Development of an environmentally friendly formulation of silk broin associated with fatty acid from Astrocaryum murumuru Mart. fat, effective against larvae of the Aedes aegypti vector

The Aedes aegypti mosquito is a vector of several diseases, such as dengue, malaria and the Zika virus. Synthetic insecticides such as chlorpyrifos and chlorothalonil have been used for plague control, despite causing damage to the environment and to humans. It is therefore important to study natural active compounds with a low environmental impact. The present paper developed an environmentally friendly formulation of silk broin (SF) associated with fatty acid esters [ethyl (FAEE-SF), propyl (FAPE-SF) and butyl (FABE-SF)] from Astrocaryum murumuru Mart. fat, which was effective against Aedes aegypti 3th instar larvae. The FABE-SF nanoemulsion induced a higher mortality rate in the larvae of the A. aegypti after 48 h (LC 50 = 18.92 µg / mL). The stabilities of the nanoemulsions were monitored for 21 days, and FABE-SF exhibited greater stability throughout the monitored period, with average particle, zeta and PDI values of around 217 ± 0.85 nm, -25.6 ± 3.24 mV and 0.338 ± 0.01, respectively. This paper reported the rst effective action of fatty acid esters from A. murumuru Mart. associated with silk broin against A. aegypti. The FABE-SF solution also had a low hemolytic index, suggesting that treatment may be safe for animal and human use.


Introduction
The effective and sustainable control of mosquito populations and vectors of diseases is currently a challenge (Baldacchino et al. 2015;Benelli and Mehlhorn 2016). For many years, the control of mosquito populations has involved the preferential use of various synthetic insecticides, such as organochlorines, organophosphates and pyrethroids (Rodrigues et al. 2019). However, the indiscriminate and frequent use of such substances has caused selected populations of mosquitoes to become more resistant, as well as resulting in environmental pollution caused by their non-selective inhibitory enzymatic action (Gilliom and Hamilton 2006), that sets humans and other non-target organisms at risk (Sarwar et al. 2009). An alternative is the search for an effective low-cost insecticide, with lower environmental risk (Baldacchino et al. 2015;Benelli and Mehlhorn 2016). studies about the effects of natural products on insects, when used as larvicidal agents (Sharma et al. 2011).
Emulsi cation methods using co-polymers and proteins have become a viable alternative for enhancing the dispersion of essential or xed oils in aqueous vehicles, as mosquitoes and other disease vectors need an aqueous environment to develop their life cycle (Echeverría and Albuquerque, 2019). Emulsions are formed and stabilized by surfactants or by amphiphilic polymers, which aim to reduce interfacial tension when adsorbed at the oil/water interface (Kaci et al. 2017;Sarquis et al. 2020). In comparison, silk broin is as an alternative to the various types of surfactants used in the formulation of emulsions, due to its low production cost, low cellular and environmental toxicity and high biodegradability, as well as being a biomaterial based on proteins (Ferreira et al. 2017a).
During the last decade, nanotechnology has presented a wide scope of applications in different areas such as medicine, electronics, catalysis and agriculture (Pavoni et al. 2019). With its advent, nanopesticides or nanoformulations that are thin lms or encapsulation of the active ingredient of the pesticide in nanostructures have emerged, the main advantage of these nanostructures is the slow and controlled release of their active principles (Bilia et al. 2014). These make them environmentally safe and non-hazardous compared to chemical pesticides (Mehlhorn 2016).
In this context, the researchers have been developing different types of nanopesticides, such as nanoencapsulated formulations, nanoemulsions, nanogels, nanospheres, among others (Kah and Hofmann, 2014). In this way, nanoparticles can help in the production of new pesticides, insecticides and repellents and nanoemulsions can guarantee an e cient and controlled release of the active ingredients (Athanassiou et al. 2018). With this, the nanotechnology can revolutionize insect management, as it can provide innovative tools for the controlled and safe delivery of pesticides (Rai and Ingle, 2012).
Silk broin is a natural polymer produced by various insects, and is therefore subject to great diversity in its sequence, structure, and properties (Zhang et al. 2007). The study of silks has been carried out from the cocoon of the domesticated silkworm Bombyx mori and the Nephila clavipes spider (Nou 1995). Silk broin is a natural amphiphilic block of hydrophobic and hydrophilic co-polymers that collectively mix, giving silk broin exibility and robustness (Tanaka et al. 1999;Zhang et al. 2007). Due to its excellent mechanical properties, high biocompatibility, biodegradability, and low cost (Pham and Tiyaboonchai 2020), silk broin is used in tissue engineering, and in the delivery system of bioactive compounds, such as drugs, peptides and proteins, among others (Kundu et al. 2010;Koh et al. 2015), in addition to a variety of biomaterials, such as nanoparticles, nanospheres, hydrogels and lms (Cheng et al. 2017).
Our research group has been investigating the action of emulsions prepared with silk protein and larvicidal actives. Recently, our Group has developing biopesticides based on the use of silk broin as a vehicle co-polymeric. Araújo (Araújo et al. 2020) evaluated the larvicidal action of the hydroethanolic, methanolic, and hexane extracts of Acmella oleracea leaves solubilized in silk broin (2%), and Sarquis (Sarquis et al. 2020), that reported the use of free fatty acids from Carapa guianensis Aubl oil associated with silk broin, and its activity against A. aegpyti larvae, and found excellent LC 50 results after 48 h of 16.7 µg.mL − 1 .
Therefore, continuing the study of silk protein as a vehicle for active larvicides, the present study aimed to evaluate the preparation of potentially sustainable, stable, and low-cost larvicidal emulsions containing fatty esters from A. murumuru fat, associated with silk broin.

