Larvicidal and Oviposition Activity of Against Vector Aedes Aegypti and Molecular Docking Studies of Metabolites from the Crude Extract of the Endophytic Fungus Aspergillus Sp. Isolated from Bertholletia Excelsa Humn. & Bonpl.

This work showed the crude extract of the endophytic fungus Aspergillus sp, isolated from the almonds of Bertholletia excelsa Humn & Bonlp collected in the Brazilian Amazon, oviposition deterrent, and larvicidal activity of against Aedes aegypti. In the oviposition deterrence test was observed that females able to lay eggs preferred the control oviposition sites (46.6%), suggesting the extract also could repel the oviposition. Futhermore, the extract showed larvicidal activity with LC 50 26.86 µg/mL at 24 hours and 18.75 µg/mL at 48 hours. Molecular docking studies were carried out to elucidate the mechanism of action of the compounds identied against the enzyme acetylcholinesterase. The compound Aspergillol B was a potent larvicide with potential for inhibition for the acetylcholinesterase enzyme (-7.74 Kcal/mol). These unprecedented results reported indicate that the secondary metabolites obtained from crude extract of Aspergillus sp. present useful biological potential against vectors of public health importance and antibiotic-resistant bacteria.

The content of mortality count was carried out in periods of 24 and 48 hours after exposure to the larvae solutions. They were considered dead larvae were not able to reach the water surface (Rodrigues et al., 2014) and using the readings was possible to establish the lethal concentrations (LC 50 and LC 90 ) by probit analysis. All bioassay experiments were conducted according to standard (World Health Organization, 2005).

Oviposition-deterrence test
The oviposition-deterrence effect of the acetyl extract of the endophytic fungus Aspergillus sp. in the laying of eggs. Ae. aegypti has been studied in 50 pregnant females (fed with blood) in 40 x 40 x 40 cm opposition cages under conditions of 25 ± 2 ° C and relative humidity of 75 ± 5% and a 12-hour photoperiod according to WHO. The cages contained dark ovitraps with extract at concentrations of 45, 35 and 20 µg / mL and control with distilled water and DMSO (0.5%). The test was done in quadruplicate and the number of eggs was rated with 48 hours.

Statistical analysis
For larvicidal activity the lethal concentrations (LC 50 and LC 90 ) were determined after 24 and 48 h of incubation and calculated using Probit analysis with StatGraphics Centurion. To evaluate the ovipositiondeterrence test, an analysis of variance (ANOVA) was performed with Turkey to determine if there is a signi cant difference between concentrations and control with GraphPad Prism 8.0.

Molecular docking simulations
In this stage, the molecules were optimized by computational method DFT B3LYP 6-31G** (Braga and Valle, 2007;Lo et al., 2017;Siegel et al., 2017;Warren et al., 2016) and used as an input le for molecular docking simulations, in order to evaluate the score of the energy function through the free-energy (ΔG) of molecule interaction from virtual ligand-based screening as well as the analysis of conformations, mode of interaction and binding a nity with the selected receptors.
The X, Y and Z coordinates of the receptors were determined according to the average region of the active site. The coordinates used for the center of the grid can be seen in Table 1.

Biological Activity Predictions of the molecules
The Prediction of Activity Spectra for Substances (PASS) were performed while using the webserver http://www.pharmaexpert.ru/passonline. PASS makes it possible to relate effects of the molecule based entirely on the molecular formula using Multilevel Neighbors of Atoms (MNA) descriptors, suggesting that biological activity is in function of its chemical structure (Ferreira et al., 2017;Kirchmair et al., 2015). Only molecules with insecticidal and anticholinesterase activities were selected at this stage.

