Contact action of neem extract (Azadirachta indica A. Juss) on Tetragonisca angustula Latreille (Hymenoptera: Apidae) under controlled conditions

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1 Contact action of neem extract (Azadirachta indica A. Juss) on Tetragonisca angustula Latreille (Hymenoptera: Apidae) under controlled conditions Antônio Queiroz Barreto 1 ; Suely Xavier Brito 2 ; Carlos Alfredo Lopes de Carvalho 3 ; Antônio Souza do Nascimento 4 ; Carlos Alberto da Silva Lêdo 4 1 Instituto de Meio Ambiente e Recursos Hídricos do Distrito Federal, Brasília-DF. aqbarreto@hotmail.com. 2 Agência Estadual de Defesa Agropecuária da Bahia, Laboratório de Epidemiologia e Tecnologia de Informação Aplicada à Sanidade. Av. Adhemar de Barros, nº967, Ondina, CEP.: , Salvador, BA, Brasil. suely.xavier@adab.ba.gov.br. 3 Universidade Federal do Recôncavo da Bahia, Ciências Agrárias, Ambientais e Biológicas, Cruz das Almas-BA. calfredo.carvalho@gmail.com. 4 Empresa Brasileira de Pesquisa Agropecuária - Mandioca e Fruticultura Tropical, CP 007, CEP.: , Cruz das Almas, BA, Brasil. nascimento@cnpmf.embrapa.br; ledo@cnpmf.embrapa.br. Abstract: The neem extract is an alternative to the use of synthetic insecticides in agricultural pest control due to its repellent effect, by impact and by contact, in order to minimize damage to the environment. However, there is still a lack of studies on their beneficial effects on insects, especially stingless bees, as Jataí bees (Tetragonisca angustula Latreille), widely distributed in Brazil and responsible for the pollination of many plant species of agricultural importance and forestry. Whereas toxicology studies and selectivity of insecticides on insect pollinators are becoming a major demand of agribusiness, with a view to increasing ecological awareness of society and environmental demands of consumers for safe food markets, this work aimed to evaluate the effect of the neem (Azadirachta indica A. Juss.) on workers of T. angustula. Neem extract showed the same level of toxicity that of the pyrethroid insecticide in commercial doses as a control, during the evaluation period of 24 hours. Key Words: stingless bees, Jataí bees, natural insecticide. Ação de Contato do Extrato de Nim (Azadirachta indica A. Juss) sobre Tetragonisca angustula Latreille (Hymenoptera: Apidae)em Condição Controlada Resumo: O extrato de Nim é uma alternativa ao uso de inseticidas sintéticos no controle de pragas agrícolas pelo seu efeito repelente, de impacto e de contato, visando minimizar as agressões ao meio ambiente. Contudo, ainda faltam estudos sobre a sua ação sobre insetos úteis, especialmente as abelhas sem ferrão, como a abelha jataí (Tetragonisca angustula Latreille), de ampla distribuição no Brasil e responsável pela polinização de muitas espécies de plantas de importância agrícola e florestal. Considerando que estudos de toxicologia e seletividade de inseticidas aos insetos polinizadores têm se constituído numa importante demanda do agronegócio, tendo em vista a crescente consciência ecológica da sociedade e as exigências dos mercados consumidores por alimentos seguros, este trabalho teve por objetivo avaliar o efeito do extrato de Nim (Azadirachta indica A Juss) sobre operárias de T. angustula. O extrato de Nim apresentou o mesmo índice de toxidez que o inseticida piretróide em dosagem comercial utilizado como controle, no período de avaliação de 24 horas. Palavras chave: abelhas sem ferrão, abelha jataí, inseticida natural. Introduction The Stingless bees are part of a group of about 400 species, with up to 50 genders, distributed in Central and South America (Velthuis, 1997), around 300 of these species are located in the Brazilian territory (Nogueira- Neto, 1997) these bees are responsible for 40% to 90% of the polinization in the tropical forests (Kerr et al., 1996; Aidar, 2010), this assure it s ecological potential. Among the stingless bees, the Jataí bee (Tetragonisca angustula Latreille) is one of most common species in Brazilian ecosystems, being found in meliponary (Carvalho et al., 2003) and visiting flowers of different species of economic interest (Carvalho et al., 1999; Carvalho and Marchini, 1999, Machado and Carvalho, 2006).

