may be equally or more important than lethal effects. Most investigations are based on the acute lethal action triggered by compounds, but the effects of natural products on insects are variable and may be toxic, repellent, cause sterility, modify behavior and development, or reduce feeding activity (ARNASON et al., 1990; BELL et al., 1990). Assessments of population growth, reproductive capacity and behavioral parameters need to be further evaluated to determine the biological activity of compounds that have the potential for use in insect pest management. Extreme cases, such as leaf-cutting ants, deserve mention. The use of bait to control ants is based on a behavioral response in which worker ants carry the bait to the nest, where it then expresses its control potential. As mentioned, concern about the use of synthetic insecticides has stimulated interest in the study of plants that possess insecticidal properties. 12 Plants such as Azadirachta indica, Trichilia pallida, Melia azadarach, Chrysanthemum cinerariaefolium, Chrysanthemum cineum, Lonchocarpus spp., Derris spp., Schoenocaulon officinale, Ryania speciosa, Nicotiana tabacum, Citrus spp., Piper spp., Allium sativum, Eucaliptus citriodora, Lycopersicon spp. and Manihot esculenta have been identified as toxic to insect pests. (NAIR, 1994; ISMAN, 2006). In addition to these identified plants, various plants are anecdotally known to have insecticidal properties, and these are important in the search for products with biological activity. However, these plants must be further studied to confirm and characterize their effects. Brazilian biomes have a great potential to provide a source of natural compounds with pesticidal properties because of their abundance and diversity of plant species. However, much remains to be discovered about this resource. It has been estimated that the chemical compositions of only 8% of Brazilian plants have been studied, and the number of plants that have had their biological properties characterized is small (SIMÕES, 2003). Meanwhile, several animal and plant species have become extinct. Further, flora and fauna are constantly smuggled by transnational organized crime networks for research purposes and to create patented goods. These factors increase the need to conduct research at sites of origin, especially on the pesticidal aspects of plants, which are not analyzed when research is focused on developing drugs. In this context, studies that aim to select plants that contain large amounts of compounds with insecticidal properties are extremely important to the management of pests. Additionally, the toxicity of new substances, their effects on the biology and behavior of insects, and their selectivity to natural enemies should be studied. This study was undertaken with the following objectives: i) to screen plants with insecticide activity against important agricultural pests; ii) to 13 evaluate the biological activity of compounds present in Acmella oleracea (L.) R.K. Jansen, the plant selected in the bioassay screening process, against Tuta absoluta Meyr. (Lepidoptera: Gelechiidae), Ascia monuste Latr. (Lepidoptera: Pieridae), Diaphania hyalinata L. (Lepidoptera: Crambidae), and Plutella xylostella L. (Lepidoptera: Plutellidae), and to evaluate the selectivity of these compounds to the predator Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) and to the pollinator Tetragonisca angustula (Latreille) (Hymenoptera: Apidae: Meliponinae); iii) to assess the effects of extracts of Amazonian plants on the survival of the leaf-cutting ants Atta sexdens rubropilosa Forel, Atta laevigata Smith and Acromyrmex subterraneus molestans Santschi (Hymenoptera: Formicidae) and to assess their effect on the mobility of these species; and iv) to evaluate the toxicity of A. oleracea extracts in the aphids Myzus persicae Sulz. and Lipaphis erysimi Kalt. (Hemiptera: Aphididae), the aphid parasitoid Diaeretiella rapae McIntosh (Hymenoptera: Braconidae), and the predator Orius insidiosus Say (Hemiptera: Anthocoridae). 14 2 THEORETICAL BACKGROUND 2.1 Plant secondary metabolism: a source of bioactive compounds The beneficial actions of plant materials typically result from combinations of secondary products in the plant. Multiple chemical compounds often act together through additive or synergistic action at single or multiple target sites associated with a physiological process (BRISKIN, 2000). The bioactivity of plants is unique to particular plant species or groups, according to the concept that combinations of secondary products in a particular plant species are often taxonomically distinct (DIXON, 1999; WINK, 1999). This is in contrast to primary products, such as carbohydrates, lipids, proteins, heme chlorophyll, and nucleic acids, that are common to all plant species and are involved in the primary metabolic processes of building and maintaining plant cells (KAUFMAN et al., 1999; WINK, 1999). Although secondary products have historically been defined as chemicals that do not appear to have a vital biochemical role in the process of building and maintaining plant cells, current research has shown that these chemicals play a pivotal role in the ecophysiology of plants. In this respect, secondary products can have a defensive role against herbivory, pathogen attack, and interplant competition or an attractant role toward beneficial organisms such as pollinators or symbionts (DIXON, 1999; KAUFMAN et al., 1999; WINK; SCHIMMER, 1999). Plant secondary products can also exhibit protective properties in relation to abiotic stresses such as those associated with changes in temperature, water status, light levels, ultraviolet (UV) exposure, and mineral nutrients. Furthermore, recent research has indicated potential roles of secondary products at the cellular level as plant growth regulators and modulators of gene expression and in signal transduction (KAUFMAN et al., 1999). 15 To promote plant survival, the structures of secondary products have evolved to interact with molecular targets that affect the cells, tissues, and physiological functions of other competing organisms. In this respect, some plant secondary products may resemble endogenous metabolites, ligands, hormones, signal transduction molecules, or neurotransmitters (e.g., those of the central nervous system or endocrine system) (DIXON, 1999; KAUFMAN et al., 1999). Wink (1999) referred to this development of structural similarity between plant secondary products and the endogenous substances of other organisms as “evolutionary molecular modeling”. Secondary metabolites are numerous and widespread, especially in higher plants. The total number of plant secondary metabolites for which structures have been elucidated is around 50,000, and this is likely only a small fraction of all plant secondary metabolites. Because fewer than 20% of all plants have been studied in depth, it is likely that the number of secondary metabolites in the plant kingdom exceeds 100,000 (SCHWAB, 2003; WINK, 2007). Toxic secondary metabolites are present in plants at low concentrations, generally less than 2% of dry matter. The amount of secondary compounds in an organism is the result of an equilibrium among synthesis, storage, and degradation. Regulation of secondary metabolism is complex. The onset of secondary metabolism is linked to the developmental stage of an organism and, often, to morphological and cytological changes (MAKKAR; SIDDHURAJU; BECKER, 2007). The chemical structures of secondary plant products are more complex than those of primary products. This is partially explained by the fact that many, though not all, secondary products are derived from amino acids or nucleotides. Most of the compounds found in plants belong to a limited number of families of substances. Minor chemical modifications, such as methylations, 16 hydroxylations, and intercalations with metal ions, produce a wide spectrum of functionally distinct substances (SEIGLER, 2002). Interest in plant secondary metabolites has increased dramatically in recent years because of their diverse effects, including antioxidant, antiviral, antibacterial, anticancer and pesticidal effects. The search for new active compounds has been termed bioprospecting, the search for biological gold. Understanding the physiology, biochemistry, and ecology of secondary metabolism is essential to exploit bioactive plant chemicals in a rational way in medicine and agriculture. 