Endophyte species identi cation
The ITS fragments and the beta-tubulin gene were successfully ampli ed and sequenced from the samples' genomic DNA. The fungus sample Biorg 09 ( Fig. 1) named CPQBA 1929/19 DRM 03 was sequenced and identi ed as Aspergillus sp. as the partial sequence of the beta-tubulin gene had 100% similarity with the corresponding sequences from the Aspergillus genus deposited in GenBank and CBS-KNAW databases. The genetic distance analysis retrieved the CPQBS 1929/19 DMR 03 sample in a low resolution cluster (68%) with the type strain of the species A. violaceofucus CBS 123 and A. japonicus CBS 11451, the latter being a synonym for the rst, thus, the results of the analyzes carried out in the databases and phylogeny suggest the nal identi cation as Aspergillus sp., within the nigri section (Fig. 2).
The Aspergillus genus is found in all areas throughout the world and has a high economic and social impact. In this genus are found species used in biotechnology for the production of metabolites with useful applications, such as antibiotics, organic acids, medicines or enzymes, and food fermentation agents (Samson et al., 2014).
The nigri section of the Aspergillus genus, commonly known as black Aspergillus, was formerly identi ed and classi ed based on morphology, according to Yokoyama et al. (Yokoyama et al., 2001). However, this section is one of the most challenging groups for identi cation, and several taxonomic schemes have already been proposed. Currently, molecular methods are used for this purpose (Varga et al., 2011). Ferranti et al. (Ferranti et al., 2018), studying the fungal diversity on the surface of Vitis labrusca and hybrid grapes, reported 13.9% of the Aspergillus genus, and species of the nigri section were found in most samples. The molecular characterization of Aspergillus in grapes showed a genetic divergence between strains of this genus and reported an ochratoxigenic strain, often isolated from grapes, called A. tubingensis (Martínez-Culebras et al., 2009). It is observed that the nigri section presents a high diversity, as was also observed in this study.

LC-MS/MS analysis
The LC-MS/MS is used for the obtention of spectra from the products' ions to assess the chemical structure of its compounds. It can analyze neutral losses and simultaneous product ions from a single dataset (Renaud et al., 2017). Employing this technique, we identi ed several metabolites produced by Aspergillus sp. isolated from Bertholletia excelsa, as shown in Table 2.
More than 330 species of fungi are known in the Aspergillus genus, found in different ecosystems; this genus has a high impact on the health of humans, animals, and plants. The species of this genus produce a remarkable number of secondary metabolites with the most varied biological activities reported (Soltani, 2016).
The crude acetyl extract of Aspergillus sp. (Biorg 09) isolated from B. excelsa presented several molecules, some of them already reported to have biological activities with potential applications in the treatment of infectious diseases, diabetes, Alzheimer, physiological disorder and are a source of antioxidant agents (Mikawlrawng, 2016). Some of the metabolites found, like enniatins, are characteristic of Fusarium species (Jonsson et al., 2016).
Cyclodepsipeptides (enniatin) were described more than 60 years ago, and also have broad biological activities like antitumor, anthelmintic, insecticide, antibiotic, antifungal, immunosuppressant, antiin ammatory and antimalarial (Sivanathan and Scherkenbeck, 2014). It is important to note that this class of metabolite is identi ed mainly in fungi of the genus Fusarium (Renaud et al., 2017). Some of their biological activities are attributed to the capacity to increase the ow of alkali metal ions through biological membranes (Ivanova et al., 2006).
Aspernolide, an aromatic butenolide compound, is a metabolite found in some endophytic species, such as in the marine endophytic fungus A. terreus (Parvatkar et al., 2009). Ibrahim et al., 2015 evaluated the antimicrobial activity of butyrolactones isolated from this endophytic fungus against S. aureus, and the and anti-leishmanial activity against the promastigote phase of Leishmania donavi. Another relevant substance identi ed in Aspergillius sp. was aspulvinone, already reported to be an inhibitor of the viral neuraminidase H1N1 (Gao et al., 2013), and a potent antioxidant agent (Zhang et al., 2015).
The compound paclitaxel, known as taxol, is a well-known and effective drug used in the chemotherapy of cancers in the head, neck, breast, lung, bladder, ovary, and cervix. It is a substance produced originally by plants from the genus Taxus. However, the symbiosis between plant and fungi made some endophytic fungi also produce this molecule (Kumar et al., 2019).
According to Zhu (Zhu and Chen, 2019), the extraction of paclitaxel from endophytic fungi has proven to be an effective alternative way to get the drug. Among endophytic fungi, several species, including Aspergillus fumigatus, Pestalotiopsis microspora, Alternaria brassicicola, are reported to produce this compound (Gill and Vasundhara, 2019;Kumar et al., 2019;Subban et al., 2019;Zhu and Chen, 2019), and our results indicate that Aspergillus sp. isolated from Brazil-nut tree also can.

