113 RBCIAMB | n.42 | dez 2016 | 113-120 Ana Carla Knakiewicz Biological Science Student, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Junir Antonio Lutinski Ph.D. in Animal Biodiversity. Professor of the Graduate Program in Health Science, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Carin Guarda Graduation in Biological Science, Master degree student in Health Science, Community University of Chapecó Region(Unochapeco) – Chapecó (SC), Brazil. Ariane Paris Biological Science Student, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Alencar Belotti Agronomist, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Maria Assunta Busato Ph.D. in Biology. Professor of the Graduate Program in Health Science, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Walter Antonio Roman Junior Ph.D. in Pharmaceutical Sciences. Professor of the Graduate Program in Health Science, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Daniel Albeny Simões Ph.D. in Entomology. Professor of the Graduate Program in Environmental Sciences, Community University of Chapecó Region (Unochapeco) – Chapecó (SC), Brazil. Corresponding address: Junir Antonio Lutinski – Rua Beija‑Flor, 254 E, Efapi, 89809‑760 – Chapecó (SC), Brasil – E‑mail: junir@unochapeco.edu.br ABSTRACT Aedes aegypti is the main vector of dengue, Chikungunya and Zika virus worldwide. The strategies for the population control involve the use of synthetic insecticides, which may cause toxicity problems and environmental contamination. This study evaluated the larvicide effect of hydroalcoholic extracts of leaves and fruits Ilex paraguariensis and Ilex theezans on A. aegypti. The bioassays were conducted under controlled conditions. Fresh and dry fruits of I. paraguariensis showed the higher larvicidal activity on A. aegypti than the leaves of the same species. Mosquito larvae showed increased susceptibility from dried leaf extracts, fresh fruit and leaf of I. theezans. It was also observed a variation in larval mortality during periods of exposure. The larvicide efficiency of I. theezans extracts on A. aegypti are promising results and widen the possibility for further studies of the use of extracts of this plant. Keywords: vector control; dengue; yerba mate; larvicide; natural products. RESUMO Aedes aegypti é o principal vetor da dengue, febre Chikungunya e Zika vírus em escala global. As estratégias para controle populacional deste vetor recorrem à utilização de inseticidas sintéticos, que podem acarretar problemas de intoxicação e contaminação ambiental. Este estudo avaliou o efeito larvicida de extratos hidroalcoólicos de folhas e frutos de Ilex paraguariensis e de Ilex theezans sobre A. aegypti. Os bioensaios foram conduzidos sob condições controladas. Os frutos in natura e secos de I. paraguariensis demonstraram maior atividade larvicida sobre A. aegypti em relação às folhas da mesma espécie. As larvas do mosquito demonstraram maior susceptibilidade frente aos extratos de folhas secas, in natura e frutos verdes de I. theezans. Também foi observada variação na mortalidade larval nos períodos de exposição. A eficiência larvicida dos extratos de I. theezans são resultados promissores e abrem a possibilidade para estudos subsequentes do uso de extratos desta planta. Palavras‑chave: controle vetorial; dengue; erva‑mate; larvicida; produtos naturais. DOI: 10.5327/Z2176-947820160177 LARVAL SUSCEPTIBILITY OF AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE) TO EXTRACTS OF ILEX PARAGUARIENSIS AND ILEX THEEZANS SUSCETIBILIDADE LARVAL DE AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE) AOS EXTRATOS DE ILEX PARAGUARIENSIS E ILEX THEEZANS https://www.facebook.com/ariane.paris.7 Knakiewicz, A.C. et al. 114 RBCIAMB | n.42 | dez 2016 | 113-120 INTRODUCTION The epidemiological situation of dengue, Chikungun‑ ya fever and Zika virus in Brazil has been increasing‑ ly complex as its main vector. The mosquito Aedes aegypti (Linnaeus 1762) is spread by almost all the na‑ tional territory, where epidemics have been recurrent (BARRETO & TEIXEIRA, 2008). The A. aegypti is one of more than 500 species of the Aedes genus (Diptera: Culicidae), considered as the main vector of these viruses and also of urban Yellow Fever. It uses water tanks to lay its eggs