Aedes aegypti (Lin, 1762) is the vector mosquito that has the highest current dispersion in urban areas over the world and the medical importance of its vectorial capacity is to disseminate four serotypes of dengue virus as well as yellow fever. The insect’s diurnal haematophagic activity, the synanthropic behavior and anthropophilic habits associated with the physical complexity of urban centers have hampered its combat (Bessera et al., 2009). Each year infections with dengue virus are estimated to be responsible for more than 100 million of classic cases and more than 500 thousand cases of dengue hemorrhagic fever worldwide (Halstead et al., 2007). In Brazil Dengue fever has reached about 3 million cases since 1986.

Source of plant material

The CNSL was provided by Kardol Chemical Industry, Campo Grande, Mato Grosso do Sul - Brazil, from the endocarp of the A. occidentale. The extraction of LCC is occurred with company standard and patented methodology.

High performance liquid chromatography (HPLC)

The CNSL was further submitted to HPLC (Varian 210, Diode Arrangement Detector) and software Star WS (workstation) was used. The columns used were reverse phase C-18 (Phenomenex) and the pre-column C-18. The elution process was carried out with a graded program of solvent A: acetonitrile/water/acetic acid; and B: 100% of tetrahydrofurane. The pump flow rate was 1 mL min^-1 and 20 μL were injected (22°C).

Identification of the compound with the aid of DAD detector scanning in the spectral range of 200 to 800 nm did not reveal interferences in retention time of the sample in HPLC by the developed elution method. Standard was easily identified and quantified based on their absorption spectra in the UV region and in retention time. Standard found in extracts were unambiguously identified by performing co-injection experiments in which aliquots of the extract and standard were mixed and diluted to a known volume, and analyzed through HPLC. The content estimation of the anacardic acid (≥85%, Sigma Aldrich) in the CNSL was performed by external calibration.

Bioassays

The experiments were performed in the Laboratories of Entomology of Dom Bosco Catholic University (UCDB), Campo Grande, MS. Larvae of late 3^rd and early 4^th instar of A. aegypti were used in the tests, from mass rearing of eggs aged from two to three months after oviposition, obtained from successive generations in vitro maintained with blood meal for one hour and half every two alternate days in the presence of partial light and temperature between 25 and 30°C according to a validated method of Porto et al. (2013) for conditions established by the use of biological oxygen demand (BOD).

In order to confirm, susceptibility tests were performed with concurrent positive control of Rotenone concentrations ranging from 0.005 to 0.020 mg mL^-1. The larvicidal biotest was performed in quadruplicates using four different concentrations of CNSL: 1.0, 0.1, 0.01 and 0.001 mg mL^-1, in batteries of tests at different times, because the product is only 69% of anacardic acid and a mixture of several minor components. Twenty-five larvae were exposed to each specimen in the test solutions (25 mL) for 24 h, this is an acute test; after this period, the number of dead insects and survivors through stimulation with Pasteur pipette was observed. Individuals that did not move after stimulation were considered dead. Concentrations with the highest mortality were submitted to calculate the concentrations of larvicide Lethal Doses (LD).

Statistical analyses

The results obtained were statistically analyzed by the Probit method (Mclaughlin et al., 1991), using software Leora® (Polo 9735947870655352). The following standard doses prior dilution 1:10 considering the highest and the lowest toxic dose and four doses between 0 and 100% effect were used. Then, parametric analysis was performed by the probit method, using a random

Composition of the CNSL and calculation of lethal concentrations (LC)

The product was provided by Kardol, in which HPLC analysis showed the presence of 69% of anacardic acid and the remainder as a pool of substances according to benchmark analysis. From the larval mortality curve (Y=28.635ln(x) + 138.8, R² = 0.9181) in relation to increasing concentrations of CNSL (Figure 1), the minimum concentration capable of producing mortality (LD₁₀) was 0.01 mg mL^-1 and the maximum concentra-tion of toxicity (LD₉₀) was 0.13 mg mL^-1. The LC₅₀ was calculated in the dilution range of about 0.07 mg mL^-1 (Figure 2). Tests carried out with diluted CNSL caused mortality of dilution from 0.01 to 0.1 mg mL^-1, and the confidence interval for mortality is a significance level of P <0.05. Anacardic acids and derivatives have been reported in a number of works as having insecticidal action, particularly for A. aegypti (Lomonaco et al., 2009). The results of this study demonstrate that A. aegypti larvae and pupae are highly susceptible to 0.012 mg mL^-1 CNSL. Larvae and pupae of Anopheles subpictus, however, are susceptible to 0.038 mg mL^-1 Cardanol/CNSL solution (Mukhopadhyay et al., 2010). The cardol (LC₅₀ = 14.20 ± 0.62 mg mL^-1) and cardanol (LC₅₀ = 32.90 ± 0.2 mg mL^-1) obtained from CNLS showed activity for A. aegypti (Lomonaco et al., 2009). According to Oliveira et al. (2011), the three components of CNSL demonstrated good larvicidal activity against A. aegypti (LC₅₀ = 12.40 mg L^-1 for anacardic acid; 10.22 mg L^-1 for cardol; and 14.45 mg L^-1 for cardanol).

Histological analyses

The third instar A. aegypti larvae in the control group showed elongated and wormlike external appearance. The histological sections showed a normal morphology of stomodeum stomach and intestinal cecum (Figure 3AB). In the middle region of low cylindrical cells, acidophilic cytoplasm with variable areas, central spherical nucleus and clear nucleolus were seen. The brush border is thicker and marked (previous) and the peritrophic matrix (PM) is evident (Figure 3C). Morphological analysis in the treated group based on different concentrations of CNSL showed concentration of 0.001 mg mL^-1 in three regions, cell shedding in the midgut, food debris and fragmentation of MP. The changes were apparent in gastric cecum, such as loss of brush border, and the absence of material ingested by the larvae in the lumen (Figure 4A). In the middle midgut, lining cells were rounded, with lots of side crossings and scaling, with a decrease in brush border and profuse secretion in the lumen (Figure 4B). The epithelium hindgut cells completely lost the lateral junction of flooring and base, resulting in detachment of the cells to the lumen of the hindgut. The brush border was evidenced in these cells (Figure 4C).

The transition zone between the anterior and medial regions were formed by throttling midgut with intestinal obstruction at a concentration of 0.1 mg mL^-1 (Figure 5A); in the posterior region, the cells had elongated brush border. The deleterious changes included total or partial destruction of the lining cells, high vacuolization of the cytoplasm and increased subperitrophic space with accumulation of acidophilic materials (Figure 5C). The Culicidae midgut of the larvae comprises three regions (previous, middle and further) and is formed by a layer of epithelial cells lining integral supported by a cylindrical basement membrane. Like other insects, insect abdominal region, in addition to its digestive function, provides chemical defense function, mechanical protecttion and defense against pathogens (Roel et al., 2010). The first change caused by anacardic acid in A. aegypti larvae was related to movement, contrasting the control group that showed great mobility and locomotion in liquid medium. The larvae subjected to the highest concentrations of anacardic acid showed low resistance, lethargy and immobility, with an overall mortality after 24 h of exposure. By observing the tissue section, it was possible to detect the disconnection of cells lining the basement membrane and loss of intercellular junctions.