Larvicidal effect of the extract and oviposition deterrence test against Aedes aegypti
In the larvicidal test against Ae. aegypti, it was possible to observe that the extract had effective larvicidal activity in 24 and 48 hours. With a mortality of 84% at 45 µg/mL and 8% at 5 µ/mL in 24 hours; 96% at 45 µ/mL and 26% at 5 µg/mL in 48 hours. Thus demonstrating the extract presents a set of compounds with potential use for the control of this vector of public health importance.
We further evaluated the values of lethal concentrations (LC) 50% and 90% for 24 and 48 hours (Table 4). For 24 h, the LC 50 was 26.86 µg/mL, and the CL 90 was 47.55 µg/mL; for 48 h, the LC 50 was 18.75 µg/mL, and the LC 90 was 38.93 µg/mL. These values show a remarkable e ciency of the extract that is timeand dose-dependent.
As stated by Geris et al., 2008, plants' secondary metabolites have a range of biological activities due to their structural diversity. These molecules are a continuous source of inspiration for drug discovery. Nevertheless, fungi also produce secondary metabolites with the most varied activities reported, such as antimalarial (Isaka et al., 2007), antibacterial (Hu et al., 2008), antiviral (Linnakoski et al., 2018), antifungal (Hoffman et al., 2008), and leishmanicidal (Cota et al., 2018).
De acordo com Balumahendhiran et al. (2019) also tested the larvicidal activity of the crude extracts from fungi of the Aspergillus genus (A. avus and A. fumigatus). The extract was tested against three diseasetransmitting mosquito species: Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti. For the extract from A. avus, the authors reported the following (against Ae. aegypti): for 24 hours, the LC 50 was 18.01 µg/mL, and the LC 90 was 31.05 µg/mL; for 48 hours, the LC 50 was 50.39 µg/mL, and the LC 90 was 82.26 µg/mL. For the extract from A. fumigatus, in turn, the authors reported: for 24 hours, the LC 50 was 14.79 µg/mL, and the LC 90 was 35.40 µg/mL; for 48 hours, the LC 50 was 35.37 µg/mL, and the LC 90 was 73.30 µg/mL. Overall, the results of these authors corroborate our's and emphasize the potential of using fungi metabolites for the control of public health importance vectors.
An ethyl acetate extract from the mycelium of the entomopathogenic fungus Beauveria bassiana was tested against the four larval stages (LI, L2, L3, and L4) of Ae. aegypti by Ragavendran et al. (Ragavendran et al., 2017). The authors observed the larvae mortality within 24 hours of exposure to different concentrations (50, 100, 150, 200, 250, and  According to Montenegro et al. (Montenegro et al., 2006), extracts show promising activity when the mortality is equal or higher than 75% at 250 µg/mL. For Cheng et al. (Cheng et al., 2003), potential substances need LC 50 values lower than 100 µg/mL to be considered a good larvicidal agent. Here, 84% of mortality was observed within 24 hours at 45 µg/mL. The discovery of new compounds to ght Ae. aegypti is attractive since, in the last 15 years, populations of this mosquito developed resistance to the main organophosphate and pyrethroid insecticides in several countries (Rocha et al., 2015). Another disadvantage of the current insecticides is the toxicity caused to the environment.
The insecticidal activity of fungal products is not always attractive for commercial use. For instance, Pradeep et al. (Pradeep et al., 2015) evaluated the larvicidal activity of the isoquinoline-type pigment produced by the fungus Fusarium moniliforme against Ae. aegypti and An. stephensi. They reported LC 50 of 237.0 µg/mL and 335.6 µg/mL in 24 hours, respectively; these are considered high values for vector control.
In addition to using metabolites produced by endophytic fungi, another approach is using a suspension of conidia, or an association of conidia and neem oil activity against Ae. aegypti larvae (Gomes et al., 2015). This demonstrates that using fungi conidia and metabolites, either isolated or associated, can be a valuable tool for vector control and other applications.