Synthesis of ethyl, isopropyl and butyl esters of A. murumuru Mart. fat catalyzed by Amano AK lipase from Pseudomonas uorescens
The enzymatic transesteri cation of fat was performed separately for ethyl, isopropyl and n-butyl alcohols to generate the respective esters (FAEE, FAPE and FABE), as described by Ferreira with some modi cations (Ferreira et al. 2017b).
Transesteri cation was performed in a 50 mL reaction ask containing 1.0 g of fat, 3 mL of respective alcohol and 0.1 g (10%) of Lipase from Pseudomonas uorescens. The reaction mixtures were magnetically stirred for 24 h at room temperature. Afterwards, the reaction solutions were ltered, the organic phases were dried with anhydrous sodium sulfate and ltered, and the solvents were removed by vacuum under reduced pressure. The products were puri ed by silica gel column chromatography, with the mobile phase being a mixture of n-hexane and ethyl acetate (9:1). The isolated products were characterized by their spectroscopic data (GC-MS and FTIR).

Gas chromatography-mass espectrometry (GC-MS)
The samples (FAEE, FAPE and FABE) were analyzed by gas chromatography mass spectrometry. The gas chromatography-mass spectrometry (GC-MS) analyses were performed in a Shimadzu GC2010 with a mass selective detector (Shimadzu MS2010plus) in electron ionization mode (EI, 70 eV). The GC-MS (equipped with a 30m×0.25mm×0.25µm RTX-5MS column) conditions were: the oven temperature started at 130°C and was kept for 2 min, increased to 290°C at 5°C min − 1 , maintained for 2 min. The total analysis time was 36 min. The injector and detector temperature was maintained at 210 o C; 1 µL was injected with 1:15 split and helium was used as the carrier gas at a 1.0 mL min − 1 ow rate. The ions were monitored from 3 to 36 min in the m/z 40-500. The components present in the samples were identi ed through comparison of spectral data with those in the Wiley library.

Infrared spectroscopy analysis
A Fourier transform infrared spectrophotometer (Shimadzu FTIR IRTracer-100) recorded the spectra of FAT (A. murumuru), FAEE, FAPE and FABE using a potassium bromide beam splitter. The KBr pellet method was employed, and the background spectrum was collected. The range was set from 400 to 4000 cm − 1 with 16-cm − 1 resolution.

