Interactions of Schinus terebinthifolius (Anacardiaceae) essential oil against Aedes aegypti (Diptera: Culicidae) larvae Interações do óleo essencial de Schinus terebinthifolius (Anacardiaceae) contra larvas de Aedes aegypti (Diptera: Culicidae) Interacciones del aceite esencial de Schinus terebinthifolius (Anacardiaceae) contra larvas de Aedes aegypti (Diptera: Culicidae)

Essential oils arouse the interest of research for insect control. Schinus terebinthifolius is described in the literature for being bioactive against Aedes aegypti larvae. However, studies are scarce to fully assess the larvicidal potential of this species. This study aimed to evaluate the chemical composition, bioactivity, time of death and bioavailability of the essential oil from different parts of S. terebinthifolius obtained from the Brazilian cerrado on Ae. aegypti larvae. For this, plants grown in the city of Goiânia-GO were used and the elucidation of the chemical composition of essential oils was carried out by means of gas chromatography coupled with mass spectrometry. Ae. aegypti larvae were used in the bioassays to assess larvicidal activity, determine the time of death and bioavailability of the essential oil in solution. In addition, the interference of essential oil in the activity of the enzyme acetylcholinesterase was also investigated. Based on the results obtained, it was observed that the most promising essential oil for the development of larvicidal formulations is that of fruits, based on having higher yield, greater bioactivity, time of death similar to synthetic insecticides. An inhibitory interaction of acetylcholinesterase was also observed. However, the essential oil had low bioavailability, so it is necessary to develop formulations to increase its bioactivity period.


Introduction
Essential oils (OE) are compounds derived from the secondary metabolism of plants, which are characterized for being volatile and hydrophobic liquids with various applications. This is because EOs are composed of complex mixtures formed mainly by monoterpenes and sesquiterpenes (Bakkali et al., 2008). These compounds are widely used by the pharmaceutical and food industries mainly due to their antimicrobial, antioxidant, and organoleptic properties (Bhavaniramya et al., 2019;Mishra et al., 2020;Goudjil et al., 2020;He et al., 2020). They are promising products in the research and development of bioinsecticides because they interact with the nervous system of insects due to anticholinesterase activity, inhibition, or antagonism of gamma-aminobutyric receptors (Braga et al., 2007) or acting on the digestive system (Camaroti et al., 2018). Therefore, they play an important part in the control of agricultural pests (Deb et al., 2020), urban vectors (Bouabida et al., 2020;Zeghib et al., 2020), and insecticide formulations, since they present low toxicity and are alternatives to the use of synthetic insecticides (Sharma et al., 2020;Amado et al., 2020).
The Anacardiaceae family has approximately 81 genera subdivided into 800 species, present mainly in tropical and subtropical regions (Pell, 2011). It is a family with plants known for food consumption, but many of these species have insecticidal potential against mosquitoes vectors of diseases such as Anacardium occidentale L. (Vani et al., 2018;Kala et al., 2019) and Spondias mombin L. (Famuyiwa et al., 2020;Ajaegbu et al., 2016), known as cashew and yellow mombin, respectively. In addition to these, the genus Schinus has species widely distributed throughout the Brazilian territory, among them, Schinus molle L., which is investigated in the control of urban disease vectors such as Culex pipiens (Diptera: Culicidae) for example (Zahran et al., 2017).
Schinus terebinthifolius, popularly known as rose pepper, "aroeira", red "aroeira", beach "aroeira", among other denominations, is widely described in the literature due to its diverse bioactivity, among them studies aimed at the control of agricultural pests and vectors of urban pathogens. That is a promising species against Anticarsia gemmatalis (Lepidoptera: Noctuidae), also known as velvetbean caterpillar, which affects various crops such as sugar cane, cotton and cauliflower (Vicenço et al., 2020); Plutella xylostella (Lepidoptera: Plutella), popularly known as diamondback moth, responsible for affecting rice, potato, and cotton crops among others ; Bemisia tabaci and Trialeurodes ricini (Hemiptera: Aleyrodidae) known as sweet potato whitefly and castor bean whitefly respectively (Hussein, 2017). 13 in addition, this plant is widely studied in the fight against mosquitoes vectors such as Cx. pipiens, vector of encephalitis, avian malaria, and filarial virus (Zahran et al., 2017) and Aedes aegypti, vector of dengue, chikungunya and zika virus (de Campos Bortolucci et al., 2019;Procopio et al., 2015). Thus, it is a promising species against agricultural pests causing numerous damages in the productive sector, and urban vectors, carriers of high incidence and high prevalence pathologies in tropical and subtropical regions.
Considering that S. terebinthifolius is a plant known for its bioactivity against several vectors of urban diseases, among them the Ae. aegypti, and there are no studies in the literature that investigate the comparative larvicidal activity between leaves, fruits and seeds, it is necessary to analyze these EOs to determine the plant part with the most promising biological activity. Thus, this paper aims to analyze the chemical composition and larvicidal activity of the EOs, as well as determine the time of death, and the bioavailability of the EO in solution. Its interference with the activity of the enzyme acetylcholinesterase was also investigated. These studies aim to support the development of natural formulations for vector control.

