Screening of plant extracts and fractions on Aedes aegypti larvae found in the state of Mato Grosso do Sul ( linnaeus , 1762 ) ( culicidae )

The constant use of chemical insecticides for Aedes aegypti control has caused resistance in the mosquito populations. Thus, the objective of this study was to analyze the larvicidal potential of extracts and fractions of plants on A. aegypti larvae. The analysis included sixty one extracts and twenty five fractions of fifty botanical species at concentrations of 0.25; 0.12; 0.06 to 0.03 mg mL; 4 replications and one negative control of dechlorinate water and 1% DMSO; and a positive control with rotenone. The toxicity index in descending order with LC50 for the most active of the extracts selected were ethanol extract of Ormosea arborea (0.111 mg mL) seeds and ethanol extracts of leaves such as Piper hispidum (0.169 mg mL), Solanum variabile (0.188 mg mL), O. arborea (0.238 mg mL), Turnera umifolia (0.242 mg mL) and Piper hispidum (0.567 mg mL). For plant fractions, the most active were chloroform (0.192 mg mL) and hexane (0.342 mg mL) P. aduncum leaves, hexane fraction (0.415 mg mL) and methanol extract (0.625 mg mL) of Spermacocea latifolia leaves. Regarding the extract of T. umifolia single species, there is no bibliographic report on their degree of efficiency as an insecticide.


INTRODUCTION
Brazil is considered an endemic area for dengue, which is transmitted by the mosquito Aedes aegypti (L.) (Culicidae) (Maciel et al. 2008).Dengue fever in Brazil, since 1986, has reached about 3 million cases.Only in the State of Mato Grosso do Sul, the year 2007 witnessed the largest outbreak of dengue fever (69,378 reports), with the city of Campo Grande recording the largest number of cases per capita ever reported in a state capital (44,695 cases), meaning one case per 16 residents (Oliveira et al. 2009).
Each year it is estimated that infections with dengue virus are responsible for more than 100 million of classic cases and more than 500 thousand cases of hemorrhagic dengue fever worldwide (Halstead 2007).In Brazil, from January to July 2010, 942,153 cases were reported in the country.In the same period of the previous year, 593,669 cases were reported, representing an increase of 58.7%.The states with the highest incidence were Acre (3,619.5 cases per 100,000 inhabitants), Mato Grosso do Sul (2,521.1 cases per 100,000 inhabitants), Goiás (1,353.1 cases per 100,000 inhabitants), Rondônia (1,256.4cases per 100,000 inhabitants), Roraima (1,146.9cases per 100,000 inhabitants) and Mato Grosso (1,095.5 cases per 100,000 inhabitants).These six states make up 75% of cases in Brazil (Oliveira et al. 2011).
The safest way to combat or prevent a virus is immunization.However, other measures have been employed in the control and against vector spread, such as environmental sanitation, epidemiological surveillance, laboratory and research support, and education (Teixeira et al. 2009).
A current form of control is fogging insecticides such as organophosphate and pyrethroid, but their continuous use has resulted in resistance of populations (Guirado and Bicudo 2009) and exposure of operators (Vilela et al. 2010) to the compounds.These effects have been reported in Latin American countries, decreasing the effectiveness of this strategy in mosquito control (Rodríguez and Fernández 2007).
Chemical control with organophosphates proved to be inefficient in combating the mosquito.Among the organophosphates, temephos was reported as Aedes-resistant by Melo-Santos et al. (2010).Malathion and fenitrothion, which are used for controlling the adult stages of Aedes, are encountering resistance in different degrees (Pontes et al. 2005).
Concerns about the possible impacts on the environment and population health have allowed the development of products based on plants as an alternative to the use of synthetic chemicals (Oliveira Filho 2008).Studies have been developed to obtain products from plants with insecticidal properties (Furtado et al. 2005, Garcez et al. 2013).
In this study, we analyzed the insecticidal potential of plant extracts and fractions found in Mato Grosso do Sul on larvae of A. aegypti.

PREPARATION OF EXTRACTS
The plant material (leaves, stems, roots and fruits) were dried in an oven at 45 °C.The extraction with different solvents occurred by continuous maceration until exhaustion of the plant extract, the solvent was then removed via rotary evaporator and concentrated to obtain a homogeneous emulsion.Part of the extracts were dissolved in methanol/ water and partitioned with solvents of different polarities (hexane, dichloromethane, chloroform and/or ethyl acetate).
The extracts and fractions of the species are presented in Table I.The descriptions are established in accordance with the updated nomenclature by the List of Species of the Brazilian Flora (2013).