Preparation of the silk broin solution
The silk broin solution was prepared based on the method developed by Ferreira (Ferreira et al., 2014). Silkworm cocoon (3.0 g, from Bratac, Brazil) were degummed in boiling a (2%, w/v) Na 2 CO 3 solution for 30 min. The resultant bers were ltrated and washed with distilled water (3 x 500 mL). Subsequently, silk ber were dissolved in a ternary solution (50 mL) of H 2 O:EtOH:CaCl 2 (8:2:1 molar proportions) at 30ºC for 4 h. This mixture was then dialyzed (cellulose tube with an exclusion limit of 16 kDa, from Viskase, Brazil) for 3 days at room temperature, and water changed every 24 hours. The broin solution was centrifuged (6000 rpm for 10 min) to remove impurities and larger particles. The concentration of the silk broin solution was adjusted to 2% (w/w).

Preparation of ester/silk broin emulsions
The emulsions were produced using a spontaneous emulsi cation process that occurs when an aqueous phase and an organic phase are mixed. In summary, deionized water was added to a mixture of esters (FAEE, FAPE or FABE) and the silk broin solution (2%). The 10 mL solution contained 94% silk broin solution (2%), 1% active compounds and 5% of a mixture of ethanol and isopropanol (1:1). The emulsions were prepared according to Sarquis et al. (2020), with slight modi cations.
Initially, a mixture of ethanol and isopropanol was added to the esters (FAEE, FAPE or FABE) under constant magnetic agitation (300 rpm) for 30 min. Next, the aqueous phase containing silk broin solution was added with continuous agitation for 5 min in vortex.
Emulsions were stored under 4 ºC and evaluated from 1 to 21 days following preparation. The droplet size, polydispersity index and zeta potential of the emulsions were determined using a ZS zetasizer (Malvern, United Kingdom). Each sample was diluted with distilled water (1:10) for analysis.
Measurements were made in triplicate. The average droplet size was expressed as mean diameter. All analyzes were performed at 25 ºC.

Larvicidal activity
Esters combined with silk broin emulsions (FAEE-SF, FAPE-SF and FABE-SF) were prepared in different concentrations (7.5, 15, 25, 50 and 75 µg.mL − 1 ) for the larvicidal tests in A. aegypti. Five replicates with asset, and dichlorvos solution (6.25 ng.mL − 1 ) was used as the positive control. The larval mortality rate was determined after 24 h and 48 h of incubation at a temperature of 25 ºC and a humidity of 75%. The larvae were considered dead when they did not respond to any stimulus or did not move on the surface of the solution, in contrast to those observed in the control. The bioassay was conducted according to WHO guidelines (2005).

Morphological analysis on larvae
After treatment, larvae were xed in 10% formalin. Their external morphology was then analyzed under an optical microscope (with 6 V DC and 20 W output) and was photographed using a digital camera (MDCE -SC USB 2.0) with the ScopeIm age software 9.0 package.

Hemolysis assay
Potential membrane injuries were evaluated according to the hemolysis assay protocol (Kang et al. 2009). Mouse blood was collected and centrifuged (3000 rpm/5 min), and erythrocytes were washed and suspended at 2% with 0.85% saline solutions. The FAEB-SF and negative control was used at nal concentrations of 5, 10, 25, 50 and 75 µg.mL − 1 , and the cells were incubated for 1h at room temperature. 0.1% Triton X-100 was used as the positive control. Samples were centrifuged (3000 rpm/5 min), and the hemoglobin released was measured at 450 nm. The results were expressed in % of hemolysis compared to the positive control.