Plant material and obtaining the essential oil
The branches, leaves, fruits and seeds were collected in the municipality of Goiânia, State of Goiás, Brazil. A voucher specimen was stored in the herbarium of the conservation unit at the Federal University of Goiás for identification purposes under number 66.444. The samples of the leaves and fruits were separated, and the seeds were peeled manually. They were then desiccated in a forced air convection oven at 40ºC for three days. Afterwards, they were crushed in Ika® A11 processor and immediately after processing underwent hydrodistillation in Clevenger apparatus for two hours. The resulting essential oil was desiccated with sodium sulfate anhydrous and stored in an amber vial under refrigeration at -22°C. The yield of the extractive process was calculated from the ratio between the mass of oil obtained and the crushed sample (Farmacopéia Brasileira, 2019).

Essential oil chemical composition determination
An aliquot of the EO obtained underwent gas chromatographic analysis, coupled to mass spectrometry (GC-MS) in a Shimadzu GC-QP2010A apparatus, with silica capillary column DB-5 (30m × 0.25mm × 0.25m with 5%-Phenylmethylpolysiloxane). Heating ramp programmed in the following scheme: starting with 60-240°C at 3°C/min, then 280°C at 10°C/min, ending with 10 min at 280°C. Carrier gas was helium with a flow of 1 mL/min. Injection port has been set to 225°C.
Mass spectrometer operating with interface temperature of 240°C; electron ionization at 70 eV with scanning mass range of 40-350 m/z and sampling rate of 1 scan/s. Chemical components of essential oil were identified by comparing the mass spectra Research, Society andDevelopment, v. 10, n. 10, e315101018892, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18892 and retention indices with those reported in the literature for the most common components of essential oils (Adams, 2007).

Bioassays
Bioassays with larvae of Ae. aegypti were performed at the Laboratory of Insect Biology and Physiology (IPTSP/UFG), under controlled conditions of climate control. In the biological chamber, the insects develop and are tested at a temperature of 28°C ± 1°C, relative humidity of 85% ± 5% and 12-hour photophase of light-dark. The larvae used in the tests grew in basins with water supplied by the public supply network and fed with cat food. Tests were carried out in polystyrene containers with a capacity of 50mL.

Assessment of larvicidal activity
To assess the larvicidal potential of fruit, leaves, seeds, and branches, aliquots of S. terebinthifolius EO were dispersed with surfactant Tween 80 (v/v) to produce an aqueous solution at 100µg/mL. A total of 20 third-stage larvae of Ae. aegypti were exposed to 20mL of test solution in serial dilutions of 20-100µg/mL. A solution of water + surfactant was used as negative control and temephos (Abate® Basf Chemical Group) at 0.012µg/mL as the positive control, according to the methodology proposed by WHO (WHO, 2005). Mortality was quantified after 24-hour exposure to treatments and confirmed by lack of response to mechanical stimulus and stiffening of the cephalic capsule. Three repetitions were performed for each assay. Subsequent tests for larvicidal activity were performed only with the EO considered more promising according to the lower lethal concentration (LC) and higher yield in the extractive process. Lethal time (Lt) determination to determine the mean time required for larval mortality, the assay proposed by Aguiar et al., (2015) was conducted, with some adaptations. In this sampling, 20 larvae of Ae. aegypti in the third stage were exposed to the EO solution of fruits of S. terebinthifolius at LC90.
After exposure, the larvae were monitored and classified as living, lethargic or dead every 40 minutes. For classification, the larvae were observed in stereomicroscope (Leica M50) augmented 20-fold. Death was confirmed with the absence of contraction in the respiratory and circulatory muscles. The evaluation was performed in triplicate.