2.2 The classes of secondary metabolites Although the structures of secondary metabolites may seem to be bewilderingly diverse, the majority of these compounds belong to one of a limited number of families, each of which has particular structural characteristics based on the ways in which they are biosynthesized (HANSON, 2003). Secondary metabolites can be divided into two groups: those without nitrogen and those with nitrogen in their structures (WINK, 2007). Nitrogen- containing compounds include alkaloids, amines, nonprotein amino acids, cyanogenic glycosides, glucosinolates, alkamides, protease inhibitor peptides and lectins. Nitrogen-free compounds include terpenoids (e.g., mono-, sesqui-, di-, tri- and tetraterpenes), polyketides, phenolics (e.g., flavonoids, tannins and lignans), and polyacetylenes. Currently, there are 17 classes of secondary metabolites. Table 1 gives an overview of the known classes and number of structures that belong to each class. 17 Table 1 Structural types of secondary metabolites and known structures. Class Number of structures With nitrogen Alkaloids 29,000 Non-protein amino acids 700 Amines 100 Cyanogenic glycosides 60 Glucosinolates 100 Alkamides 150 Lectins, peptides 800 Without nitrogen Monoterpenes (including iridoids) 2,500 Sesquiterpenes 5,000 Diterpenes 2,500 Triterpenes, steroids, saponins 5,000 Tetraterpenes 500 Phenylpropanoids, coumarins, lignans 2,000 Flavonoids, tannins 4,000 Polyacetylenes, fatty acids, waxes 1,500 Polyketides (anthraquinones) 750 Carbohydrates 200 Source: Adapted from Wink (2007). 2.3 Chemical ecology Many plant secondary metabolites were originally investigated because of their value as medicines, perfumes or foods. However, beginning in the latter part of the 20th century, an increasing amount of attention has been directed at the biological functions of natural products and their ecological roles in regulating interactions between organisms. Developments in instrumental methods have enabled the detection and identification of very small amounts of materials as well as the observation of their effects, particularly on insects. 18 Natural products often have an ecological role in regulating the interactions between plants, microorganisms, insects and animals. Secondary metabolites can protect plants against herbivores, microbes, or competing plants. Some secondary metabolites also function as signal compounds to attract pollinating or seed-dispersing animals. Because of their ecological role, secondary plant substances can be classified as ‘allelochemics’, which are defined as ‘non-nutritional chemicals produced by an individual of one species that affect the growth, health, behavior, or population biology of another species (WHITTAKER, 1970). Several plant-insect relationships are determined by the presence of secondary metabolites. The production of chemicals that are capable of deterring insect pests by toxic activity is an important survival strategy for plants. These compounds may be either deterrents or attractants. Plants have chemical defense systems to avoid attack from phytophagous insects, and plant products can act as insecticides, repellents and antifeedants against insect. Structurally, such toxins are usually non-volatile compounds as a result of their molecular weight or hydrophilicity. If they accumulate in the tissue of healthy plants prior to insect attack, they are considered to be constitutive (STAMP, 2003; WITTSTOCK; GERSHENZON, 2002). Alternatively, they may only be present, or present in much higher concentrations, after plants have encountered attack or after exposure to natural plant- or insect-derived defense activators. In this case, they are considered to be induced toxicants (WALLING, 2000). Induction provides economic advantages to the plant because metabolic energy is diverted from primary metabolism to produce toxins. In addition, insect herbivores are less likely to develop resistance to induced defense products because they are not frequently exposed to them. However, the balance between these two strategies may depend on the likelihood that a plant will come under attack. Plants that encounter more frequent attacks by pests may be 19 forced to rely more heavily on constitutive rather than induced defenses, despite the greater energetic cost to the plant (MCKEY, 1979). The distribution of defense metabolites is often restricted both spatially and temporally, and plant organs associated with survival or reproduction tend to contain the highest concentrations of constitutive defense metabolites (WITTSTOCK; GERSHENZON, 2002). They may be developmentally regulated, being present at the highest concentrations when the plant is young and less able to protect itself against predators, or they may be concentrated around the region of contact with the invader. 2.4 Botanical insecticides Botanical insecticides, occasionally referred to as “botanicals”, are derived from plants. Many botanical insecticides have been used for hundreds of years but have recently been displaced by synthetic insecticides. The use of botanical insecticides in agriculture in China, Egypt, Greece, and India dates back at least two millennia (THACKER, 2002; WARE, 1883). Nicotine (from Nicotiana tabacum), piretrins (from Tanacetum cinerariifolium) and rotenone (from Derris and Lonchocarpus) are examples of plant compounds that were used long ago to control agricultural pests. In Europe and North America, the documented use of botanicals extends back more than 150 years, predating discoveries of the major classes of synthetic chemical insecticides (e.g., organochlorines, organophosphates, carbamates, and pyrethroids) in the mid- 1930s to 1950s. Synthetic insecticides have effectively displaced botanicals from their important role in agriculture and relegated them to an essentially trivial position in the marketplace of crop protectants. However, recent history shows that the overzealous use of synthetic insecticides has led to numerous problems unforeseen at the time of their introduction: the disruption of natural biological 20 control and pollination; the acute and chronic poisoning of applicators, farm workers, and even consumers; the destruction of fish, birds, and other wildlife; extensive groundwater contamination that is potentially threatening to human and environmental health; and the evolution of resistance to pesticides in pest populations. In response to these events, many countries have reassessed the risks of using synthetic insecticides and banned products from use in agriculture, especially those developed before 1980. Consequently, there is an increased impetus to discover and develop alternative pest management products, including insecticides derived from plants. 2.4.1 Chemical synergisms in the ecological function of secondary products and the benefits of botanical insecticides In contrast to synthetic insecticides based on single chemicals, many phytochemicals exert their toxic effects through several chemical compounds that act additively or synergistically at single or multiple target sites associated with a physiological process (PESSARAKLI, 2001). This synergistic or additive effect can promote pesticidal effectiveness without the problematic effects associated with the predominance of a single xenobiotic compound. For example, with the use of multiple compounds, insects are less likely to develop resistance to insecticides. The additive or synergistic interaction of multiple chemicals probably originated in the functional role of secondary products in promoting plant survival (WINK; SCHIMMER, 1999). The additive or synergistic effects of a mixture of chemicals at multiple target sites not only ensures effectiveness against a wide range of herbivores or pathogens but also decreases the chance 21 that these organisms will develop resistance or adaptive responses (KAUFMAN, 1999; WINK, 1999). 