and consequently develop their larvae. An important evolutionary feature that al‑ lowed the conquest of a large proportion of the globe is that its eggs are resistant to desiccation, remain‑ ing viable in the absence of water for more than one year (TAUIL, 2002; POWELL & TABACHNICK, 2013). When it is in contact with water, the larvae emerges, going through four stages (L1, L2, L3 and L4) and sub‑ sequently originating the pupae and, soon after, adult mosquitoes (NEVES, 2011). Among the strategies for population control of this vec‑ tor, there is also the use of synthetic insecticides, such as Pyrethroids Deltamethrin and the Carbamate Ben‑ diocarb (GUARDA et al., 2016). The use of these insec‑ ticides may cause toxicity problems and environmen‑ tal contamination, leading to risks to local biodiversity and human health. Synthetic insecticides have low se‑ lectivity and can select genes that cause resistance in populations of Aedes (CALDAS, 2000; SANTIAGO et al., 2005). The vector management with the use of natural products is less intrusive than using the conventional insecticides (synthetic), having a more rapid degrada‑ tion, resulting in lower occupational exposure and less environmental pollution (VALLADARES; DEFAGO; PALA‑ CIOS, 1997; BARRETO, 2005). Studies have shown the effectiveness of natural prod‑ ucts for the control of embryonic development, larval, pupa and adult emergence of A. aegypti (BRUNHEROT‑ TO & VENDRAMIM, 2001; ROSSI et al., 2007; BUSATO et al., 2015). A perspective arises from the study of Busato et al. (2015) successfully testing the larvicide efficiency to the ethanol extracts of leaves of Ilex para- guariensis A. St.‑Hill (Aquifoliaceae) on A. aegypti. The species I. paraguariensis is from South America, popularly known as yerba mate or congonha, being grown and consumed as tea or mate in Argentina, Brazil, Paraguay, and Uruguay. After processing, its leaves and branches make the drinks appreciated by the population of these countries (SOUZA, 2009; LORENZI & MATOS, 2002). In the botanical descrip‑ tion of Kricun (1983) and Giberti (1995), it is an ev‑ ergreen tree, with height about 3 to 5 m (in growing forests) and up to 12 m high and 70 cm in diame‑ ter, in a native forest environment. Besides its cul‑ tural and gastronomic aspects, the great commercial interest in I. paraguariensis is due to the presence of caffeine and theobromine, recognized for exercis‑ ing stimulating effects on cardio‑circulatory systems as coffee, cocoa and guarana (CASTALDELLI et al., 2011). The plant also has vitamins, amino acids and triterpene saponins of nutritional and medicinal in‑ terest in its chemical constitution (RATES, 2004; PIO‑ VEZAN‑BORGES et al., 2016). The Ilex theezans Mart species Ex Reissek (Aquifoliace‑ ae) is popularly known as Cauna and little used com‑ mercially, but common in vegetation types in the South of Brazil. It is an evergreen tree, early secondary or late secondary species. Its height reaches up to 20 m, has a diameter of 70 cm and the chemical properties of the extracts of this plant are still poorly known. In this context, this study aimed to evaluate the larvicid‑ al effect of hydroalcoholic extracts of leaves and greens and dried fruit of I. paraguariensis, native and cultivat‑ ed and I. theezans on A. aegypti. MATERIAL AND METHODS Plant material The leaves and fruits collection of I. paraguariensis, na‑ tive and cultivated and I. theezans were held in Mare‑ chal Bormann district (27°, 19’05”S,52°,65’11”W), Chapecó (SC), on December 2015. The collected plant material was identified by the curator of the Herbarium of the Universidade Comunitária da Região de Chapecó. Larval susceptibility of Aedes aegypti (I.) (Diptera: culicidae) to extracts of Ilex paraguariensis and Ilex theezans 115 RBCIAMB | n.42 | dez 2016 | 113-120 These materials were transferred to the Pharma‑ cognosy Laboratory, reduced to small fragments, and submitted to drying at room temperature, protected from direct light and moisture. Subse‑ quently, the dried plants were ground in a knife mill (Ciemlab®, CE430) selected in a filter of 425 μm (35 Tyler/Mesh), identified and stored away from the light. Extracts production For the preparation of extracts by turbolisis (5 days), fresh and dried leaves