Assessment of persistence time and bioavailability of essential oil (Bt)
At first the test was performed to verify the residual effect of the EO of the fruits of S. terebinthifolius. In this test 20 third-stage larvae of Ae. aegypti were exposed to 50mL EO solution in LC90 for 24 hours. After counting mortality events, new larvae were exposed to the solution, without any renewal of the solution, and mortality events were quantified after 24 hours of exposure. The replacement of larvae every 24 hours should occur until the nullity of the solution effect (Romano et al., 2018;Menezes et al., 2019). However, the solution showed no residual effect. Given this condition, an exposure scheme was developed to verify the bioavailability time of the EO in the test solution. Considering the volatility of essential oils, the experiment to determine the bioavailability time (Bt) was drawn from the correlation between the preparation time of the solution and the occurrence of larval mortality. For this 140mL of EO solution of S. terebinthifolius was prepared in LC90 and immediately distributed in seven containers, containing 20mL of solution each. The exposure of the larvae in the solution obeyed a temporal scheme so that the first container received 20 larvae of Ae. aegypti in third instar immediately after preparation. The second container received the larvae after 40 minutes of filling with the solution. The exposure of the larvae to the test solution obeyed this interval of 40 minutes, so that at the last exposure the larvae found a ready solution at

Enzymatic activity on acetylcholinesterase
Enzyme activity assay on acetylcholinesterase was performed according to the methodology proposed by Sugumar et al., (2014), with minor modifications. To observe the interference of EO of fruits of S. terebinthifolius on acetylcholinesterase activity, 50 fourth-stage larvae were exposed to the EO test solution in increasing solutions from 50 to 400ppm. Larvae treated with EO were macerated with 500µL PBS buffer pH 7.2 and Triton X-100 at 10% to produce the body homogenized. The homogenized was centrifuged 12,000rpm, at 4°C for 10min. Enzymatic activity was measured by the Ellman's reaction (1961).
Homogenized prepared with larvae exposed to temephos at 0.012ppm was used as a positive control and the negative control was prepared with larvae exposed to water solution and surfactant only. For the reaction, 50µL of homogenized body was incubated in 125µL of PBS buffer and 50µL of 10mM of 5,5′-Dithiobis(2-nitrobenzoic acid) and 50µL of 12.5mM of acetylcholine iodide as a substrate, for 5 min. The absorbance of the reaction was measured at 405nm.

Statistical analysis
The lethal concentration (LC) responsible for 50 and 90% of mortality was estimated by nonlinear regression (Probit) (α=0.05). The percentage of enzyme inhibition was calculated with the absorbance obtained in the enzyme assay using the formula I = (A0 -At) A0*100, where I equals the percentage of enzyme inhibition, A0 corresponds to the absorbance of the negative control and the absorbance of the test solution (Owokotomo et al., 2015). The inhibition rate was used to predict the concentration required for minimum enzyme inhibition (MI) of 50 and 90%, also calculated by nonlinear regression. All statistical analyses were performed using Statistica 12.0 software (StatSoft, 2013).