EXPERIMENTAL COLONY
Collections of A. aegypti eggs were made in five neighborhoods in the city of Campo Grande, Mato Grosso do Sul, in partnership with the Center for Zoonosis Control (CCZ) using adapted methodology (Miyazaki et al. 2009, Gomes et al. 2007).The eggs were subjected to incandescent lighting for seven days until maturity.After that, F1 generation was produced and bioassays were conducted (Barata et al. 2001, Shaalan et al. 2005, Pontes et al. 2005).
The eggs were subjected to hatching in dechlorinate water and kept in Biochemical Oxygen Demand (BOD) incubator at 27 ± 2 ° C and 14-hour photoperiod.The larvae were fed with cat food and water was daily replaced, as well as pH checked.In the adult stage, females were fed with blood supply every three days for 1.5 hours and males with 8% sugary food.BIOASSAYS Third stage larvae were selected for the testing, with larval density of 1:1 larvae/mL extract dissolved in dimethyl sulfoxide (DMSO) at 1%.A negative control group of larvae was submitted to only dechlorinate water and DMSO at 1% and a positive control group to rotenone concentration from 0.002 to 0.020 mg mL -1 for 10, 50 and 90% of the exposed population group.The analysis was performed in quadruplicate.
Samples of each extract were dissolved in 1% DMSO to obtain a solution of 0.5 mg mL -1 .It was initially used for the preliminary tests with two replications per species for a toxicity general testing and then in dilutions of 0.25; 0.12; 0.06 and 0.03 mg mL -1 in quadruplicate.The test results were checked after 24 hours of exposure.

STATISTICAL ANALYSIS
For the analysis of toxicity testing (LC 10 , LC 50 and LC 90 ), parametric regression method Probit (Mclaughlin 1991) was performed using Leoraâ software (POLO 9735947870655352).