Statistical analysis
Probit analysis was performed with a 95% con dence interval to determine the Lethal concentrations (LC 50 and LC 90 ) and Chi-square using software Statgraphics Centurion XV version 15.2.11 (Statpoint Technologies, Inc., Warrenton, VA). If the control mortality of the treated groups was between 5% and 20%, the analysis was corrected in accordance with the WHO guidelines (2005) formula mortality (%) = X -Y ∕ X × 100, where X = percentage survival in the untreated control group and Y = percentage survival in the treated sample. Results of hemolysis test were expressed as mean ± standard deviation (SD) using GraphPad Prism 8.0 software. Normality and homogeneity of variances were veri ed with Kolmogorov-Smirnov and Levene's tests, respectively. Comparisons among the Triton X-100, Murumuru fat and FABE-SF groups results were performed using one-way Analysis of variance (ANOVA) and a Tukey post-test. The minimum signi cance level was set at p < 0.05 in all cases.

Results And Discussion
The transesteri cation reaction of A. murumuru with ethanol, isopropanol and n-butanol using Amano AK lipase from P. uorescens provided a fatty acid ethyl ester (FAEE) percentage of 78.4%, a fatty acid propyl ester (FAPE) percentage of 75.1% and a fatty acid butyl ester (FABE) percentage of 63.5%, after puri cation in a chromatographic column with silica gel.
The gas chromatographic analysis of FAEE from A. murumuru showed a composition formed of saturated, monounsaturated and polyunsaturated fatty acids (Table 1). However, A. murumuru fat from the present study identi ed a highest saturated fatty acid percentage of 95.5%, with the predominant presence of lauric acid (C12:0) and myristic acid (C14:00), at 53.5% and 25.8% respectively. Other saturated fatty acids, such as palmitic (C16:0), stearic (C18:0), capric (C10:0) and caprylic (C14:00) acids, were also present in A. murumuru fat, in smaller proportions (Table 1). From the unsaturated fatty acid, oleic (C18:1) and linoleic (C18:2) acid were identi ed, at 3.8% and 0.7%, respectively. Due to a high content of short chain fatty acid, these fats are used in the chemical industry for the manufacture of detergents and soaps (Kostik et al. 2013). The chromatographic pro le of the compounds found in the samples were compared with the spectral data of those presented in the Wiley library, and exhibited high levels of similarity.
The polyunsaturated/saturated fatty acid ratio (P/S) of the A. murumuru fat used was 0.007, while several studies indicate that a higher P/S index means a reduced deposition of lipids in the body (Kang et al. 2004). According to the investigation of (Pereira et al. 2019), the lauric acid (C12:0) content was found in major proportions (47.6%), and the P/S index was 0.04. In another species, Astrocaryum aculeatum G. Mey (Pereira Lima et al. 2017), lauric acid also represented the majority of fatty acids.
The FTIR spectra (Fig. 2)  Generally, the fatty acid esters acted as an emulsion stabilizer, with different degrees of stability improvement (Park and Walsh 2019). Size distribution for DLS analysis of the emulsion (FAEE-SF, FAPE-SF and FABE-SF) from the fatty acid ester of A. murumuru associated with silk protein were recorded at the beginning of the experiment and every 7 days during storage ( Table 2). The size of all the emulsions prepared was satisfactory (Fig. 3), and no phase separation was observed in the formulation during storage at -4 ºC for 21 days, showing that silk protein could play an important role in the stability of the lipid particles from the conjunct of fatty acid esters. Watanabe (Watanabe et al. 2018) showed that sucrose fatty acid esters with HLB = 5-7 were suitable for obtaining O/W emulsions. In addition, the creaming phenomenon was inhibited for 30 days or more when fatty acids with a linear saturated alkyl chain with 14 or more carbon atoms were added. These ndings are useful for designing stable O/W emulsions for food and cosmetic products.
Silk broin/oil emulsion stability is based on a number of factors, as follows: 1) Silk broin is an amphiphilic polymer with large hydrophobic domains. These hydrophobic regions are interrupted by small hydrophilic spacers, and the N and C-termini of the chains are also highly hydrophilic (Lu et al. 2015). Therefore, the amphiphilicity of the chain organization likely plays a signi cant in the stability of the emulsions; 2) The viscosity of the aqueous phase increases with the addition of the silk broin solution, which may have inhibited droplet aggregation and gravitational separation (Feng et al. 2018). This favors Brownian motion and inhibits occulating and coalescing (Khuwijitjaru et al. 2004;Al-Sabagh et al. 2012).