In our study, it was also possible to observe external structure changes in the larvae caused by the crude extract of Aspergillus sp. (Fig. 3). It is noted a loss of color and loss of segmentation in the treated larva (Fig. 3b), unlike in the control group (Fig. 2a), where is observed segmented abdomen structures (AB) and normal coloration under the optical microscope. We suggest that the extract of Aspergillus sp. could interact with the larval chitin cuticle, causing changes that resulted in their death. It is essential to highlight that these changes were not observed in the control group, treated with water and DMSO.
According to (Ureña et al., 2019), several mechanisms may be involved in the mortality of Ae. aegypti larvae by insecticides. Some substances act directly on the central nervous system; others can lead to cell damage and, consequently, the death of the larvae because the digestive system tries to eliminate the compound, leading to extrusion of the peritrophic matrix (Valotto et al., 2011). Red seaweed extract was also reported to affect the larvae's external structure. When compared to the control, the larvae had progressive deformities in their lateral hair, anal papillae, distorted body, loss of color, and changes in the respiratory siphon (Deepak et al., 2019).
These external larval structure changes have also been reported as one of the mechanisms of larvae death in other studies, along with a darker or pale body, cuticle changes, and other larvae aberrations (Araújo et al., 2020;Kuo et al., 2015). The histopathological effect of spores from the fungus Aspergillus clavatus against Culex quinquefasciatus was also assessed (Bawin et al., 2016). The authors reported that the spores adhered to the external cuticle and the larval digestive tract; this accumulation caused gradual destruction of the digestive epithelium, muscular and connective tissues, and the epidermis right below the cuticle, which together may be accountable to larval death.
In the oviposition deterrence test, it was observed that pregnant females of Ae. aegypti preferred laying eggs (46.6%) in the control oviposition site with water and DMSO (0.5%). There was no signi cant difference among the different concentrations of the extract (ANOVA, p > 0.05). However, there was a statistical divergence between the extract doses 20, 35, 45 µ/mL compared to the control.
According to Soonwera and Phasomkusolsil (Soonwera and Phasomkusolsil, 2017), the application of oviposition repellent is an effective strategy for controlling mosquito populations. These authors tested the effect of the essential oil from Zanthoxynlum limonella Alston (Rutaceae) against the oviposition of Ae. aegypti and Cx. quinqufasciatus. They reported that a solution made of 10% of this oil dissolved in ethanol 70% had 100% repellency against Ae. aegypti and 99.53% against Cx. quinquefasciatus. In the environments receiving insecticides, these agents act as modulators of insect growth (Tilak et al., 2005), by preventing oviposition and consequent population boost, which is especially relevant against insects of medical importance, like Ae. aegypti.
In a recent study by Michaelakis et al., 2020, the authors tested an aqueous suspension of spinosad -a natural bioinsecticide toxic to pest and vectors -against Cx. Pipiens molestus larvae. However, there was no signi cant difference between spinosad compared to the control, different from our study, where the crude extract, besides being effective against the larval stage, also could repel the oviposition of pregnant females in 48 hours.
Other natural products also have shown promising results in the oviposition deterrence test against Ae. aegypti, such as the extract of Melanochyla fasciculi ora (Zuharah et al., 2015), the essential oil, and aqueous extract of Alpinia purpurata (Santos et al., 2012). Besides being repellent against the oviposition, these products also were effective against the larval stage. Such compounds can potentially be used in the water for vector control by preventing pregnant females from depositing eggs, or by killing the larvae.

