SULLAMy D. G. ACIOLE^1,2, CARLA F. PICCOLI², JONNy E. DUQUE L.^2,3, EMMANOEL V. COStA⁴, MARIO A. NAVARRO-SILVA², FRANCISCO A. MARQUES⁵, BEAtRIZ H. L. N. SALES MAIA⁷, MARIA LúCIA B. PINHEIRO⁶, and MARIA t. REBELO¹

¹ Ph. D. y M. Sc. respectivamente. Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal / CESAM - Universidade de Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.

² M. Sc. y Ph. D respectivamente. Laboratório de Entomologia Médica e Veterinária - Departamento de Zoologia, Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, 81531-990, Curitiba, PR, Brazil.

³ Grupo de Investigación en Enfermedades Infecciosas y Metabólicas (GINEM) - Facultad de Salud, Departamento de Ciencias Básicas, oficina 205. Universidad Industrial de Santander. Bucaramanga, Colombia. Correo electrónico: jonedulu@uis.edu.co; mnavarro@ufpr.br; Autor para correspondencia.

⁴ Ph.D. Laboratório de Pesquisa em Química Orgânica de Sergipe (LABORGANICS), Departamento de Química, Universidade Federal de Sergipe, Rosa Elze, 49100-000, São Cristóvão, SE, Brazil.

⁵ Ph.D. Laboratório de Produtos Naturais e Ecologia Química, Departamento de Química, Universidade Federal do Paraná (UFPR), Centro Politécnico, Jardim das Américas, 81531-990, P.O. Box 19081, Curitiba, PR, Brazil.

⁶ Ph.D. Laboratório de Produtos Naturais, Departamento de Química, Universidade Federal do Amazonas, Av. Gen. Rodrigo Otávio Jordão Ramos, 3000, Coroado, 69077-000, Manaus, AM, Brazil.

⁷ Ph.D. Laboratório de Ecologia Química e Síntese de Produtos Naturais, Departamento de Química, Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, 81531-990, P.O. Box 19081, Curitiba, PR, Brazil.

Dengue cases and their clinical complications appear in countries of tropical and subtropical regions every year, with no promising prospects for future decrease of this problem (Guzmán et al. 2006). Unplanned urbanization, demographic and climatic changes in conjunction with the fast human migrations worldwide through air and land transport facilities are increasing the spread of the dengue arboviruses and its vector, Aedes aegypti (Linnaeus, 1762) to new places (Kroeger and Nathan 2007).

In the absence of a vaccine that confers permanent immunity to the four serotypes of the DENV1-4 dengue and  their genetic variations, vector control is used as the key measure to fight the disease (Hombach 2007; Periago and Guzmán 2007). However, even with the extensive accumulated knowledge over decades on this problem and knowing that so far that the only viable possibility is the direct vector control; this is not efficient since the epidemics in countries of tropical and subtropical regions continue occurring.

The dependence on synthetic organophosphorus (OP) and pyrethroid (P) insecticides to combat both immature and adult forms of the vector mosquito has been the most frequently adopted procedure for years, despite its little impact on the reduction of dengue cases. Unfortunately, such procedure has favored the outburst of A. aegypti populationsthat are insecticide tolerant at concentrations that otherwise would cause mortality to susceptible individuals (WHO 1992). this phenomenon has been reported in several regions such as: Venezuela (Mazzarri and Georghiou 1995), the Caribbean (Rawlins 1998), Singapore (Ping et al. 2001), Cuba (Rodriguez et al. 2002), Peru (Chávez et al. 2005), thailand (Ponlawat et al. 2005), Argentina, Bolivia (Biber et al. 2006), México (filores et al. 2006) and Brazil (Montella et al. 2007).

Botanical-origin products emerge as a promising alternative to control the vector of the dengue virus, after being set aside between the 30's and the 50's because the discovery of chemical synthetic insecticides (organochlorines, organophosphates, carbamates and pyrethroids). Besides have proven insecticidal effect, the plant-based products display a diversity of compounds with attractive, dislodging or repel-lent features that could be used in integrated pest management systems, as alternatives aimed at monitoring and control the mosquito populations (Isman 2006; Navarro-Silva et al. 2009).

the Annonaceae family comprises approximately 130 genera with 2.300 species of tropical and subtropical distribution (Kessler 1993). this group of plants has well known economic importance due to the trade of its fruits, byproducts, pharmacological activity, raw material for cosmetics, perfume industry, natural medicine and antimicrobial and insecticidal activity compounds (Costa et al. 2008, 2009; Boyom et al. 2003; Isman 2006). the genus Guatteria Ruiz & Pav, belongs to this family, with approximately 290 species distributed throughout Mesoamerica, the Caribbean and South America (Erkens and Maas 2008) with no study related to its insecticidal activity yet. the history of biological potential of the Annonaceae and its antimicrobial activity described in Costa et al. (2008), related with the chemical composition of essential oils of three species of the genus Guatteria, Guatteria blepharophylla (Mart.), Guatteria friesiana (W.A. Rodrigues) and Guatteria hispida (R.E. Fries) (Erkens and Maas 2008), raised the hypothesis of its insecticidal properties.