2.4.2 The potential of using botanical insecticides for the control of agricultural pests The use of insecticides against pest insects is one of the main pest management tools available in agriculture (COOPER; DOBSON, 2007; EDWARDS-JONES, 2008), but attitudes and behaviors regarding the use of these compounds are steadily changing as safety demands increase (MATSUMURA, 2004; MATTHEWS, 2008). New compounds have been developed to answer such demands (NAUEN; BRETSCHNEIDER, 2002; NICHOLSON, 2007), but the concern remains about their overuse and their effects as pollutants. Among the newly developed and used pesticides, biopesticides or biorational pesticides have received a considerable amount of attention (ISMAN, 2006; ROSELL et al., 2008). Natural products have had and continue to have value as components of crop protection strategies, both as per se insecticides and as chemical backbones for the synthesis of new insecticidal molecules (COATS, 1994; KIDD, 2000). Phytochemicals are an attractive alternative to the currently used synthetic insecticides because they constitute a rich source of bioactive molecules (WINK, 1993). They are usually active against a limited number of specific target pests, biodegrade into non-toxic compounds, and are, therefore, potentially useful in integrated pest management programs. Accordingly, recent efforts have been directed toward the discovery of secondary metabolites that could be used as commercial insecticides or lead compounds (GULERIA; TIKU, 2009). 22 Overall, botanical insecticides can be used in the following ways to control pests: - As a powder product prepared “in natura” and as aqueous or alcoholic extracts (GUERRA, 1985; SANTOS et al., 1988). - In commercial and semi-commercial concentrated formulations (MORDUE; BLACKELL, 1993). - As purified and isolated pure compounds obtained from plant extracts. (NAIR, 1994). - As a source of molecules for the synthesis of novel agrochemicals with desirable characteristics (CUTLER, 1988; HEDIN et al., 1994; ISMAN, 1989; NAIR, 1994). - Incorporated into the genetic material of crops by genetic engineering, thus minimizing the damage caused by insect pests, microorganisms and weeds (CUTLER, 1988; ELANOVICH, 1988). 2.4.3 Advantages of botanical insecticides Many compounds with diverse chemical structures and different modes of action are classified as botanical insecticides. Therefore, presenting a detailed list of advantages or disadvantages that apply to all compounds in this category is difficult. General advantages shared by most of these compounds include the following (CLOYD, 2004; WEINZIERL; HENN, 1994): - Rapid degradation. Botanicals and insecticidal soaps degrade rapidly in sunlight, air, and moisture and are readily broken down by detoxification enzymes. This is important because rapid breakdown means less persistence in the environment and reduced risks to nontarget organisms. Many botanicals may be applied to food crops shortly before harvest without leaving excessive residues. 23 - Rapid action. Botanicals and soaps act quickly to stop feeding by pest insects. Although they may not cause death for hours or days, they often cause immediate paralysis or cessation of feeding. - Low mammalian toxicity. Most botanicals and insecticidal soaps have low to moderate mammalian toxicity. - Selectivity. Although most botanicals have a broad spectrum of activity in standard laboratory tests, in the field, their rapid degradation and the action of some as stomach poisons makes them more selective in some instances for plant-feeding pest insects and less harmful to beneficial insects. - Low toxicity to plants. Most botanicals are not phytotoxic (toxic to plants). Insecticidal soaps and nicotine sulfate, however, may be toxic to some ornamentals. 2.4.4 Disadvantages of botanical insecticides Natural insecticides are generally less stable than synthetic materials and degrade quickly in the environment, meaning that they are also less potent and have shorter residual periods than their synthetic counterparts (KÜHNE, 2008). Therefore, satisfactory arthropod pest management can only be achieved if insecticide use is integrated with other strategies, including the timing of applications to minimize harmful effects on beneficial organisms. One obstacle to the commercialization of new insecticides made of natural substances is the requirement of a large marketing base to cover the high costs associated with marketing approval (KÜHNE, 2008). Botanicals also tend to be more expensive than synthetics, and some are not as widely available. Furthermore, there are three major barriers to the commercialization of botanical insecticides: the sustainability of the botanical resource, the standardization of chemically complex extracts, and regulatory approval. Other drawbacks or 24 limitations include the slowness of their action and the lack of residual action for most botanicals (ISMAN, 2006). 2.4.5 Current botanical insecticides in use Currently, there are three major types of botanical insecticides used for pest control, pyrethrins, rotenone and azadiractins, and three others in limited use, ryania, nicotine, and sabadilla. Other plant extracts and oils (e.g., garlic oil and Capsicum oleoresin) have limited regional uses in various countries (ISMAN, 2006). Pyrethrins Pyrethrins refer to the insecticidal compounds that occur in pyrethrum. Pyrethrum is an oleoresin extracted from the dried flowers of the pyrethrum daisy, Tanacetum cinerariaefolium (Asteraceae), and is considered as the archetypical natural insecticide (GLYNNE-JONES, 2001). Pyrethrins include three esters of chrysanthemic acid and three esters of pyrethric acid. Among the six esters, those incorporating the alcohol pyrethrolone, namely pyrethrins I and II, are the most abundant and account for most of the compound's insecticidal activity. Most insects are highly susceptible to low concentrations of pyrethrins. Pyrethrins are extremely fast acting, and their insecticidal action is characterized by a rapid knockdown effect, hyperactivity and convulsions. These symptoms are a result of the neurotoxic action of the pyrethrins, which block voltage-gated sodium channels in nerve axons. As such, the mechanism of action in pyrethrins is qualitatively similar to that of DDT and many synthetic organochlorine insecticides. Pyrethrins are especially labile in the presence of the UV component of sunlight, a fact that has greatly limited their use outdoors. They 25 are the predominant botanical in use, accounting for approximately 80% of the global botanical insecticide market (ISMAN, 2006). Pyrethrins are effective as broad spectrum insecticides and are used to control pests such as aphids, whiteflies, stinkbugs, and mites (COX, 2002). They are available as dusts, sprays, and aerosols and may be mixed with synthetic pesticides or other botanicals. Rotenone Rotenone is one of several isoflavonoids produced in the roots or rhizomes of the tropical legumes Derris, Lonchocarpus, and Tephrosia. As an insecticide, rotenone has been used for more than 150 years (SHEPARD, 1951; ISMAN, 2006). It is widely used in gardens and to a lesser extent on pets. Rotenone is a powerful inhibitor of cellular respiration, the process that converts nutrient compounds into energy at the cellular level. In insects, rotenone exerts its toxic effects primarily on nerve and muscle cells, causing rapid cessation of feeding (KLAASSEN; WATKINS, 2003; TADA-OIKAWA, 2003). Death occurs several hours to a few days after exposure. Rotenone is extremely toxic to fish, and is often used as a piscicide in water management programs. Rotenone is effective against a wide range of insects and has a short residual life. It is not toxic to honeybees, but it does kill some beneficial insects (WEINZIERL; HENN, 1994). It is registered for use against a number of chewing insects on many vegetables and some fruits. Azadirachtins The azadirachtins belong to a group of tetranortriterpenoids that exhibit a variety of biological activities. This class of chemicals, extracted from the seeds of the neem tree, Azadirachta indica (Meliaceae), has generated a considerable amount of excitement with respect to insect control and safety to 26 mammals. The numerous reported effects include repellency, feeding deterrency and