and fruits, were follow protocol used as recommended by the Brazilian Pharmacopoeia (FB 5, 2010). There were 12 extracts prepared: fresh and dry leaves, as well as the fruits of I. paraguariensis (na‑ tive and cultivated); leaves (fresh and dried) and fruit of I. theezans. After their filtration through Büchner fun‑ nel, the extracts were concentrated by rotary evapora‑ tion under reduced pressure, lyophilized, weighed and stored in a freezer at ‑20°C for further assays. Experimental procedure The collection of eggs of A. aegypti was conducted from November 2015 to April 2016. There were 15 egg‑traps characterized as plastic containers of black color, with a capacity of 500 mL, with 200 mL of water into each trap and monitored every 7 days. For laying the eggs and for the collection of the field, a white crop seed germi‑ nation paper cut into strips of 29.7 x 10 cm was used (GOMES; SCIARICO; EIRAS, 2006). The cut was placed around the inside of the trap so that approximately four centimeters were submerged in the solution. The egg traps were distributed at different points of the city of Chapecó, in establishments previously known by the mosquito infestation, according to data from the Pro‑ grama Municipal de Controle da Dengue (city program for dengue control). The papers with the eggs were placed in white plastic trays 20 x 30 cm containing 2 L of water without treat‑ ment to obtain the larvae. The larvae were fed with fish feed (Holiday®) and remained in the trays until the L2 stage with a period of three days. The bioassays were carried out in laboratory conditions (Ecological Ento‑ mology Laboratory ‑ LABENT‑Eco of the Community University of Chapecó Region), with a temperature of 28 ± 3°C and a photoperiod of 12 hours. Experimental design The experimental microcosms were in transparent plastic cups with 300 mL capacity. Each microcosm had a volume of 80 mL of vegetable extracts concen‑ trations ranging from 500 to 2,000 Experimental g/mL and 20 active A. aegypti larvae of the second stage. The control microcosms received only 80 mL of un‑ treated water and 20 mosquito larvae. Each treatment was repeated three times and all larvae used in the ex‑ periment were fed with fish feed – only at the begin‑ ning of the test. Larval susceptibility was evaluated at intervals of 24, 48 and 72 h after exposure to the solu‑ tions. The living larvae were counted in each period. After the experiment ends, the remaining larvae were sacrificed in boiling water and discarded. Statistical analysis The data evaluation was performed by the analysis of variance (ANOVA, one‑way) on the number of living larvae, where the means were grouped by the Dun‑ can test at 5% probability of error. The efficiency of the treatments tested was calculated by Abbott equation (1925), used to compare the treatments with respect to control and defined by the formula %Efficiency = (T‑t)*100/T, where “T” represents the average number of living larvae in Control, and “t” is the average of sur‑ viving larvae in each treatment. RESULTS Larval susceptibility to A. aegypti was significantly af‑ fected by the treatment variable, by the time variable and also by the interaction between treatment and time (Table 1). Knakiewicz, A.C. et al. 116 RBCIAMB | n.42 | dez 2016 | 113-120 The extracts of dried leaves of I. theezans at a con‑ centration of 750 μg/mL showed the highest larvi‑ cide efficiency from the first 24 hours. There was an efficient success in extracts of fresh leaves and fruits (1,000 μg/mL) and dried fruits (2,000 μg/mL) of I. theezans. The native dried fruit extract of I. paraguariensis also showed greater than 50% activity in concentration of 1,000 μg/mL. All ex‑ tracts of I. paraguariensis matched to the control (p < 0.01) (Table 2). Table 1 – Variance analysis of the number of Aedes aegypti living larvae exposed to treatments with hydroalcoholic extracts of I. paraguariensise and of I. theezans under laboratory conditions (temperature 28°C and 12 hours photoperiod). Variation cause Degrees of freedom Average square Probability Treatment 28 0.82 p<0.01 Hours 3 3.93 p<0.01 Treatments x hours 84 0.26 p<0.01 Residue 232 0.04 Total 347 Coefficient of variation = 6.8% Table 2 – Treatments, average of A. aegypti alive after 72 hours of