2 The uses of pesticides have drastically reduced the populations of wild bees and honey production; the largest parts of indigenous bees are more susceptible to pesticides than the honey bees. According to this author, not only adult bees are poisoned by insecticides, but also the larvaes in all stages (Moraes et al., 2000; Baptista et al., 2009; Del Sarto, 2009; Sthuchi, 2009; Diamantino et al., 2010). The most common way that the bees are contaminated with pesticides could be the followings: contact, through the absorption of the tegument; ingestion, with contaminated food or by the process of hygienitization, or breathing, via the spiracles. Pesticides can kill through a single mode of action, or the combination of all these (Gallo et al., 2002; Freitas and Pinheiro, 2012). Studies about the toxicology and selectivity of pesticides and natural substances for beneficial insects have been made mainly under agribusiness requests, with the goal to increase an ecological consciousness in the society and to increase the consumers demand for safe food (Reis et al., 2002; Carvalho et al., 2005, Ferreira et al., 2006; Rocha et al., 2006). Among the neurological insecticides which affect the transmission of nerve impulses, the pyretroids that are esters derived from chrysantemic acid; as example, deltamethrin (Decis 25 CE) characterized by its rapid action on insects, usually leading to hyper excitement, convulsions, ataxia and paralysis. The pyretroid primary site of action are the sodium channel located in the bee s nervous central system (NCS) and the symptoms are: typical regurgitation (especially in the case of highly toxic pyretroids) and erratic movements, followed by inability to fly, paralysis, agony and death, all in a very short period of time (Gallo et al., 2002). These organic products, like the Neem (Azadirachta indica A. Juss) have been popular among farmers for the control of various pests. The Neem is a plant natural Asia with multiple uses in several areas like: medicine, industry, cosmetics, reforestation and forestation in urban areas and for the control of agricultural pests (Diamantino et al., 2010). In chemical composition of Neem are found: salanina, meliantrol, gedunina, Nimbolin, but azadirachtine is the main active ingredient, that acts in the insect promoting rejections, growing delay, ovicide and larvicide action; reducing the rates of fertility and inducting deformities (Martinez, 2002; Silva et al., 2004), also presents fungicide, bactericide and nematicide action (Martinez, 2002; Depieri et al., 2005). The azadirachtine is less potent per contact than by ingestion, so the effect of natural enemies on Neem is lower than in insect-pests. The Neem is virtually harmless to humans and vertebrate, it is completely biodegradable, and it s active life in the soil is approximately of 20 days (Martinez, 2002). The action of the contact azadirachtine is less potent than by ingestion, so that under the effect of natural enemies is lower than in insect-pests. Despite its importance as a natural insecticide, few useful tests have been conducted on insects, especially on Brazilian bees (Gervásio and Vendramim, 2004). This work evaluates the effect of the Neem extract (A. indica) under controlled conditions on Jataí bee workers (T. angustula). Materials and Methods To standardize the age of the insects of the test, the workers bees that fly around the colony s entrance were caught with an entomological net and packed in wooden cages of the following dimension 20 x 14 x 15 cm, covered with a white nylon fine mesh cloth, previously treated. The experimental design was completely randomized, 15 cages (experimental units) were used, divided into three treatments, with 5 repetitions each. The treatments evaluated were water (witness), extract from Neem (Nim - I-Go, at commercial dose of 3%) and a piretroid insecticide (Decis 25 CE at commercial dose of 0.3 ml / 1 liter of water). The units were treated one hour before the release of workers of Jataí bees (10 specimens / cage), using a manual pump (Guarany brand), with a capacity of 370 ml. The workers of Jataí bee were feed with a honey and water solution brought into cages after being treated with the products under study. The results were obtained from observations recorded every hour in a 24 hours period, to check the survival of insects. The data were subjected to the method of Kaplan-Meier, which gives the advantage to use of information from specimens who were lost during the monitoring, or who do not know the actual time of death, but survived to the previous period (Smeets and Duchateau, 2003; Wenseleers et al., 2005). To correlate the time of survival with the treatment data were submitted to the analysis of Log-rank (Santoro et al., 2004).

3 Results and Discussion The chemical insecticide (Decis 25 CE) caused a mortality of 72% T. angustula at the sixth hour after test began, reaching a mortality of 88% at the end of the evaluation. The Neem extract caused a mortality of 26% until the seventh hour of assessment, increasing this index to 86% at the 18 th hour, equaling the values of the chemical insecticide test at the end of the evaluations (Figure 1). Generally extracts of plants, do not have a toxic snapshot effect (Souza and Vendramim, 2005), which is confirmed in this work based on the low bee s mortality in the early hours of exposure to treatment with Neem extract, undesirable feature for a product under a farmer vision accustomed to the use of synthetic insecticides with immediate effect. The method of Log-rank found significant differences between the witness and treatments with Neem and chemical insecticide, however, not statistical difference was observed between the treatment with Neem and the chemical insecticide, when compared with each other (Table 1). Figure 1 - Curve of the Kaplan-Meier survival chart for workers Tetragonisca angustula submitted two treatments (Nim and Decis 25 CE) in a 24 hours period. Table 1 - Comparison of the survival of workers Tetragonisca angustula submitted two treatments (Nim and Decis 25 CE) in a 24 hours period. Comparisons 2 Water - Nim 68,0897** Water - Decis 25 CE 42,4222** Nim - Decis 25 CE 0,0259 ns ** significant at 1% probability by 2 Log-rank test. ns not significant at 5% of probability. The extract of Neem presented lethal toxic effect on workers bees of T. angustula when used in commercial dose using contact as the action way, in a laboratory under controlled conditions, and equaling the effect of chemical insecticide Decis 25 CE.

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