interference with growth, development, and reproduction (GULERIA; TIKU, 2009). Research has shown that azadirachtins can control more than 400 species of insects, including many key crop pests, and has proven to be one of the most promising plant ingredients for integrated pest management. Azadirachtins effectively control common pests such as thrips, whiteflies, leaf folders, bollworms, aphids, jassids, pod borers, fruit borers, stem borers, leafhoppers and caterpillars (MARTINEZ; EMDEN, 2001). Ryania The stems and roots of the South American plant Ryania speciosa (Flacourtiaceae) yield the alkaloid ryania, which is much more stable than rotenone and nicotine but not as potent as other botanical insecticides (WARE, 1892). Ryanodine was originally isolated as the active principle, but eleven ryanoids have been identified with different insecticidal activities. The mechanism of action primarily affects the Ca2+ release channel in muscle, and ryanodine acts as a muscular poison by blocking the conversion of ADP to ATP in striated muscles (GULERIA; TIKU, 2009). The acute and chronic oral toxicity of ryania in mammals is moderate. It is generally not harmful to most natural enemies, but it may be toxic to certain predatory mites. The residual activity of ryania is longer than that of most other botanicals. It has been used commercially in fruit and vegetable production against caterpillars, including European corn borers and corn earworms, and thrips (WEINZIERL; HENN, 1994). 27 Nicotine Five different families of plants produce nicotinoids that are potent insecticides in situ or when extracted from the leaves. Tobacco, Nicotiana tabacum (Solanaceae), is the primary source, and its use has been widespread for more than a century. Nicotine constitutes between 2 and 8 percent of dried tobacco leaves. Insecticidal formulations generally contain nicotine in the form of 40 percent nicotine sulfate (ISMAN, 2006). In both insects and mammals, nicotine is an extremely fast-acting nerve toxin. It competes with acetylcholine, the major neurotransmitter, by bonding to acetylcholine receptors at nerve synapses and causing uncontrolled nerve firing. This disruption of normal nerve impulse activity results in the rapid failure of body systems that depend on nervous input for proper functioning. In insects, the action of nicotine is fairly selective, and only certain types of insects are affected. It is used in greenhouses and gardens as a fumigant and contact poison to control soft-bodied sucking pests such as aphids, thrips, and mites (GULERIA; TIKU, 2009). Despite the fact that smokers regularly inhale small quantities of nicotine in tobacco smoke, nicotine in pure form is extremely toxic to mammals and is considered a Class I (most dangerous) poison. Nicotine has been responsible for numerous poisonings and accidental deaths because of its rapid penetration of both skin and mucous membranes and because it is used in a concentrated form (WEINZIERL; HENN, 1994). Sabadilla Sabadilla is a botanical insecticide obtained from the seeds of the tropical lily Schoenocaulon officinale. When sabadilla seeds are aged, heated, or treated with alkali, several insecticidal alkaloids are formed or activated. Alkaloids are physiologically active compounds that occur naturally in many 28 plants. The alkaloids in sabadilla are known collectively as veratrine or as the veratrine alkaloids. The mode of action of these alkaloids is remarkably similar to that of the pyrethrins, despite their lack of structural similarity (DENAC, 2000; ISMAN, 2006; ZLOTKIN, 1999). Sabadilla is a broad-spectrum contact poison, but has some activity as a stomach poison. Baits, dusts or sprays may be used in organic fruit and vegetable production against squash bugs, harlequin bugs, thrips, caterpillars, leaf hoppers, and stink bugs. These alkaloids break down rapidly in sunlight and air, leaving no harmful residues. However, it is highly toxic to honeybees, and should only be used when bees are not present (ISMAN, 2006). 2.4.6 Prospects of botanical insecticides According to Isman (2006), in industrialized countries, it is difficult to imagine botanicals playing a more prominent role than they currently play, except in organic food production. In conventional agriculture, botanicals face tremendous competition from the newest generation of reduced risk synthetic insecticides, such as the neonicotinoids. The benefits of botanical insecticides can be best realized in developing countries, where farmers may not be able to afford synthetic insecticides and plants and plant derivatives are traditionally used for crop protection. Even if synthetic insecticides are affordable, limited literacy rates and a lack of protective equipment result in thousands of accidental poisonings annually (FORGET; GOODMAN; VILLIERS, 1993). Pest control efficacy is only one factor in the adoption of botanicals. The logistics of production, preparation, and use can affect the uptake of botanicals (MORSE et al., 2002). It may be time to refocus the attention of the research 29 community toward the development and application of known botanicals, while continuing to screen more plants and isolate novel bioactive substances. 30 3 CONCLUSION The crude hexane extract of A. oleracea showed high insecticidal activity and can be used to control T. absoluta, A. monuste, D. hyalinata and P. xylostela, in organic or conventional crops. Quantification of LD50 values of isolated compounds showed that alkamides could serve as potent insecticides for T. absoluta, A. monuste, D. hyalinata and P. xylostela control programs. The spilanthol was the main alkamide active isolated. This alkamide is the most promising as it had the highest insecticidal activity and was selective to non- target organisms. All extracts showed some insecticidal effect on the leaf-cutting ants A. sexdens rubropilosa, A. laevigata and A. subterraneus molestans, and the A. oleracea extract was the most toxic to all ant species studied. No extract affected the walking behavior of the ants. The results of this study demonstrate the substantial effects of ethanol extracts of A. oleracea against M. persicae and L. erysimi under laboratory conditions and verify the extract selectivity to natural enemies, suggesting its potential in controlling this insect pest under field conditions. 31 REFERENCES AHUJA, I.; ROHLOFF, J.; BONES, A. M. Defence mechanisms of Brassicaceae: implications for plant-insect interactions and potential for integrated pest management. A review. 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Annual Review of Entomology, Stanford, v. 44, p. 429-455, 1999. 39 PART TWO – ARTICLES ARTICLE 1 BIOACTIVITY OF COMPOUNDS FROM Acmella oleracea AGAINST Tuta absoluta (MEYRICK) (LEPIDOPTERA: GELECHIIDAE) AND SELECTIVITY TO TWO NON-TARGET SPECIES This article was written in accordance with the standards of Pest Management Science, for which it was submitted and accepted in March 2011. 40 Bioactivity of compounds from Acmella oleracea against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and selectivity to two non-target species Shaiene C Morenoa*, Geraldo A Carvalhoa, Marcelo C Picançob, Elisangela G F Moraisb, Rogério M Pereirab aDepartamento de Entomologia, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil bDepartamento de Biologia Animal, Universidade Federal de Viçosa, 36570- 000, Viçosa, MG, Brazil *Corresponding author. Departamento de Entomologia, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil. Email: shaiene.moreno@ifrj.edu.br Abstract BACKGROUND: Tropical plants are recognized sources of bioactive compounds that can be used for pest control. Our objective was to evaluate the biological activity of compounds present in Acmella oleracea (L.) R.K. Jansen (Asteracea) against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), which is the main pest of tomato crops in Latin America. We were also interested in the selectivity of these compounds to the predator Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) and to the pollinator Tetragonisca angustula (Latreille) (Hymenoptera: Apidae: Meliponinae). RESULTS: A bioassay screening with hexane and ethanol extracts from 23 plants was performed. The