Molecular docking simulations and biological activity
Retrieving the shapes of GNT inhibitors, I40 and HJIII, it was possible to validate the molecular docking protocols, calculating the RMSD values of 0.36, 0.78 and 0.94 Å, respectively. According to (Gowthaman et al., 2008;Ramos et al., 2019), the binding mode predicted using docking indicates that when the RMSD is less than 2.0 Å in relation to the crystallographic pose of a respective ligand, the validation is considered satisfactory. The best results of the validation can be seen in Fig. 3.
The residues of active sites for GNT (PDB 4EY6) are around the α-helix between the amino acid residues 336-338 and in the β-leaf between the amino acid residues 85-87, 121-124 and 202-203. For the ligand it was possible to notice common hydrogen bonds with the residues Tyr124, Glu202 and Ser203. A hydrophobic interaction with residues Trp86, Gly121 and Tyr337 is also highlighted (Cheung et al., 2012). Generated docking pose made it possible for the ligand to also interact with the amino acid residues of the I40 active site (PDB ID 1QON) around the α-helix between the Thr369-Asp375 amino acid residues and included in the β-leaf between the amino acid residues Ile82-Trp83. In the ligand, it is possible to observe hydrophobic interactions with the vast majority of residues in Tyr71, Trp83, Tyr370, Phe371, Tyr374 and His380, results that are in agreement with the studies by (Harel et al., 2000).
In order to assess whether the interactions showed greater binding a nity than the speci c control ligands (I40, GNT and JHIII) for acetylcholinesterase from different organisms (Drosophila melanogaster and Homo sapiens) and juvenile mosquito hormone (Ae. aegypti) it was observed that of molecules submitted to docking, only Aspergillol B, Aspernolide A and Aspulvinone D presented values greater than or equal to the negative controls used. The binding a nity values of the new inhibitors at the acetylcholinesterase receptor can be seen in Fig. 4.
Inhibitors complexed with Aspergillol B (-8.2 Kcal /mol) and Aspulvinone C (-8.5 Kcal /mol) at the acetylcholinesterase receptor showed values close to the pyriproxyfen control, whereas Aspulvinone D had a higher value due to the binding a nity of -9.3 Kcal/mol. Therefore, it is suggested that the molecule is promising for insecticidal activity, since it presents interactions similar to the controls used in the docking study. Thus, comparing the a nity values of Aspulvinone D molecule to GNT control, a difference of ± 0.7 Kcal/mol is observed, and to the others a variation of ± 1.8 to ± 1.5 (Kcal/mol).
When the study was related to the organism of Drosophila melanogaster, the Aspernolide A molecule showed a binding a nity of -9.9 Kcal/mol, followed by Aspergillol B with − 9.5 Kcal/mol compared to controls I40 and pyriproxyfen (PDB ID 1QON), according to Fig. 5B. I40 exhibited a binding a nity of -13.3 Kcal/mol higher than pyriproxyfen of -9.0 Kcal/mol. The Aspergillol B and Aspernolide A molecules showed a higher binding a nity value than the pyriproxyfen (control) used in molecular docking. Thus, when comparing the Aspernolide A compound with the I40 control, a difference of ± 3.4 Kcal/mol was observed, while a variation of ± 3.8 Kcal/mol was observed in Aspergillol B, as shown in Fig. 5B.
In relation to the 5V13 complex (Ae. aegypti), the Aspergillol B and (+)-N-deoxymilitarinone showed a higher binding a nity value compared to JHIII (control) used with a value of -9.4 and − 9.7 Kcal/mol, respectively. The results of the a nity values can seen in Fig. 5. JHIII showed a binding a nity of -8.9 Kcal/mol lower than the pyriproxyfen of -10.6 Kcal/mol. However, a binding a nity variation of ± 1.2 kcal/mol of Aspergillol B and ± 0.9 Kcal/mol of (+) -N-deoxymilitarinone is observed compared to pyriproxyfen (control).
Based on the data found, we propose that the molecules present in the extract of the fungus Aspergillus sp. are able to bind to the active sites of the proposed targets. The Aspulvinone D molecule has a greater a nity for the human acetylcholinesterase active site, while Aspernolide A has a greater a nity for the active site of the acetylcholinesterase binding protein in e (+) -N-deoxyilitarinone for the youth hormone. Aspergillol B was the common molecule for both cases, considering the mode of binding in all study targets, thus the promising dual effect is observed, both for the inhibition of the enzyme acetylcholinesterase (human and insect) as well as the juvenile hormone.
Molecules with insecticidal potential can irreversibly inhibit the production of acetylcholinesterase, such an enzyme is responsible for the hydrolysis of acetylcholine (Ach) which ends the propagation of the nervous impulse. The inhibition of the enzyme acetylcholinesterase is the initial mechanism for a substance considered to have a potential insecticide in the larval phase, considering this knowledge cited by several authors it is essential to observe interactions at the active site of the acetylcholinesterase complex, in which three important characteristics are present in order to understand the mechanism of elucidation of the biological action of enzyme production.