However, the results of the present study revealed that the stability of the mixture depended on the fatty acid ester type present in the sample. For FABE-SF, the particle diameter was smaller (215.5 ± 1.57 nm and 217.5 ± 0.85 at 0 days and the 21st day, respectively) than the particle size of emulsions formed from fatty acid esters with propanol and ethanol [274.06 ± 2.41 nm (0 days) and 702.5 ± 92.43 nm (0 days), respectively] (Table 2). Alcohols, like ethanol, induce broin to transform into a Silk II crystalline (Tsukada et al. 1995). However, a decay in the particle size was observed, signi cantly for the FAEE-SF emulsion, starting at 702.5 (± 92.43) nm (0 day) and reducing to 229.4 (± 8.31) nm (21st day). It is therefore believed that the relationship between the ester structures can in uence the particle diameter in the case of an emulsion formed by ethanol, propanol, and butanol fatty acid esters from A. murumuru fat (fatty acid mixture).
The present study showed that silk broin which could play an important role in the stability of the lipid particles from the conjunct of the fatty acid esters. The polydispersity index (PDI) indicates the homogeneity and stability of the size of the droplets distributed in the emulsions. According to (Lemarchand et al. 2003), PDI values of around 0.3 or below indicate the more homogeneous size distribution of the particles dispersed in the suspension. FABE-SF exhibited an excellent PDI, ranging from 0.320 (± 0.03) to 0.338 (± 0.01), while FAPE-SF had a PDI ranging from 0.452 (± 0.02) to 0.415 (± 0.04), and FAEE-SF a PDI of 0.709 (± 0.08) to 0.396 (± 0.03). One interesting nding was the reduction in the PDI values of the FAEE-SF and FAPE-SF nanoemulsions on the 21st day, to 0.396 and 0.415, respectively ( Table 2). The results of this work indicate that the elongation of the ester carbon chain (-Ethyl; -Isopropyl and -n-Buthyl) in uenced the thermodynamic balance of the phases. Factors such as the spontaneous diffusion and evaporation of volatile material from the internal phase have been observed as a mechanism for reducing droplet size (Silva et al. 2011).
Zeta potential is the electrostatic potential at the slipping plane a few molecules away from the surface (Dalgleish 1997). For the nanoemulsion to be considered stable by electrostatic repulsion alone, a zeta potential value ± 20 is required (Honary and Zahir 2013). All nanoemulsions associated with silk protein exhibited zeta values ranging from − 53.9 (± 4.50) to -25.6 (± 3.24) mV, during the 21 days of monitoring. It is noteworthy that FABE-SF and FAPE-SF showed greater stability throughout the entire monitoring period ( Table 2). The stability of a polymer is important for the storage of a drug delivery device. The stability of these emulsions may be related to the improved balance between the dissociation of the propyl and butyl esters with the carboxylic and amino groups present in silk broin, resulting in a slight variation in the zeta potential for these emulsions, making them more stable. Electrophoretic mobility experiments have demonstrated that SF was positively charged below pH 3.9 and negatively charged above pH 3.9 (Malay et al. 2008), which is its pI (isoelectric point = 3.9).
The choice of solvents of the formulation is essential for the larvicidal activity on an aqueous medium. Initially, we evaluated the effect of the fatty esters solubilized in DMSO (5%), and the emulsions of the fatty esters associated with the silk broin solution, at concentrations of 25 and 75 µg.mL − 1 (Table 3). The results were then obtained after 24h against the A. aegypti larva of the III instar larvae.