exposure to treatment with hydroalcoholic extracts of I. paraguariensise of I. theezans followed by standard error and efficiency percentage in laboratory conditions (temperature 28°C and 12 hours’ photoperiod). Means followed by capital letters differ by Duncan test (p < 0.01). Treatments Alive larvae % Efficiency (72 h) Control 20 ± 0 A 0% I. paraguariensis fresh native fruits 750 μg/mL 20 ± 0 A 0% I. paraguariensis fresh native leaves 1,000 μg/mL 20 ± 0 A 0% I. paraguariensis fresh cultivated leaves 1,000 μg/mL 20 ± 0 A 0% I. paraguariensis fresh cultivated fruits 1,000 μg/mL 20 ± 0 A 0% I. paraguariensis fresh native leaves 2,000 μg/mL 20 ± 0 A 0% I. paraguariensis dry native leaves 1,000 μg/mL 20 ± 0 A 0% I. paraguariensis dry native leaves 2,000 μg/mL 20 ± 0 A 0% I. paraguariensis dry cultivated leaves 2,000 μg/mL 20 ± 0 A 0% I. paraguariensis dry cultivated leaves 1,000 μg/mL 19.66 ± 0.08 A 1.7% I. paraguariensis dry native fruits 1,000 μg/mL 19.33 ± 0.77 A 3.35% I. paraguariensis fresh cultivated fruits 2,000 μg/mL 18.66 ± 0.11 A 6.7% I. paraguariensis fresh cultivated leaves 2,000 μg/mL 18.66 ± 0.23 A 6.7% I. paraguariensis dry cultivated fruits 750 μg/mL 16.03 ± 0.65 AB 19.85% I. paraguariensis fresh native fruits 1,000 μg/mL 14.93 ± 0.57 AB 25.35% I. paraguariensis dry native fruits 2,000 μg/mL 13.93 ± 0.98 AB 30.35% I. theezans freen leaves 750 μg/mL 13.85 ± 0.69 AB 30.75% I. paraguariensis fesh native fruits 2,000 μg/mL 12.82 ± 0.84 AB 35.9% I. paraguariensis dry cultivated fruits 1,000 μg/mL 9.76 ± 1.37 AB 51.2% I. theezans dry fruits 1,000 μg/mL 8.78 ± 1.41 B 56.1% I. theezans fresh fruits 2,000 μg/mL 8.58 ± 1.43 BC 57.1% I. theezans fresh leaves 1,000 μg/mL 6.23 ± 2.03 CD 68.85% I. theezans fresh fruits 1,000 μg/mL 5.64 ± 1.74 CD 71.8% I. theezans dry leaves 750 μg/mL 1.88 ± 2.36 E 90.6% Larval susceptibility of Aedes aegypti (I.) (Diptera: culicidae) to extracts of Ilex paraguariensis and Ilex theezans 117 RBCIAMB | n.42 | dez 2016 | 113-120 The most effective larvicidal effect of the extracts was observed after 24 hours of exposure. Al‑ though it was observed activity in the period be‑ tween 0 and 24 hours for the fresh leaf extract of I. theezans (1,000 μg /mL), the stronger activity of the other was observed in the period between 24 and 48 hours and less intense between 48 and 72 hours (Figure 1). DISCUSSION The results showed that the hydroalcoholic extracts of leaves and fruits, both fresh or dry of I. theezans showed larvicidal activity at all concentrations test‑ ed, and concentrations above 750 μg/mL were the most efficient. The extract of dried leaves of I. th- eezans at a concentration of 750 μg/mL was the most efficient (Table 2). In 72 hours, it showed the efficien‑ cy of 90.6%. The fresh fruits extract of this species presented 71.8% efficiency. The highest activity was observed in the period between 24 and 48 hours of exposure (Figure 1). Ilex theezans is an early successional species, consid‑ ered one of the most frequent in Mixed Rain Forest and according to Viani and Vieira (2007) it usually co‑ exists with I. paraguariensis. The I. theezans leaves are mixed in the mate to increase the bitterness, having a higher concentration of saponins. The main biological activities of saponins are related to their active tense, complexing, haemolytic and toxic properties (MAHA‑ TO; SARKAR; PODDAR, 1988). The fact that I. theezans had larvicide effectiveness against A. aegypti may be related to the presence of a higher concentration of saponins usually used to differentiate species of Ilex. However, they would require additional studies on their composition. It was noticed that the extracts of I. theezans showed the highest efficiency between 24 and 48 hours. After this period, the activity was still observed, although with a reduction in the effect of the extracts on A. aegypti larvae. For Garcia (2014), a product is commercially ef‑ ficient and recommended if it shows lethality inferior to 80%, so that there is a selection of genes causing resis‑ tance. One of the treatments reached the parameter of the extract of dried leaves of I. theezans (750 μg/mL). Figure 1 – Survival of A. aegypti larvae exposed to different concentrations of I. theezans extracts, evaluated every 24 hours under laboratory conditions (temperature 28°C and 12 hour photoperiod). 