In the Aspulvinone C and Aspulvinone D molecules used in the docking study, similar interactions were detected in the control molecules in relation to the acetylcholinesterase active site, located around the αhelix between the amino acid residues Ser203, Tyr337 and Phe338 and in the β-leaf between the Trp86, Gly121 and His447 residues, as shown in Fig. 6. According to (Meriç, 2017) at the AChE active site, the catalytic triad (Ser203, Glu334 and His447) is found in the lower portion of the active site, surrounded by three important parameters for catalytic activity: the acyl sac (residues of Phe295, Phe297 and Phe338), the oxy-anion channel (nitrogen from the main chain of residues Gly121, Gly122 and Ala204) and the choline binding site (Trp86 eTyr337). The most signi cant contributions of the interactions in the docking study were observed in the Aspulvinone C molecule, in which the contribution of the catalytic triad represented by His447 and the connection with the Trp86 hill is notable. Other not so common interactions were also observed with the Tyr72 and Tyr341 residues, these interactions end up stabilizing the ligand in the active center of receptor. The increase in binding a nity, in turn, ends up inactivating the enzyme acetylcholinesterase by competition with an active site with GNT.
The interactions observed in controls I40 and pyriproxyfen in the docking study were also similar in the Aspergillol B and Aspernolide A molecules in relation to the acetylcholinesterase site, located around the α-helix between the amino acid residues Tyr370-Tyr374 and the β-leaf in the residue Trp83, as shown in Fig. 7.
Interactions of potential inhibitors with the amino acid residues Trp71, Trp83, Tyr370, Phe371 and His480 of acetylcholinesterase are similar to those reported in the literature (Fournier et al., 1993;Gnagey et al., 1987). The best evaluated inhibitors in binding a nity parameters were Aspergillol B and Aspernolide A, considering that the interactions were similar to those observed in controls I40 and pyriproxyfen for Tyr71, Trp83, Tyr370 and Tyr374 residues, contributing to the increase in binding a nity. The less common interactions between the inhibitors were Glu80, Gly150 and Phe371, and these electrostatic contributions help stabilize the protein's active site.
Aspergillol B molecule with a free energy of -7.74 Kcal/mol has a hydrogen bond with the amino acid residue Tyr370 and an π-π interaction with Trp83, the latter being also similar at I40. Aspernolide A molecule with the greatest potential for insecticidal activity has greater free energy than the pyriproxyfen control (-9.35 Kcal/mol) and with π-π interaction with a similar Tyr370, however, the interactions are more intense in the inhibitor favored by the reduction the distance. The conformation of inhibitors at the active site is in uenced by the distances of interactions with amino acid residues.
In the juvenile hormone receptor complexed with JHII, the Aspergillol B and (+) -N-deoxymilitarinone molecules had interactions similar to the control molecules present in the amino acid residues located around the α-helix between the amino acid residues Tyr33, Leu37, Val51, Val68 and Tyr129 and β-leaf between Trp53 and Phe144 residues, as shown in Fig. 8 The Aspergillol B molecule with a free energy of -8.57 Kcal/mol has hydrophobic interactions with the Tyr33, Val51, Val65 and Ser69 residues and π-π interactions with Trp33 and Trp53. The molecule with the greatest potential to inhibit the enzyme acetylcholinesterase is (+) -N-deoxymilitarinone, as it has signi cant similar interactions and free energy of -11.79 Kcal/mol compared to the controls used. There are less common interactions for the Tyr129 and Tyr155 residues which contribute to greater stability of the molecule at the receptor.
In the complex of the juvenile hormone protein, the ligand JHIII is present in the binding pouch of the Nterminal domain, the conformation of the crystal being identical to the three chains of the protein. In JHIII, the presence of an epoxy group located in the center of the domain is observed and a methyl ester is oriented towards the surface. The epoxy group forms hydrogen bonds with the phenolic hydroxyl of Tyr129 and the rest of the isoprenoid chain is surrounded by hydrophobic side chains including those of Phe144,Tyr64,Trp53,Val65,Val68,Leu72,Leu74,Val51 and Tyr33 [48].
Predicting the biological activity of molecules submitted via the web PASS server (Harel et al., 2000) resulted in the data shown in Table 3. The reference molecules (pyriproxyfen, I40, GNT and JHIII) showed insecticidal or similar activity, corroborating the results of the literature (Braga and Valle, 2007;Harburguer et al., 2009;Kim et al., 2017;Olmstead and LeBlanc, 2003;Paul et al., 2006;Sullivan and Goh, 2008).
In the molecules analyzed in the biological activity prediction study, only Aspergillol B showed a satisfactory prediction compared to the controls used in the molecular docking study, in which Pa was 0.187 and similar to other bioactive molecules known for insecticidal activity, acetylcholinesterase and acetylcholine inhibitor when Pa > Pi.

Conclusion
Results of this study suggest that metabolic from crude extract of endophytic fungus Aspergillus sp. isolated from Bertholletia excelsa was effective against drug-resistant bacteria strains of S. aureus, demonstrating that the crude extract of Aspergillus sp. can also be used as an antimicrobial agent. Moreover, the metabolites present in the extract are potential natural larvicides to control the vector Ae. aegypti, the ndings of the molecular docking study demonstrated the mechanism of action of the compound Aspergillol B by inhibiting the enzyme acetylinesterase (-7.74 Kcal / mol). Therefore, this compound can serve as a new class of product with environmentally friendly larvicidal activity.      Results of binding a nity of the compounds with (A) human acetylcholinesterase; (B) insect acetylcholinesterase.

Figure 5
Results of binding a nity of the compounds with the juvenile hormone receptor. Interactions of controls I40 (A), Pyriproxyfen (B) and potential inhibitors Aspergillus B (C) and Aspernolide A (D) with insect acetylcholinesterase active site.