For at concentrations of 25 and 75 µg.mL − 1 tested, the results showed that all fatty acid esters (FAEE, FAPE and FABE) from A. murumuru fat associated with silk broin nanoemulsion have greater larvicidal activity against the mosquito than normal fatty acid esters in DMSO (Table 3). The FAPE-SF and FABE-SF emulsions, for example, exhibited 92% mortality at a concentration of 75 µg.mL − 1 , while the FAEE-SF emulsion yielded a mortality rate of 68% at the same concentration. This result highlights the carrying capacity of the emulsion from broin and its positive impact on biological activity. It is likely that nanoemulsions associated with silk broin solution interact inside the larva cells, leading to a more rapid death than with the DMSO solution, due to changes in the active release kinetics (Table 4). Additionally, the hydrophobic interactions showed to be the main cause of interactions between SF and the fatty acid esters from A. murumuru fat. Similarly, previous reports by Sarquis (Sarquis et al. 2020) proposed that the nanoemulsion containing 75% silk broin solution (2%), 5% fatty acid from C. guianensis Aubl. and 24% ethanol, shown be effective against A. aegypti (III instar larvae) with an LC 50 94.45 µg.mL − 1 at 24 h and 16.79 µg.mL − 1 at 48 h.
One of the great advantages of silk broin in the emulsion system is the biodegradation process, which occurs via enzymatic degradation, the production of non-toxic by-products, and the controllable degradation rate, setting it apart from other synthetic or natural polymers (Nguyen et al. 2019). Table 4 Here In view of the preliminary results, other concentrations were tested to determine LC 50 and LC 90 at 24 and 48 h of nanoemulsion larvae A. aegypti larvae. Among the nanoemulsions in broin solution, FABE-SF exhibited the highest mortality percentage. After 24 h, the mortality rate was 94% at 75 µg.mL − 1 and 4% at 7.5 µg.mL − 1 , the lowest concentration tested. At 48 h, the mortality rate was 100% at 75 and 20% at 7.5 µg.mL − 1 (Fig. 4A and B). In contrast, a negative control consisting of broin solution only exhibited no larvicidal action. This result demonstrates the lack of toxicity of the broin solution towards A. aegypti.
From these results, it was also possible to establish values for the lethal concentrations LC 50 and LC 90 ( The present study found that around 78% of mortality was observed at a concentration lower than 75 µg.mL − 1 for the FAEE-SF and FAPE-SF nanoemulsions, after 24 h of exposure of the larvae to nanoemulsions. The FABE-SF nanoemulsion at 24 h exhibited mortality of 92% at a concentration of 75 µg.mL − 1 , and 100% mortality after 48 h. These results make it clear that emulsions of different esters with silk broin represent promising larvicidal agents against Ae. aegypti.
Although the toxicity mechanism of these nanoemulsions is not very well known, we suggest that the larvicidal properties of nanoemulsions, especially FABE-SF, come from its hydrophobic character/interaction with the silk protein, enhanced by the increase in the alkyl grease chain.
A set of methyl esters, from long saturated chains, extracted from the leaves of Vitex trifolia, exhibited good larvicidal activity against the C. quinquefasciatus mosquito, exhibiting LC 50 and LC 90 values of 9.26 and 21.28 µg.mL − 1 , respectively, after 24 h treatment. It should be stated that this species had a high composition of lauric acid (Kannathasan et al. 2008 However, it should be mentioned that most research with emulsions, nanoemulsions or solutions, even when obtained from plant extracts, use solvents or emulsi ers of signi cant toxicity, which can interfere with biological processes or cell maintenance. Toxicity is therefore an important factor in the choice of surfactants, which are potentially irritating or poorly tolerated, as they have nonspeci c effects on biological membranes. In general, cationic surfactants are more toxic than anionic surfactants, which in turn are more toxic than nonionic surfactants (Pouton and Porter 2008). In view of the above, silk protein represents a highly viable alternative in the use of these surfactants, as it is a biomaterial that does not exhibit cellular toxicity (Fig. 5).
The effects of FABE-SF and the solution of A. murumuru fat in silk broin were evaluated on erythrocytes isolated of the blood from rats in function of incubation time (1 h) at the following concentrations (5, 10, 25, 50 and 75 µg.mL − 1 ). The FABE-SF emulsion was chosen for the hemolysis test as it exhibited better larvicidal activity results.