hexane extract of A. oleracea was the most active of the extracts and was selected for further study. The following three alkamides were isolated from the hexane extract of the aerial parts of A. oleracea: spilanthol, undeca-2E-en-8,10-diynoic acid isobutylamide and (2E)-N-(2- 41 methylbutyl)-2-undecene-8,10-diynamide. All of the isolated compounds showed insecticidal activity with spilanthol being the most active (LD50 = 0.13 µg mg-1) against T. absoluta. The alkamides were selective to both beneficial species studied. CONCLUSION: The crude hexane extract of A. oleracea showed high insecticidal activity and can be used to control T. absoluta in organic or conventional crops. Quantification of LD50 values of isolated compounds against T. absoluta showed that alkamides could serve as potent insecticides for T. absoluta control programs. The spilanthol was the main alkamide active isolated. This alkamide is the most promising as it had the highest insecticidal activity and was selective to non-target organisms. Keywords: botanical pesticide; insect control; secondary metabolites; bioactive alkamides, tomato leafminer 1 INTRODUCTION The tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a neotropical oligophagous insect that attacks solanaceous crops.1 Since the 1960s, it has become one of the key pests of tomato crops in most South American countries.2 Recently, T. absoluta has also become a serious threat to the tomato production in the Mediterranean region.3 Following the detection in the Spanish tomato-growing area at the end of 2006, T. absoluta spread quickly to other European and northern African countries.4,5 The larvae attack tomato plants during all growth stages, producing large galleries in their leaves, burrowing stalks, apical buds, green and ripe fruits. Chemical control has been the main method of control used against T. absoluta. Horticultural growers have attempted to decrease the damage caused by T. absoluta by applying insecticides more than two times a week during a 42 single cultivation period.2 T. absoluta control is a challenge due to the nature of the damage it causes and its rapid ability to develop resistance toward conventional insecticides.1,6 Thus, there is an urgent need to develop safe alternatives to conventional insecticides for the protection of tomato plants against T. absoluta. The use of eco-friendly and easily biodegradable plant products with natural insecticidal activity has increased in recent years. To control pests without disturbing the environment, natural products have been screened for potential sources of insecticides. Plant materials with insecticidal properties have been used to kill insects throughout the world for generations. These plants are considered an alternative to conventional pesticides because of their low toxicity to warm-blooded mammals as well as their high volatility. Botanical insecticides may be safer for the environment than synthetic insecticides, and they are usually easily processed and used by farmers and small industries.7 Tropical plants are recognized sources of bioactive compounds, but less than 1% have been chemically investigated.8 They can be used for pest control as plant extracts, horticultural oils or as a source of molecules for pesticide synthesis such as pyrethroids and neonicotinoids. Acmella oleracea (L.) R.K. Jansen is an annual plant of the family Asteraceae (Compositae) originating in tropics of Brazil. The distribution covers tropical and subtropical areas around the world, and it is known in English as toothache plant or paracress and in Portuguese as jambú.9 The inflorescence is composed of yellow flowers, and the leaves have a pungent flavor accompanied by tingling and numbness. The plant has been used in cooking and in popular medicine, mainly for stammering, toothache, stomatitis and throat complaints. The plant contains alkamides including spilanthol, which is the principal pungent compound. This chemical compound is known for having several chemical and pharmaceutical applications. It has shown anti-inflammatory, 43 antibacterial, antifungal, diuretic, sialagogic and larvicidal properties.9 The activity of A. oleracea has been studied extensively. However, only few studies have assessed the insecticidal activity of compounds from this plant. 10-13 Furthermore, the majority of these studies have focused on human health pests such as insect vectors of pathogens.10-13 These studies show that compounds of A. oleracea have high insecticidal activity and that the potential use of this plant species for management of agricultural pests requires further investigation. 10-13 New compounds should provide selectivity to non-targets species, especially predators and pollinators, in addition to efficiency against insect pests. Attack by natural enemies are the most frequent source of mortality for phytophagous arthropods in agroecosystems and the conservation of these organisms is an essential component in Integrated Pest Management (IPM).14 Furthermore, pollination is central for successful reproduction in most plants. Thus, pollinators should be preserved because they support the maintenance of biodiversity in the ecosystems they inhabit and because they are known as keystone species in many terrestrial habitats.15 In agroecosystems, there are many species that are part of these groups, including the predator Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) and the pollinator Tetragonisca angustula (Latreille) (Hymenoptera: Apidae: Meliponinae). The predation by S. saevissima has played an important role in reducing pest insects in agricultural systems. Way & Khoo16 cited species of the genus Solenopsis as important agents of biological control in the tropics and subtropics. T. angustula is one of the most common stingless bees in the neotropical region. Stingless bees are generalist foragers and are efficient native pollinators of the American flora.15 Considering the potential of tropical plant species for pest control and the importance of T. absoluta, the aims of this study were to screen plants with insecticide activity to T. absoluta. The goal was to isolate, identify and assess the bioactivity of insecticide compounds present in the bioactive plant against 44 this key insect pest of tomato crops. Furthermore, we wanted to investigate the selectivity of these compounds to the beneficial insects S. saevissima and T. angustula. 2 MATERIALS AND METHODS 2.1 Insects The bioassays were performed with second-instar larvae of T. absoluta and adults of S. saevissima and T. angustula. Larvae of T. absoluta were obtained from a laboratory rearing located at the campus of the Universidade Federal de Viçosa (UFV), Viçosa, Minas Gerais State, Brazil. Adults of S. saevissima and T. angustula were collected from nests located around the campus of the UFV. 2.2 Plant screening 2.2.1 Plant extracts preparation Table 1 describes the plants that were used for extraction and toxicity bioassays. The plants were chosen based on available literature, popular or indigenous knowledge and chemotaxonomy. The plant material was identified in the botanical park of the Federal University of Acre. Samples of 1.0 kg from the canopy of each plant species were collected in Rio Branco, AC, Brazil (plants of the Amazon Biome) and in Viçosa, MG, Brazil (plants of the Cerrado and of general occurrence). Each sample was lyophilized and the dried material was crushed and placed in a 2 L Erlenmeyer flask, with enough hexane to submerge the plant material. After 48 hours, the solvent was removed under filtration. Ethanol extraction was performed by grinding the samples with the solvent and waiting for 48 hours. The hexane and ethanol extracts were concentrated under low pressure and reduced temperature (45-50°C) using a rotary evaporator. The yield for each extract is shown in Table 1. The plant extracts were stored at low temperature for subsequent bioassays. 