Introduction

Severe human tropical diseases such as malaria, dengue and filariasis are transmitted by the bites of infected female mosquitoes belonging to the genera Aedes Meigen, Anopheles Meigen, Culex L. and Haemagogus L. (Diptera: Culicidae) (Pohlit et al., 2011). Dengue fever is an acute febrile viral disease characterized by sudden onset, of fever for 3-5 days, intense headache, myalgia, anthralgic retro-orbital pain, anorexia, GI disturbances and rash. Dengue viruses are flaviviruses and include four different serotypes 1, 2, 3 and 4. These viruses are also responsible for the lethal Dengue Hemorrhagic Fever (DHF). The viruses are transmitted to man by the bite of infected mosquitoes, mainly Aedes aegypti. The incubation period is 4-7 days (range 3-14 days). This disease is now endemic in most tropical countries. DHF caused by the same viruses, is characterized by increased vascular permeability, hypervolemia and abnormal blood clotting mechanisms. Dengue and dengue hemorrhagic fevers threaten an estimate of 2.5 billion people – 40% of the world population – and approximately 50 million people contract the disease per year. Most of patients, whom are children, require hospitalization each year and around 2.5% of those affected dies (WHO, 2011).

Another serious tropical disease is filariasis or elephantiasis. Filariasis is caused by infections by several round worm species of which Wuchereria bancrofti Cobbold (Filariidea: Onchocercidae) is the most important and is transmitted by the bites of the common house mosquito Cx. pipiens L. complex, Cx. quinquefaciatus Say, Aedes and Anopheles spp (WHO, 2011).

In recent years, numerous scientific reports have been published on plants which are useful for the control of arthropod vectors of tropical diseases (Pohlit et al., 2011). In Brazil, many plants are used in the form of crude extracts or infusions to treat common infections (Omena et al., 2007).

Plants may be a source of alternative agents for control of mosquitoes because they are rich in bioactive chemicals. For instance, the mosquito larvicidal properties of leaf and seed extract of plant Agave americana (Dharmshaktu et al., 1987); the mosquito larvicidal activity in the extract of Tagetes minuta flowers against Aedes aegypti (Green et al., 1991); the methanolic fraction of leaves of Mentha piperita, Phyllanthus niruri, Leucas aspera, and Vitex negundo against larvae of Culex quinquefasciatus (Pandian et al., 1994); the methanolic extracts of Solanums uratense, Azadirachta indica, and Hydrocotyle javanica exhibited larvicidal activity against Culex quinquefasciatus (Muthukrishnan et al., 1997); the benzene and methanol extracts of Artemisia vulgaris has shown repellent activity against Aedes aegypti (Yit et al., 1985).

The Amazon biodiversity has many unexplored potentialities, among them is its large diversity of fruits that are very appreciated by the local population. Some of these fruits have striking shapes, tastes and aromas, associated to their chemical composition. Herein we described the larvicidal activity of some edible fruits found in the West Brazilian Amazon, Oenocarpus bataua (pataua), Oenocarpus bacaba (bacaba), Endopleura uchi (uxi-amarelo), Leopoldinia piassaba (piaçava) and Astrocaryum aculeatum (tucuma). It is the first report on the larvicidal activity of these species and none of them have chemical studied, but E. uchi (Nunomura et al., 2009).

1 Results and Discussion

The activity of the extracts and fractions in the Ae. aegypti and Cx. quinquefasciatus bioassay confirm their potential as vector control method of the larvae populations. Exposure of different extracts and fractions in mosquito larval diet reduced larval survivability and increased mortality in the larval third-instar after 48 h of contact. The results of the present study indicate that the mortality rate of the tests in the larval stages of Cx. quinquesfasciatus and Ae. aegypti was significant at p <0.05 at concentrations evaluated at 24 and 48 hours exposure. The LC50 values of extracts and fractions against the vectors are shown in Table 1.

Thirteen other extracts and fractions showed no activity at doses as high as 500 μg.mL^-1 in the Ae. aegypti assay. Solely the chloroform fraction of methanol extract of pulp of piassava (LC₅₀ 198.9 μg.mL^-1) was more effective after 48 h of exposure.