RESULTS AND DISCUSSION
The results of toxicity testing for larvae of A. aegypti at 0.5 mg mL -1 in 61 extracts, 25 fractions and 50 plant species are presented in Table I.
The extracts of S. grisebacei, B. dracunculifolia and G. americana showed larvicidal activity capable of causing death in 30% of the population at concentration of 0.5 mg mL -1 .However, these extracts did not result in mortality at lower concentrations and hindered the calculation of LC 50 .
The  total mortality at a concentration of 0.5 mg mL -1 (Table I) and lethal concentrations (LC) at lower dilutions (Table II).
Derivatives of the emulsion composed of different species of Copaifera showed insecticidal activity against vectors of A. aegypti and A. darlingi.The lethal concentrations for A. aegypti of Copaifera sp.LC 50 showed values of 47 mg L -1 and LC 90 91 mg L -1 .For C. guianensis, the values were  LC 50 136 mg L -1 and LC 90 551 mg L -1 (Prophiro et al. 2012, Trindade et al. 2013).
The second major toxicity was obtained in ethanol extract of P. hispidum leaves.Piper has been described as effective insecticide.Studies with P. aduncum showed larvicidal activity of essential oil on A. aegypti (Oliveira et al. 2013).
The extracts of leaves and roots of P. aduncum and extracts of leaves, fruits and branches of P. tuberculatum at 0.5 g L -1 caused total mortality in A. aegypti larvae (Pohlit et al. 2004).The essential oils of P. gaudichaudianum, P. permucronatum, P. humaytanum and P. hostmanianum against A. aegypti larvae were analyzed.The most active oil was P. permucronatum LC 50 0.36 mg mL -1 , followed by P. hostmanianum LC 50 0.54 mg mL -1 (Morais et al. 2007).
According to Misni et al. (2008), the essential oil of P. aduncum acts as an excellent repellent for A. aegypti.Subsequent studies showed that the essential oil at 8 and 10% led to significant mortality higher for A. aegypti (80%) than for A. albopictus (71.6%) (Misni et al. 2011).
The essential oil of P. marginatum inflorescences did not affect the oviposition when at concentration of 0.05 mg mL -1 , but was effective in mortality of A. aegypti larvae at LC 10 and LC 50 of 0.0138 and 0.02 mg L -1 , respectively (Autran et al. 2009). Sousa et al. (2008) reported low toxicity when analyzing the action of Piper species on A. aegypti larvae.
The grandisin lignan isolated from P. solmsianum caused larval mortality at LC 50 150 mg mL -1 .Histological analyzes reveal that the larvae had changes in the digestive tract, in the anterior and middle intestine with a tissue disorganization followed by death (Leite et al. 2012).
According to the third LC 50 , the most toxic extract was obtained from S. variabile (Table I).
The larvicidal action on A. aegypti may be related to its antioxidant activity, causing inhibition of esterase, glutathione transferases or monooxygenases.This action was cited by Guirado et al. (2009) as one of the defense mechanisms of vectors and a common condition observed in resistant populations found in the national territory.
Preliminary phytochemical analysis of Capsicum frutescens var.longum showed high content of tannins, alkaloids, steroids and glycosides present in leaves and fruits (Vinayaka et al. 2010).They may be used as blockers of enzyme action since one of the mechanisms for larval mortality is related to the metabolism of esterases and other monooxygenase.Gupta et al. (2011) evaluated the larvicidal potential of seed α-amylase inhibitor of Macrotyloma uniflorum in larvae, pupae and adults of A. aegypti and found adulticidal and larvicidal effect at a concentration of 0.2 mg mL -1 .
To analyze the larvicidal potential of Cestrum nocturnum extract, Jawale et al. (2010) classified as active with 100% larvae mortality in 24 hours, setting LC 100 12 mg L -1 and LC 50 6 mg L -1 .In accordance with the results obtained for the extracts of Plumbago zeylanica and C. nocturnum, the presence of bioactive phytochemicals influenced life cycle of A. aegypti by inhibiting the development of pupae and adult emergence (Patil et al. 2011).
The extract of T. umifolia leaves in this study was the only one with no reports of insecticidal activity.The genus Turnera (Turneraceae), known as chanana, has distribution in Brazil and South America.Santos et al. (2010) analyzed of the extract of T. ulmifolia leaves and observed the molluscicidal activity against Biomphalaria glabrata, and cytotoxicity with Artemia salina.The species has also been effective in controlling infectious forms of Trypanosoma cruzi and Leishmania (Santos et al. 2012).
Reports from Nazar et al. (2009) and Dhanasekaran et al. (2013) describe the larvicidal and ovicidal potential and the repellent in ethanol extract of S. hispida against Anopheles stephensi, A. aegypti and Culex tritaeniorhynchus.The mortality of A. stephensi occurred at LC 50 89.45mg L -1 and 100% repellency against female adult mosquitoes.Over 75% mortality was obtained from ethanolic extract of Spermacocea verticillata at concentration of 250 mg L -1 on the larvae of A. aegypti (Souza et al. 2013).
The data obtained in this study from larvicidal activity and literature information enabled the characterization of the species potential.The family(ies) evaluated proved its potential compared to the literature.However, it is recommended to continue the monitoring studies of sub-lethal dosages and fractionation of greater efficiency extracts for identification of main active principles which can be used as efficiency markers for other species.

CONCLUSIONS
This study shows the insecticide potential in plants found in Mato Grosso do Sul, Brazil.Forty-eight botanical species were analyzed and showed potential insecticide of O. arborea, S. latifolia, S. variabile, P. hispidum, P. aduncum and T. umifolia in order of efficiency for causing toxicity at lower concentrations.
Fifty plant species were collected from 1999 to 2012.Forty-eight came from different regions of the Cerrado and Pantanal in Mato Grosso do Sul and two exotic species from Campo Grande -MS (Melia azedarach L. e Nerium Oleander L.).The voucher specimens were sent to identification to the herbarium of Anhanguera -Uniderp University (by Eloty Justina Dias Schleder) and Federal University of Mato Grosso do Sul (by Vali Joana Pott and Arnildo Pott).

TABLE I (
continuation) An Acad Bras Cienc (2017) 89 (2) *30%: percentage of minimum mortality for testing with toxicity indicative in plant extracts at different concentrations as established by McLaughlin (1991).

TABLE II Lethal Concentration (LC) in mg mL -1 for the different extracts of Aedes aegypti larvae.
of leaves.B= Ethanolic extract of seeds.C= Hexane extract of leaves.D= Methanolic extract of leaves.E= Chloroform extract of leaves.