Research has been carried out to determine the potential efficacy of derivates from plants in vector control programs. the environmentally safe and biodegradable botanical insecticides could be an alternative method of control, owing to the growing incidence of the insect resistance to synthetic insecticides. In view of the abovementioned and the records of the insecticide action of the Annonaceae Family and the antimicrobial activity of the genus Guatteria, the essential oils of these species were evaluated for their effect against A. aegypti larvae under laboratory conditions.

Sample Collection. With the purpose to observe some variance in the chemical constituents on the essential oils of the species of Guatteria (G. blepharophylla, G. friesiana, and G. hispida) the collection was made in the same months, as well as, of the same species used by Costa et al. (2008), but three years later. Leaves of G. blepharophylla were collected in January 2008, on the campus of the Federal University of Amazonas (UFAM); Leaves of G. friesiana were collected in January 2008, at the Experimental Farm of the Federal University of Amazonas (UFAM), and leaves of G. hispida were collected in July 2008, at the Adolpho Ducke Forest Reserve. Voucher specimens were deposited in the Herbarium of the Department of Biology, UFAM, Manaus, AM, Brazil, under registration numbers 7340, 7341 and 7707, respectively. Leaves were obtained from fowered plants.

Extraction of essential oils. the leaves (250g) of the three Guatteria species were randomly collected dried at room temperature for three days, grounded and subjected to hydrodistillation for 4 hours, using a modifed Clevenger-type apparatus. the oils were dried over anhydrous sodium sulphate (Na₂SO₄) and the percentage content was calculated based on the dry weight. the extraction was repeated three times.

Gas Chromatography (GC-FID) analysis. the GC analyses were carried out using a Shimadzu GC-17A ftted with a fame ionization detector (FID) and an electronic integrator. Separation of the compounds was achieved employing a ZB-5MS fused capillary column (30m x 0.25mm x 0.25µm film thickness) coated with 5%-phenyl-arylene-95%-methylpoly-siloxane. Conditions of injection were performed according to Costa et al. (2008): injector temperature 240^oC; oven temperature program of 60^oC-300^oC at a rate of 3^oC/min; split 20:1 during 1.50 min, carrier gas He: 1 mL/min, constant fow; sample volume 0.5 µL.

Gas Chromatography - Mass Spectrometry GC-MS analysis. the GC-MS analyses were performed on a Shimadzu QP5050A GC/MS system equipped with an AOC-20i auto-injector. A J&W Scientific DB-5MS (coated with 5%-phe-nyl-95%-methylpolysiloxane) fused capillary column (30m x 0.25mm x 0.25µm film thickness) was used as the stationary phase. the conditions of injection were the same as described above and according to Costa et al. (2008). the mass spectrometer was operated at 70eV. the constituents of the essential oils were identifed by comparison of their mass spectral pattern and retention indices (RI) with those given in the literature (Adams 2007). the retention indices (RI) were calculated according to Van Den Dool and Kratz (1963).

1D/2D ¹H and ¹³C Nuclear Magnetic Resonance analysis (NMR). the crude essential oils of these species were analyzed by Nuclear Magnetic Resonance (NMR) of ¹H and ¹³C 1D/2D. Nuclear Magnetic Resonance (NMR) spectra were recorded in a Bruker Avance 400 spectrometer operating at 9.4 tesla, observing ¹H at 400 MHz and ¹³C at 100 MHz. Chloroform was used as the deuterated solvent. Chemical shifts values were given in parts per million (ppm) relative to the tetramethylsilane (tMS), used as internal reference standard (d 0.00).

Determination of insecticidal activity. the larvae from the Rockefeller Colony - CDC (Center of Disease Control) were kept in plastic trays (35.5cm x 21.5cm x 6.5cm) containing 3.000mL of dechlorinated water under controlled temperature (25ºC±1), humidity (70%±10) and photoperiod (12:12) conditions in a climatized chamber Model 347 CDG, at the Laboratory of Medical and Veterinary Entomology. thus they remained there until reaching the stage of final 3^rd instar and initial 4^th instar, a change observed from the exuviate. the latter did not receive any food or chemical treatment.

After reaching the larval stage described above, the larvae were counted, separated and transferred with a Pasteur pipette to disposable plastic glasses with a 50mL capacity, containing 20mL of the same dechlorinated water, in a total of 10 larvae per glass. then, these larvae were exposed to different concentrations of essential oils from the Guatteria spp. (12, 15, 20, 35, 40, 50, 60, 65, 80, 85, 95 and 120 ppm). the amount of oil for each concentration was placed in plastic containers of 330mL capacity containing Dimethyl sulfoxide (DMSO) 1% (BIOtEC® brand, with 99% purity) and 80mL of mineral water, in a final volume of 100mL.