On the other hand, eight of the extracts and eight fractions showed significant larval mortality after 48 h of exposure in Culex assay. The methanol extract of pulp of tucuma, the chloroform fraction of the methanol extract of pulp of bacaba, the hydroalcoholic fraction of methanol extract of pulp of piassava, the methanol extract of pulp of pataua, and hydroalcoholic fraction of methanol extract of pulp of pataua, showed activity below 500 μg.mL^-1 after 24 h of contact with larvae of Culex. These extracts and fractions were the most active after 48 h against Culex larvae with LC₅₀ values lower than 100 μg.mL^-1. The most active extracts and fractions were the chloroform fraction of methanol extract of pulp of piassava (LC₅₀ 29.7 μg.mL^-1), the EtOAc fraction of the methanol extract of pulp bacaba (LC₅₀ 31.9 μg.mL^-1), the fraction of hydroalcoholic extract of methanolic pulp of bacaba (LC₅₀ 32.5 μg.mL^-1), the methanol extract of pulp of bacaba (LC₅₀ 36.5 μg.mL^-1), the methanol extract of pulp of tucuma (LC₅₀ 37.8 μg.mL^-1), the chloroform fraction of the methanol extract of pulp bacaba (LC₅₀ 38.5 μg.mL^-1), the hydroalcoholic fraction of methanolic extract of pulp of pataua (LC₅₀ 40.9 μg.mL^-1), the methanol extract of the seed of bacaba (LC₅₀ 41.6 μg.mL^-1), the methanol extract of pulp of piassava (LC₅₀ 45.1 μg.mL^-1), the methanol extract of pulp pataua (LC₅₀ 47.1 μg.mL^-1), the methanol extract of the seed of piassava (LC₅₀ 48.2 μg.mL^-1), the EtOAc fraction of the methanol extract of pulp of pataua (LC₅₀ 61.2 μg.mL^-1), the fraction hydroalcoholic of methanol extract of pulp piassava (LC₅₀ 70.9 μg.mL^-1), the methanol extract of the seed of pataua (LC₅₀ 99.8 μg.mL^-1), the methanol extract of pulp of uchi (LC₅₀ 139.3 μg.mL^-1) and the aqueous pulp wine of piassava (LC₅₀ 157.2 μg.mL^-1). It is important to notice that no mortality was observed in the C. quinquefasciatus assay after 48 h in the negative control (solvents only), indicating that active principles are present in the mentioned active extracts and fractions. It is interesting to note that one of the most active extracts was the aqueous extract of the pulp of piassava, from the “wine” that is a local drink very appreciated in the Brazilian Amazon. This extract may be the basis of a new product for vector control of C. quinquefasciatus in the region.

Some of the active extracts and fractions in the C. quinquefasciatus assay can also be promising sources for new larvicidal compounds, since very few studies describing the chemical composition of these species can be found in the literature.

2 Materials and Methods

2.1 Plant material

The selected fruits are all edible ones well-known in the region. They were collected in the Adolpho Ducke Reserve, located approximately 23 km away from Manaus or acquired in local trade markets in the city. The fresh fruits were separated into seeds and pulps and dried in an oven at 60°C. The dried seeds and pulps were powdered mechanically using a commercial electrical stainless steel blender.

2.2 Preparation of extracts

The powdered seeds and pulps were extracted first with hexane (3 times for 6 h) and then with MeOH (3 x 6 h) in a Soxhlet apparatus. The extracts were evaporated under reduced pressure in a rotary evaporator (Fisatom 802A). The resulting crude extracts were stored in a freezer at - 20°C until before assayed. The MeOH extracts of pataua, piassava and bacaba were fractionated by liquid-liquid partition with chloroform and ethyl acetate. All obtained extracts and fractions were submitted to the larvicidal assays.

2.3 Bioassays

The bioassays were carried out with the mosquito species Aedes aegypti and Culex quinquefasciatus. Larvae of A. aegypti and C.quinquefasciatus were obtained from an insect, maintained at temperature of 23-27°C and relative humidity of 50-70% in the Laboratory of Malaria and Dengue of the National Institute of Amazonian Research. In the bioassays, third-instar larvae of Aedes aegypti and C. quinquefasciatus were exposed to different extracts and fractions at different concentrations. The extracts and fractions were all dissolved with dimethyl sulfoxide (DMSO) and were prepared at final concentrations of 500, 250, 100, 50 and 25 μg.mL^-1 in a final volume of 5 mL. The assays were carried out in plastic cups containing distilled water and feed for the larvae. A minimum amount of 10 larvae was used for the experiments, which were conducted in triplicate following the Standard Procedure of WHO. The larval mortality for the treatment and the control groups were recorded after 24 h and 48 h of treatment. Pure solvent (DMSO or water) was used as negative control and a solution of Temephos® as positive control (LC₅₀=39 ng.mL^-1).