45 2.2.2 Screening bioassay A set of screening bioassays was performed to identify the bioactive plant extracts to T. absoluta. The stored extracts were diluted with acetone to a dose of 10 µg mg-1 body mass. The average weight was obtained by measuring the mass of ten groups containing ten insects each, on an analytical balance. The experimental design was completely randomized with six replications. Each experimental unit consisted of a glass petri dish (9.5 cm x 2.0 cm) containing ten insects. The bioassays were conducted by topical application. For each insect a 10 µl Hamilton micro syringe was used to add 0.5 µL of a solution of the test extract. In a control experiment under the same conditions, 0.5 µL of hexane was applied on each insect. After the application, the insects were kept in individual Petri dishes containing tomato leaflets (cv. Santa Clara) as food. The Petri dishes were placed in an incubator at 25 ± 0.5°C, 75 ± 5% relative humidity, with a photoperiod of 12 h. The mortality counts were made after 6, 12 and 24 hours of treatment. Mortality included dead individuals as well as those without movements. Mortality data were subjected to analysis of variance and the averages were compared by the Scott-Knott grouping analysis test (P < 0.05). 2.3 Bioactivity of compounds from A. oleracea 2.3.1 Extract preparation of A. oleracea The hexane extract of A. oleracea, which showed the highest insecticidal activity in the screening bioassay, was selected for isolation and structure elucidation of its bioactive compounds. A total of 2.0 kg of dried and powdered aerial parts of A. oleracea was used for this purpose. The solvent (hexane) was changed every two days for 45 days. The extraction continued until the solvent 46 was colorless. The filtered extract obtained was concentrated in a rotary vacuum evaporator under low pressure and reduced temperature (45-50°C). 2.3.2 Isolation and structural elucidation of bioactive compounds Fractionation of the hexane extract (28 g) was performed by column chromatography (Silica Gel 60, 70-230 mesh) using hexane with increasing amounts of ethyl acetate and finally with methanol as the eluting solvents. Thin layer chromatography (TLC, Silica gel 60 F254 0.25 mm) was used to identify fractions containing similar compounds. The TLC spots were detected under UV (254 and 365 nm) as well as by heating the plates to 100ºC after spraying with phosphomolybdic acid/ethylic alcohol. Eight fractions (A-H) were collected and subjected to bioassay with T. absoluta using the same methods as described in section 2.2.2. The most toxic fractions were purified by preparative TLC of Silica gel 60 F254, Merck (20 x 20 cm plates, 0.75 mm adsorbent). The IR spectra of isolated compounds were recorded on KBr in an infrared spectrometer Paragon 1000 FTIR (Perkin Elmer, Wellesley, MA, USA) from 600 to 4000 cm- 1. GC-MS was conducted with a Shimadzu QP5050A gas chromatograph-mass spectrometer. To identify the isolated compounds, 1H NMR and 13C NMR were recorded in a Varian Mercury 300 spectrometer (Varian Inc., Palo Alto, CA, USA) using CDCl3 as a solvent and TMS as an internal standard. 2.3.3 Dose-mortality bioassays The isolated compounds and the hexane extract of A. oleracea were subject to toxicity bioassays against T. absoluta, S. saevissima and T. angustula. The insecticidal activity of neem (Azadirachta indica A. Juss) seed kernel hexane extract and of permethrin (92.2% purity, Syngenta), a synthetic derivative of the natural pyrethrins recommended for T. absoluta control, were also evaluated and used as positive controls. The experimental design was completely randomized 47 with six replications. Each experimental unit consisted of a glass Petri dish (9.5 cm x 2.0 cm) containing ten insects. The average weight of each insect species was obtained by measuring the mass of ten groups containing ten insects each, on an analytical balance. Initially, four doses of each compound were tested to identify the range of concentrations that would provide mortalities greater than zero and less than 100%. Once the range of concentration was defined, other doses were tested for each compound. The number of doses used to obtain the dose-mortality curves varied from five to eight. Bioassays were conducted by topical application. For each insect a 10 µl Hamilton micro syringe was used to apply 0.5 µL of a solution of the test compound, dissolved in acetone. In a control experiment, carried out under the same conditions, 0.5 µL of acetone was applied to each insect. After application, the insects were kept in individual Petri dishes containing the appropriate food. T. absoluta were fed tomato leaflets (cv. Santa Clara) while S. saevissima and T. angustula both received a mixture of honey (50%) and pure water (50%). The mixture of honey and water were supplied in plastic containers that were 1.5 cm in diameter and 1.0 cm high. The Petri dishes were placed in an incubator at 25 ± 0.5°C, 75 ± 5% relative humidity, with a photoperiod of 12 h. The mortality counts were made after 24 h. Mortality included both dead individuals and those that were no longer moving. Dose-mortality data were subjected to probit analysis using SAS software (PROC PROBIT; SAS) to estimate dose-mortality curves.17 We accepted curves which had probabilities greater than 0.05 by the χ2 test.18 2.3.4 Risk assessment to non-target insects To determine the magnitude of selectivity of the compounds to the beneficial insects, we calculated the selectivity ratio using the formula SLR50 = LD50 of the 48 insecticide for the beneficial insect per LD50 of the insecticide for T. absoluta. Values of 1 and <1 indicate that the chemical is non-selective to the beneficial insect. Values >1 indicate that the chemical is selective and/or harmless to the beneficial insect.19 Using the dose-mortality curves, mortalities caused to beneficial insects by the doses of the compounds that caused 80% mortality in T. absoluta were also estimated. 3 RESULTS 3.1 Bioactivity of plant extracts (plant screening) The hexane extract from aerial parts of A. oleracea exhibited the highest activity of all extracts, causing 100.0 ± 0.0% (N = 60) mortality ± standard error (SE) in T. absoluta after six hours of exposure. The mortality caused by the solvents was zero (0.0%) in all of the bioassays (Table 2). The ethanol extract of A. oleracea also showed high activity (88.6% mortality) against T. absoluta, and was the second most active extract. The hexane and ethanol extracts of the remainder of the plants tested showed low insecticidal activity toward T. absoluta (Table 2). On the basis of these results, the hexane extract of A. oleracea was selected for isolation and structure elucidation of its bioactive compounds. 3.2 Isolation and structural elucidation of compounds from A. oleracea To obtain bioactive compounds, hexane extract (28 g) was fractionated by a bioactivity guided fractionation approach and eight fractions, A-H, were obtained. The eight fractional groups were evaluated for their insecticide activity against T. absoluta larvae. Fractions F and G eluted with hexane-ethyl acetate (1:1) were biologically active, causing 100% mortality 6 hours after administration of a dose of 10 µg mg-1 body mass. The remainder of the fractions (A, B, C, D, E and H) caused mortalities less than 40%. 