All the experiments were repeated four times, including a control treatment exclusively with DMSO and mineral water. Finally, seven concentrations were tested (12, 15, 20, 35, 40, 60 and 85ppm).

the yields of essential oils were 0.6% for G. friesiana, 0.5 for G. hispida, and 0.3 for G. blepharophylla. these results of the GC-FID and GC-MS analyses are similar to those obtained by Costa et al. (2008) (table 1). the analyses of ¹H and ¹³C 1D/2D NMR spectral data of the crude essential oils of G. friesiana and G. blepharophylla, along with the analysis of these crude essential oils by GC-FID and GC-MS (Table 1), confrm the results reported by Costa et al. (2008) that G. friesiana are dominated by a-eudesmol (15.1%), beudesmol (52.0%) and g-eudesmol (24.0%) (Fig. 1A-C), respectively, while G. blepharophylla is dominated by caryo-phyllene oxide (70.0%) (Fig. 1D). From the actual ¹H and ¹³C NMR spectral along with GC-FID and GC-MS (table 1) analyses of the crude essential oil of G. hispida, the three major compounds, a-pinene (31.0%), b-pinene (36.0%) and (E)-caryophyllene (21.0) (Fig. 1E-G respectively), were confirmed after extensive analysis by Barero et al. (1995); Lee (2002); Hall et al. (2005), and suggested by the GC-MS performed by Costa et al. (2008). the ¹H and ¹³C NMR data obtained for compounds a-, b-, geudesmols, caryophyllene oxide, a-, b-pinenes, and (E)-caryophyllene in the crude essential oils were in accordance with Barero et al. (1995); Lee (2002); Hall et al. (2005) and Costa et al. (2008).

Mortality tests with the A. aegypti larvae under laboratory conditions using essential oils of the three Guatteria species indicated a strong larvicida action, with the highest rates being recorded for LC₅₀,₉₅ and ₉₉ for G. hispida, and the lowest rates for G. friesiana (table 2). By comparing the confidence intervals (CI) of each lethal concentration (LC) of the three species, it becomes evident that G. hispida is not in the same category as of the others. However, G. blepharophylla and G. friesiana were similar according to IC. the same behavior of insecticide action was observed in relation to the Angular Coefficient (AC) of concentrations and mortality response for the three species. Finally, all the experiments were significantly adjusted to the Probit (p < 0.05) model (x² = 0.318, 3.20 and 3.58, table 2).

Considering the increase of A. aegypti insecticide-resistant populations, mosquito control requires candidates to replace organophosphates (OP) and pyrethroids (P) chemical pesticides with products of proven insecticide effective-ness while these are environmentally safe. New plant records with biological action emerge every day, standing as possible substitutes to be incorporated into the fight against Culicidae.

The extraction yield of vegetable essential oils is a factor to consider in botanic products with biological action. When compared to the yield of oils extracted from other Annona-ceae published in literature (Boyom et al. 2003), it becomes evident that the Guatteria species analyzed produced higher yields, especially considering that only 250g of leaves from each species were used for the entire extraction process. the type of compound and its chemical characteristics are fundamental factors to determine whether an extract or essential oil can act as an insecticide. According to Costa et al. (2008), the essential oils of the three Guatteria species are formed by mono and sesquiterpenes. According to NMR data the major compounds in G. friesiana are b-eudesmol, geudesmol, and aeudesmol (Fig. 1A-C respectively); in G. blepharophylla, caryophyllene oxide (Fig. 1D), and in G. hispida a-pinene, b-pinene, and (E)-caryophyllene (Fig. 1E-G respectively), agreeing with the results of Costa et al. (2008). Determining the relation between the insecticide activity and the chemical composition of the essential oils is a significant challenge, because the possibility of synergic activities is always present, which makes difficult to establish an efficient model for this purpose. However, contrasting analyses of oil composition, its major compounds and the abundance of its constituents are often carried out in an attempt to assign the mortality action observed.

The comparison between LC₅₀ and LC₉₅ indicates that diterpenes could have an even more toxic effect than sesquiterpenes for this mosquito species, as shown with Copaifera reticulata Ducke (Fabaceae) for diterpenoid acid 1[(-)-3b-acetoxylabdan-8(17)-13-dien-15-oic] (LC₅₀=0.8ppm and LC₉₅=8.2ppm) and acid 2{alepterolic [(-)-3b-hydroxylab-dan-8(17)-13-dien-15-oic ]} (LC₅₀ = 87.3ppm and LC₉₅ =128.8ppm) (Geris et al. 2008). More interesting is the fact that tetranortriterpenoids (azadirachtin) have presented less effect than monoterpenes, sesquiterpenes and triterpene ac-cording to the LCs for A. aegypti (Wandscheer et al. 2004). By observing the results in Furtado et al. (2005) and Caval-canti et al. (2004) (table 3), only the essential oils of a few plants present the LCs with values close to the determined by G. friesiana, and G. blepharophylla which places the essential oils of these species among the most active against A. aegypti larvae. Finally, we recommend conducting studies to reveal the mechanisms of action of the major compounds of these oils in the mosquito.

The authors thank Rodrigo F. Chitolina for his help on the experimental work and also CNPq, CAPES FAPItEC/SE, and Fundação Araucária for financial support. J. E Duque thanks Prodoc/Capes for the postdoctoral fellowship in the period of 2008-2010. F.A. Marques and E. V. Costa thanks CNPq/ INCt - Controle Biorracional de Insetos Pragas - Estudos Integrados, for financial aid.

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