0 5 10 15 20 0 24 48 72 Av er ag e liv e la rv ae Time after application (hours) I. theezans fresh fruits 2,000 μg/ml I. theezans fresh leaf 1,000 μg/ml I. theezans fresh fruits 1,000 μg/ml I. theezans dried leaf 750 μg/ml Knakiewicz, A.C. et al. 118 RBCIAMB | n.42 | dez 2016 | 113-120 Fruit and fresh and dry leaves extracts of I. paraguar- iensis, native and planted, from the concentration of 1,000 μg/mL showed a relatively low larvicide efficien‑ cy when compared to extracts of I. theezans (Table 2). The fact of being native or planted did not result in dif‑ ferences in this study. For more favorable, shaded and with specific microclimatic condition environments, such as intensity and duration of sun radiation, and the predetermined genetic variations (COELHO; MARIATH; SCHENKEL, 2002) and even the presence or absence of endophytic microorganisms (MELO & AZEVEDO, 1998), differences in the activity of the extracts were expect‑ ed. Studies regarding the phytochemical composition of yerba mate demonstrated that their compounds may vary in quantitative and qualitative terms, such as the type of crop, climate, agronomic conditions, plant age, methodology analysis and industrial processing (DUTRA, 2009). In the study of Busato et al. (2015), in a bioassay using a hydroalcoholic extract of I. paraguar- iensis leaves, using the drying method in an oven for 48 hours, larvicide efficiency was checked on A. aegypti, with 100% mortality at a concentration of 2,000 μg/mL. Comparing the results of Busato et al. (2015), it is possi‑ ble to infer that the extracts of the preparation method may have influenced the results for I. paraguariensis. The fresh and dry fruits of I. paraguariensis showed greater activity than the leaves of the same species. The fresh fruits of yerba mate, compared to the leaves, showed higher concentrations of saponins, which are present as a defense against herbivores and other forms of consumption and predation (PAVEI et al., 2007). Af‑ ter ripening, there is a predominance of other nutrients, which facilitate the use and dispersal of seeds, such as sugar, proteins, fats and carbohydrates (RAVEN; EVERT; EICHHORN et al., 1996). Probably these saponins in fresh fruits of I. paraguariensis were lethal to A. aegypti lar‑ vae. The effect of this saponin in fruits of various families of plants is known to be deleterious to the development of pests of maize monocultures such as Spodoptera fru- giperda (Smith, 1797) and Helicoverpa zea caterpillars (Boddie 1850) (DOWD et al., 2011). The great commercial interest in I. paraguariensis as well as its cultural and gastronomic aspects is due to the presence of xanthine bases. According to Borges et al. (2013), one of the main constituents of yerba mate leaves used for teas are methylxanthines, caf‑ feine, theobromine and theophylline traits. These results open perspectives in replacement of syn‑ thetic insecticides by natural products in the control of A. aegypti, by their abundant presence in southern Brazil. These results are promising, creating the possi‑ bility for further studies regarding the use of extracts of these plants as larvicides of A. aegypti, as an alter‑ native to the synthetic products. For the treatments carried out, the method of extraction and preparation of I. paraguariensis extracts proved to be little efficient. One possibility is that not all compounds were released during the process of preparation. For the species I. theezans, the method was efficient, widening the per‑ spective for further studies of this yet little used species. ACKNOWLEDGMENT Research supported (scholarship) by CNPq/PIBITI (Notice 180/Dean/2015) and the Universidade Comunitária da Região de Chapecó ‑ UNOCHAPECÓ. REFERÊNCIAS BARRETO, C. F. Aedes aegypti – Resistência aos inseticidas químicos e as novas alternativas de controle. Revista Eletrônica Faculdade Montes Belos, v. 1, n. 2, p. 62‑73, 2005. BARRETO, M. L.; TEIXEIRA, M. R. Dengue no Brasil: situação epidemiológica e contribuições para uma agenda de pesquisa. Estudos Avançados, v. 22, n. 64, p. 53‑72, 2008. BORGES, A. C. P.; DARTORA, N.; RIL, F. T.; GONÇALVES, I. L.; VALDUGA, A. T. 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