2.4 Statistical analysis

The lethal concentration (LC₅₀) was calculated using Probit analysis in the POLOPC program (Finney, 1971). The percentage mortality was calculated by using the formula: Percentage of mortality = Number of dead larvae / Number of larvae introduced x 100. The 95% confidence intervals, values and degrees of freedom of the χ2 goodness of fit tests, and regression equations were recorded. The data were analyzed by a chi-square test, and the regression coefficients, fiducial limits and relative potency of the data were also evaluated.

Acknowledgements

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support (CT-Amazonia Proc. N. 575503/2008-4 e Proc. N. 575625/2008-2; Rede Malaria and Rede Bionorte) and also to Fundação de Amparo a Pesquisa do Estado do Amazonas (FAPEAM) (PPP Processo n° 3007/10).

References

Dharmshaktu N.S., Prabhakaran P.K., and Menon P.K., 1987, Laboratory study on the mosquito larvicidal properties of leaf and seed extract of plant Agava Americana, The Journal of Tropical Medicine and Hygiene., 90: 79-82

Finney D.J., 1971, Probit Analysis, third ed. Cambridge University Press, London, UK, p.38

Green M.M, Singer J.M, Sutherland D.J, and Hibbon C.R., 1991, Larvicidal activity of Tagetes minuta (Marigold) towards Aedes aegypti, Journal of the American Mosquito Control Association, 7: 282-286

Muthukrishnan J., Pushpalatha E., Kasthuribhai A., 1997, Biological effect of four plant extracts on Culex quinquefasciatus say larval stages, International Journal of Tropical Insect Science, 7: 389-394

https://doi.org/10.1017/s1742758400019238

Nunomura R.C.S., Oliveira V.G., da Silva S.L., Nunomura S.M., 2009, Characterization of bergenin in Endopleura uchi bark and its anti-inflammatory activity, Journal of the Brazilian Chemical Society, 20: 1060-1064

https://doi.org/10.1590/S0103-50532009000600009

Omena M.C., Navarro D.M.A.F., Paula J.E., Luna J.S., Lima M.R.F., and Sant’ana A.E.G., 2007, Larvicidal activities against Aedes aegypti of some Brazilian medicinal plants, Bioresource Technology, 98: 2549-2556

https://doi.org/10.1016/j.biortech.2006.09.040
PMid:17137781

Pandian R.S., Abraham M.G, Manoharan A.C., 1994, Susceptibility of the larvae of Culex quinquefasciatus Say to extracts of medicinal plants, Environmental pollution, 1: 109-22

Pohlit A.M., Lopes N.P., Gama R.A., Tadei W.P., Neto V.F.A., 2011, Patent literature on mosquito repellent inventions which contain plant essential oils – A review, Planta Medica, 77: 598-617

https://doi.org/10.1055/s-0030-1270723
PMid:21328177

Pohlit A.M., Rezende A.R., Baldin E.L.L., Lopes N.P., Neto V.F.A., 2011, Plant extracts, isolated phytochemicals, and plant-derived agents which are lethal to arthropod vectors of human diseases tropical – A review, Planta Medica, 77: 618-630

https://doi.org/10.1055/s-0030-1270949
PMid:21432748

WHO Media Centre, 2011, Dengue. World Health Organization (WHO) Media Centre, Available at http://www.who.int/mediacentre/factsheets/fs117/en/ Accessed September 10

WHO Media Centre, 2011, Filariasis. World Health Organization (WHO) Media Centre, Available at http://www.who.int/topics/filariasis/en/ Accessed September 10

YitHs Ku-Hua W.V., Kumato J.H, Mulla M.S., 1985, Isolation and identification of mosquito repellent in Artemesia vulgaris. Journal of Chemical Ecology, 11: 1297-1306

https://doi.org/10.1007/BF01024117
PMid:24310391