49 The bioactive fraction F was purified by preparative TLC (hexane-ethyl acetate, 6:1) to yield the following three major bands: I (725 mg, Rf 0.65), II (56 mg, Rf 0.45), and III (21 mg, Rf 0.25). Band I was biologically active and was further purified by preparative TLC (hexane-ethyl acetate, 1:2) to give compounds 1 and 2 (320 and 210 mg, respectively). The bioactive fraction G was purified by preparative TLC (hexane-ethyl acetate, 3:1) to yield compound 3 (27 mg). Compound 1, (2E,6Z,8E)-deca-2,6,8-trienoic acid N-isobutyl amide or spilanthol (Fig. 1), was isolated as a colorless oil. The IR spectrum showed the presence of a secondary amide group (3340, 1636, and 1550 cm-1), a double bond conjugated to an amide carbonyl group (1678 cm-1), and a conjugated diene group with the Z, E or E, Z configuration (987 and 953 cm-1). The MS spectrum had the molecular ion peak at m/z 221, which indicates the molecular formula C14H23NO. GC-EIMS 70 eV, m/z (rel. int.): 221 [M]+ (20), 206 (3), 141 (100), 126 (23), 98 (23), 81 (87), 69 (10), 53 (10). The 13C NMR (CDCl3) and the 1H NMR (CDCl3) spectra showed the spilanthic acid (Tables 3 and 4). On the amine moiety, the typical signals at δ 3.15 (2H, t, H-1’), 1.78 (1H, m, H-2’), and 0.93 (6H, d, H-3’, 4’) in 1H NMR and δ 46.9 (C-1’), 28.6 (C-2’) and 20.1 (C-3’,4’) in the 13C NMR indicated the presence of a isobutylamino group. All of the spectral data were in agreement with those of spilanthol (1) in literature.20 Compound 2 was isolated as a colorless crystal. In the 1H NMR spectrum characteristic signals at δ 3.18 (dd, H-l’), 1.79 (m, H-2’) and 0.91 (d, H-3’ and H-4’) indicated the isobutylamide moiety. This compound was identified as undeca-2E-en-8,10-diynoic acid isobutylamide (Fig. 1) by comparing its 1H NMR spectral data (Table 3) with published values.21 The 13C NMR spectrum (Table 4) was consistent with the published data.10 Compound 3, (2E)-N-(2-methylbutyl)-2-undecene-8,10-diynamide (Fig. 1), was isolated as a colorless oil. The IR spectrum presented absorption bands 50 attributable to a triple bond (2225 cm-1) in addition to a secondary amide group (3299, 1627 and 1554 cm-1) and a double bond conjugated with an amide carbonyl group (1669 cm-1). The 1H NMR spectrum revealed signals at δ 5.77 (d, J = 15 Hz) and 6.80 (dt, J = 15 Hz and 7 Hz) which have been attributed to olefinic protons, H-2 and H-3, respectively (Table 3). On the amine moiety, a pair of 1H ddq signals at δ 1.17 and 1.41 are attributed to methylene protons of C-3’ and a pair of ddd signals at δ 3.14 and 3.27 are attributed to methylene protons of C-1’ due to the presence of asymmetric carbon at C-2’ (Table 3). The 13C NMR spectrum gave rise to 16 carbon signals. Five carbon signals at δ 45.2, 35.1, 27.1, 11.3 and 17.2 confirmed a 2-methylbutylamine moiety (Table 4). The 13C NMR and 1H NMR signals correspond well with the literature.20 3.3 Bioactivity of isolated compounds The dose-mortality results from insecticide application in larvae of T. absoluta showed low χ2 and high P values (<7.7 and >0.103 respectively), indicating the suitability of the probit model for fitting the dose-response curves and consequently obtaining estimates of the mortality parameters LD50 and LD80 (Table 5). Compound 1 (spilanthol) exhibited the highest toxicity to T. absoluta, with the lowest LD50. Furthermore, the spilanthol (1) was approximately five times more toxic than permethrin and approximately 321 times more potent than A. indica extract (Table 5). The compounds undeca-2E-en-8,10-diynoic acid isobutylamide (2) and (2E)-N-(2-methylbutyl)-2-undecene-8,10-diynamide (3) showed insecticidal activity similar to the commercial insecticide permethrin. In comparison with the extract of A. indica, compounds 2 and 3 were about 62 and 52 times more toxic to T. absoluta, respectively (Table 5). 51 The A. oleracea extract was less toxic than the isolated compounds, but it showed good insecticidal activity. It was 23-fold more toxic than the neem extract (Table 5). 3.4 Selectivity of isolated compounds The dose needed to kill 50% of the test population (LD50) was determined for the beneficial insects (Table 6) and used to calculate selectivity ratios of the insecticides for the two beneficial insects (Table 7). The A. oleracea extract and the compounds 1, 2 and 3 were selective to the predator S. saevissima and the pollinator T. angustula relative to T. absoluta, with a selectivity ratio (SLR50) greater than 1.0 (Table 7). For the A. oleracea extract, the doses that caused 50% mortality of T. absoluta larvae were 36% and 39% lower than the doses that caused the same mortality to S. saevissima and T. angustula, respectively. The estimated mortality of S. saevissima and T angustula by the LD80 of this extract to T. absoluta were 56% and 55%, respectively (Table 7). For compounds 1, 2 and 3, the doses that caused 50% mortality of T. absoluta larvae were 38%, 39% and 64% lower than the doses that caused the same mortality to S. saevissima, respectively. Furthermore, the doses were 169%, 37% and 35% lower than the doses that caused the same mortality to T. angustula, respectively. The estimated mortality of S. saevissima and T angustula by the LD80 of these compounds to T. absoluta ranged from 55% to 68%. However, the LD50 of permethrin for T. absoluta was 15.4-fold and 2,366.7-fold higher than the LD50 for S. saevissima and T. angustula, respectively. These results indicate that permethrin is harmful to the beneficial insects. The estimated mortality of S. saevissima and T. angustula by the LD80 of this insecticide to T. absoluta was 100% (Table 7). 52 Based on the SLR50, the neem extract was selective to S. saevissima. However, the DL80 of neem extract to T. absoluta caused a mortality of 84% and 98% to S. saevissima and T. angustula, respectively (Table 7). 4 DISCUSSION The plant species showing higher insecticide activity in our study was the toothache plant A. oleracea. Furthermore, the activity was higher in the hexane extract than in the ethanol extract. The bioactivity of A. oleracea is due to alkamides present in the plant. The main active amide in the plant is an isobutylamide, (2E, 6Z, 8E)-deca-2,6,8- trienoic acid, commonly known as spilanthol.22,23 These alkamides have a pungent effect and have been studied for various purposes. The flowers and leaves of A. oleracea are used in cooking and in popular medicine, mainly as an analgesic for toothache. The spilanthol is known for having several chemical and pharmaceutical applications in addition to the analgesic for toothache already mentioned. It is used for the treatment of aphtha and herpes, for stomatitis and infections in the throat, in treatment of tuberculosis, as a synagogue, as a fungistat and fungicide against Aspergillus spp., as an antimutagenic agent and as a cicatrizant.24-26 The results also showed that the alkamides evaluated in this study have the potential to control arthropods of agricultural importance. Three alkamides were identified in the bioactive fractions of the hexane extract of A. oleracea (spilanthol, undeca-2E-en-8,10-diynoic acid isobutylamide and (2E)-N-(2- methylbutyl)-2-undecene-8,10-diynamide). This study evaluated the effect of A. oleracea on T. absoluta, an important pest of tomato in the world. The results showed that all of the compounds isolated had high insecticidal activity, which was at least as toxic as permethrin, a pyrethroid recommended for control of T. absoluta. Furthermore, the compounds were far more toxic than the neem 53 extract. The high efficiency of these compounds combined with the ready availability from natural sources and the friendlier environmental footprint makes this plant an excellent candidate as a future natural insecticide. The results from this study showed the alkamides 1, 2 and 3 were selective to S. saevissima and T. angustula. The tolerance of beneficial insects to alkamides could be related to lower rates of insecticide penetration through the integument, higher rates of insecticide break down, and relative insensitivity of the target site in beneficial insects compared with T. absoluta.27,28 Penetration rates of insecticides in the insect integument are associated with the physicochemical properties of the insecticide and the insect cuticle, including cuticle thickness and biochemical composition.29-31 Soft-bodied insects such as T. absoluta have a thinner cuticle than S. saevissima and P. sylveirae, which supports this hypothesis. The selectivity of alkamides may also be associated with higher rates of metabolization in beneficial insects by detoxification enzymes such as P450-dependent monooxigenases. These enzymes transform lipophilic xenobiotics into polar metabolites that are then excreted.18 This hypothesis is based on the high lipophilic character of these alkamides (spilanthol has a log Pow of 3.4 and it is practically insoluble in water, 18.63 mg/L).32 The selectivity provided by alkamides to S. saevissima and T. angustula, suggests that the use of these compounds to control T. absoluta present a low risk to these beneficial insects. Furthermore, the results from this study showed that all compounds had a lower toxicity than permethrin (insecticide already used to control T. absoluta) to all non-target species studied. This finding indicates that the alkamides are less harmful to the beneficial insects. Thus, to preserve the predaceous and the pollinator investigated in this study, the use of these compounds for pest control can be recommended as a strategy to manage these beneficial insects. 