The hemolysis percentage results are shown in Fig. 5. Both the emulsion and the fat solution of A. murumuru in silk broin exhibited a low capacity for hemolysis at the tested concentrations, of less than 6% even at the highest concentration (75 µg.mL − 1 ), compared with the 0,1% Triton X-100 solution (positive control). The silk broin solution used as a negative control did not exhibit erythrocyte lysis, suggesting excellent biocompatibility, behaving like a highly sustainable emulsion. Amino acid-based surfactants have the ability to interact with the lipid bilayer of cell membranes. The action of ve amino acids derived from anionic lysine type N α , N ℇ -dioctanoyl lysine and three cationic surfactants derived from arginine (methyl ester of N α -lauroyl-L-arginine, methyl ester of N α -myristoyl -L-arginine and derivatives of methyl N α -acyl-arginine), both surfactants, exhibited strong anti-hemolysis action (Sánchez et al. 2007).
The action of surfactants based on amino acids in plasma membranes is complex. A possible relationship between these surfactants, and their effects on the cell membrane may be due to the amphiphilic character of these surfactants, since they are interspersed in the lipid bilayer of the membrane. So the hydrophilic region is located at the hydrophilic/hydrophobic interface of the membrane and the hydrophobic region in the core of the bilayer (Zachowski and Durand 1988).
The results of the present study also suggest that there is no interaction between the FABE-SF nanoemulsion and erythrocytes, since the presence of silk broin in the negative control did not cause cell lysis. Silk broin, as well as surfactants based on amino acids, has an amphiphilic character; in addition, repulsion forces can occur between the negative surface charge of the silk broin and the negative charges present in the erythrocyte membranes.
Optical microscopy images of A. aegypti larvae after 48 h of exposure in the nanoemulsions (FAEE-SF, FAPE-SF and FABE-SF) revealed particles of the emulsions precipitated in the larvae, which causes lesions in the cuticles of the initial segment of the larvae, such as the head and thorax. In addition to adhering to its lateral bristles, while darkening and torsion in the larvae body was also observed (Fig. 6a). The morphological damage in the larvae is the lethality effect of the nanoemulsions. Sarquis (Sarquis et al. 2020) observed similar changes when using a nanoemulsion with free fatty acid from C. guianensis and silk broin, which was able to cause changes in the anal papilla and in the digestive and respiratory systems of A. aegypti larvae. Araújo (Araújo et al. 2020) observed morphological damage in the respiratory siphon and anal papilla in A. aegypti larvae when using hexanic extract from the leaves of Acmella oleracea solubilized in silk broin. There were no changes in the structures of the larvae exposed to the control (Fig. 6b).
It FABE-SF nanoemulsion is based on global trends towards sustainable development stimulate the use of renewable and biodegradable raw materials, as esters of murumuru, with higher biological performance in comparison to a synthetic-based vehicle, this case DMSO, to mosquito larval control, based drug delivery system.

Conclusion
This is the rst study to use different esters associated with silk broin as larvicidal agents. The esters (FAEE, FAPE and FABE) associated with the silk brin solution exhibited strong larvicidal activity, especially the FABE-SF nanoemulsion, which exhibited a CL 50 value of 18.92 µg.mL − 1 , after 48 h of larvae exposure. It displayed greater temporal stability throughout all 21 days of monitoring, with an average particle value of 217 (± 0.85) nm, zeta potential of -25.6 (± 3.24) mV and a PDI of 0.338 (± 0.01). The FABE-SF exhibit both a hydrophobic and hydrophilic character, increasing the biodistribution and bioavailability controlled of the fatty acid esters in the aqueous medium. It also observed that the nanoemulsions caused structural changes in the larvae, affecting their development and survival. This mortality rate shows that the formulations can be used as biopesticides.

Declarations
CRediT authorship contribution statement

Consent for publication
Not applicable.

Availability of data material
All data generated or analysed during this study are included in this published article (and its additional les).

Declaration of Competing Interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the study reported in this paper.

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