54 Physiological selectivity is based on the use of insecticides that are more toxic to the target pest than the natural enemies and should always be considered when controlling pests. Furthermore, the principles of ecological selectivity should also be considered.33,34 The ecological selectivity is related to the different methods of applying insecticides as a means to minimize exposure of natural enemies to the insecticide.33 It is of utmost importance to use selective insecticides to preserve the beneficial species in the ecosystem, and it is necessary to resort to strategies that will enable the achievement of ecological selectivity even if it is not possible. With the ecological selectivity, an insecticide can be applied with a methodology designed to make it selective. The low stability of botanical pesticides and consequent rapid degradation in the environment is a characteristic that favors ecological selectivity, because it reduces the exposure time of beneficial organisms to toxic compounds. The mechanism of action of active alkamidas found in A. oleracea has not yet been determined. It appears to affect the nervous system as evident by abnormal movement such as uncoordinated muscular activity. This effect suggests that the compounds disturb nerve conduction somewhere. The analgesic activity of spilanthol in humans has been attributed to an increased GABA release in the temporal cerebral cortex,35 while other bioactive alkylamides are acting on voltage-gated sodium channels.36 The mortality after short exposure to the compounds indicate that alkamides greatly disturb the ongoing processes of histolysis of larval tissues. Saraf and Dixit11 observed rapid mortality of pupae of Aedes aegypti Linn, Anopheles culicifacies Giles and Culex quinquefasciatus Say (Diptera: Culicidae) when exposed to spilanthol. These results suggest that spilanthol interferes in histolysis and histogenesis processes. Further research is needed to address this question. Overall, the results of this research indicate that the A. oleracea extract is the most promising among the plant extracts studied. The active alkamides 55 from A. oleracea can be a potential alternative for controlling T. absoluta and should be studied further for other agricultural pests. All compounds presented high insecticidal activity for the insect pest T absoluta and selectivity for beneficial insects S. saevissima and T. angustula. Given the vital need for environmentally friendly chemicals that represent new insecticide groups with novel mechanisms of action, low persistence in the field and low toxicity to mammals and non-target species, the feasibility and impacts of using natural chemicals in pest management programs require further attention. We must remember, however, that the biological activity of a chemical is a function of its structure rather than its origin. The biological properties of a chemical depend on its structure and the way in which the chemical is used. Bioactive alkamides from A. oleracea have been found harmless to the majority of vertebrates and lethal to invertebrates.37,38 Because A. oleracea is widely used as both food and folk medicine in their region of origin, it is assumed that the toxicity to humans is extremely low. However, the actual risks of using these natural products should be identified. Therefore, to assess the feasibility and impacts of using the A. oleracea alkamides in agriculture more research on the effects on humans and the environment should be performed. 5 CONCLUSION The hexane extract of A. oleracea showed high insecticidal activity and can be used to control T. absoluta in organic or conventional crops. Quantification of LD50 values of isolated alkamides of A. oleracea against T. absoluta showed that alkamides could serve as potent insecticides for T. absoluta control programs. The spilanthol was the main alkamide active isolated. This alkamide is the most promising as it had the highest insecticidal activity and was selective to non- target organisms. Therefore, spilanthol and the others alkamides isolated are potential pest management tools likely to have they insecticide activity improved 56 through organic synthesis guided by studies of quantitative structure-activity relationship. ACKNOWLEDGEMENTS The authors are grateful to the Minas Gerais State Foundation for Research Aid (FAPEMIG), the National Council of Scientific and Technological Development (CNPq), and the CAPES Foundation of the Brazilian Ministry of Education for financial support. REFERENCES 1 Suinaga FA, Picanço M, Jham GN and Brommonschenkel SH, Causas químicas de resistência de Lycopersicum peruvianum (L.) a Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). An Soc Entomol Brasil 28:313-321 (1999). 2 Souza, JC, Reis PR and Salgado LO, Traça-do-tomateiro: histórico, reconhecimento, biologia, prejuízos e controle. Belo Horizonte, Empresa de Pesquisa Agropecuária de Minas Gerais, 15 pp. (1983). 3 Seplyarsky V, Weiss M and Haberman A, Tuta absoluta Povolny (Lepidoptera: Gelechiidae), a new invasive species in Israel. 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Academic Press, New York. 434 pp. (1960). 35 Rios MY, Aguilar-Guadarrama AB and Gutierrez MD, Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae), J Ethnopharmaco 110:364-367 (2007). 36 Gertsch J, Immunmodulatory lipids in plants: plant fatty acid amides and the human endocannabinoid system, Planta Med 74:638-650 (2008). 37 Pandey V and Agrawal V, Efficient micropropagation protocol in Spilanthes acmella L. possessing strong antimalarial activity. In Vitro Cell Dev Biol Plant 45:491-499 (2009). 38 Watt PM and Brayer-Brandwijk MC, The medicinal and poisonous plants of Sourthern and Eastern Africa. E&S Livingstone, Edinburgh, 1457 pp. (1962). 61 Table 1. Identification of plants used in screening bioassays (scientific name and family) and yield of hexane and ethanol extracts obtained from 1.0 kg of the plants aerial parts Yield (g) Nº Scientific name Family Hexane extract Ethanol extract Plants of the Amazon Biome 1 Acmella oleracea L. Asteraceae 10.74 15.04 2 Banara guianensis Aubl. Flacourtiaceae 22.74 26.54 3 Banara nitida Spruce ex Benth. Flacourtiaceae 4.2 10.49 4 Clavija weberbaueri Mez. Theophrastaceae 5.42 35.88 5 Copaifera duckei Dwyer Caesalpinioideae 7.42 18.44 6 Eugenia egensis DC. Myrtaceae 7.42 18.44 7 Mayna parvifolia Sleumer Flacourtiaceae 16.95 9.23 8 Piper aduncum L. Piperaceae 7.6 11.58 9 Piper augustum Rudge Piperaceae 7.86 21.33 10 Ryania speciosa Vahl. Flacourtiaceae 8.96 77.34 11 Siparuna amazônica Mart. ex A. DC. Monimiaceae 10.43 36.3 Plant of the Cerrado Biome 12 Curatela americana L. Dilleniaceae 13.26 19.87 Plants of general occurrence 13 Ageratum conyzoides L. Asteraceae 12.00 25.24 14 Allamanda cathartica L. Apocynaceae 5.31 4.51 15 Argemone mexicana L. Papaveraceae 5.98 6.48 16 Artemisia vulgaris L. Asteraceae 4.47 6.81 17 Bauhinia variegate L. Caesalpinioideae 11.48 32.02 18 Bougainvillea glabra Choisy Nyctaginaceae 7.56 9.63 19 Calendula officinalis L. Asteraceae 4.30 5.81 20 Chenopodium ambrosioides L. Chenopodiaceae 4.75 6.38 21 Coriandrum sativum L. Apiaceae 5.59 7.52 22 Spathodea campanulata P. Beauv. Bignoniaceae 12.70 28.76 23 Tropaeolum majus L. Tropaeolaceae 6.32 7.21 62 Table 2. Contact toxicity of plant extracts at concentration of 10 µg of extract per mg of insect against Tuta absoluta 6, 12 and 24 hours after topical application Mean percent mortalitya 6 hours after topical exposure 12 hours after topical exposure 24 hours after topical exposurePlants Ethanol extract Hexane extract Ethanol extract Hexane extract Ethanol extract Hexane extract Acmella oleracea 88.3 (± 1.5) Ab 100.0 (± 0.0) Aa 88.3 (± 1.5) Ab 100.0 (± 0.0) Aa 88.3 (± 1.5) Ab 100.0 (± 0.0) Aa Ageratum conyzoides 26.7 (± 1.9) Bb 45.0 (± 2.0) Ba 35.0 (± 3.1) Bb 48.3 (± 2.8) Ca 35.0 (± 3.1) Bb 51.7 (± 3.1) Ca Allamanda cathartica 21.7 (± 2.8) Ba 18.3 (± 2.1) Da 21.7 (± 2.8) Ba 23.3 (± 1.9) Ea 25.0 (± 3.4) Ba 23.3 (± 3.3) Ca Argemone mexicana 25.0 (± 3.1) Ba 20.0 (± 2.4)