Essential oil chemical composition determination
The chemical composition of the EOs of branches, leaves, fruits and seeds is shown in Table 01. Results show that the components found in fruits and seeds are formed predominantly by monoterpenes. Experiments carried out by Barbosa et al., (2007) and Cavalcanti et al., (2015) evaluated the extraction kinetics of the volatile components of S. terebinthifolius and found the composition of the EO of the fruits formed predominantly by monoterpenes during the first hours of extraction, just as in this study. The 17 monoterpenes are described in the literature for their satisfactory larvicidal activity, being the main compounds of interest evaluated in this study (Kweka et al., 2016).
After the extraction of the EOs, the yield of the extractive process was determined to verify which EO is most viable for large-scale use in the production of larvicides. The results obtained in this study are shown in Table 1. Thus, it was possible to verify that the yield of fruits and seeds are higher than that of leaves and branches, which have low yield that disfavors their use in the development of formulations. The seeds, despite having higher yields, have an additional step (peeling) in the acquisition process, which would require a higher production cost, whether with equipment or labor. Therefore, when considering the yield factor alone, it can be said that the essential oil extracted from the total fruit is the most viable. Source: Authors.

Assessment of larvicidal activity and enzymatic activity on acetylcholinesterase
The evaluation of the larvicidal activity of the leaf, fruit, seed, and branch samples is shown in Table 2. The mean lethal concentration (LC) is the lowest concentration necessary for a population exposed to a given substance, in a preestablished period, to die (Minho et al., 2017). When analyzing the samples, it is possible to see that the seeds and fruits have greater lethality against larvae of Ae. aegypti. However, for large-scale production of bioinsecticide, the additional steps in obtaining the essential oil from seeds can drive up costs. Therefore, the fruits are considered as the most promising sample for the evaluation of larvicidal potential. In studies by Bortolucci et al., (2019), the larvicidal activities of the essential oil of fruits of S. terebinthifolius collected in Juranda, Paraná (Brazil) were determined in third-stage larvae of Ae. aegypti and found a LC50 of 374mg.L -1 . Variations in the composition of essential oil influence the larvicidal activity and are explained due to differences in seasonality, temperature, water availability, light incidence, among other factors that alter both the presence and concentration of certain metabolites in the plant (Gobbo-Neto & Lopes, 2007). However, due to the high concentration required for the inhibition of 90% of enzyme activity, it can be assumed that enzyme activity is not the only path for activation of lethality.
In addition to enzymatic activity on acetylcholinesterase, other probable mechanisms of action are described in the literature in distinct species of vector and agricultural pests. Studies involving Essential Oil of S. terebinthifolius showed promising larvicidal activity against fourth-stage larvae of Cx. pipiens which suggests the probable mechanism of death is enzymatic activity (Zahran et al., 2017). Bilobol, an isolated alkylresorcinol, obtained from the leaves presented results of LC50 7.67mg.L -1 , considered a potent candidate for natural larvicide (Schulte et al., 2021). Plant extracts produced from leaves of S.
terebinthifolius collected in Recife, Pernambuco (Brazil) were tested in fourth-stage larvae of Ae. aegypti, thus observing a larvicidal effect caused by damage to their midgut (Procópio et al., 2015). In addition, studies using samples also collected in Recife, Pernambuco (Brazil) demonstrated interference in intestinal enzymes of Sitophilos zeamais (Coleoptera: Curculionidae), a major pest of corn and other stored grains (Camaroti et al., 2018).

Determination of lethal time (Lt) and assessment of persistence and bioavailability (Bt) of essential oil
To assess the time of death of larvae after exposure to the EO of the fruits, the time in which there was 50% mortality (LT50) was determined in 183.31 minutes and 90% mortality (LT90) in 349.83 minutes using the lethal concentration of 90% (LC90). From Figure 1 it is possible to analyze that the percentage of live and lethargic larvae reduces over time, as well as an increase in the percentage of mortality. Source: Authors.
In the literature, there are no scientific studies that analyze the time of death of larvae of Ae. aegypti using EO of S.
terebinthifolius. These data are fundamental to understand how long a potential larvicide produced from the EO of the fruits of Pink Pepper will take to reach the mortality of the exposed population. In Lethal time studies involving EO of Siparuna guianensis Aubl (Siparunaceae) in third-stage larvae of Ae. aegypti, a LT50 of 12.11 min was obtained using samples collected in Gurupi, Tocantins, Brazil (Aguiar et al., 2015). Studies with EO of Plectranthus amboinicus (Lour.) Spreng. (Lamiaceae) collected in Taiwan found a LT50 of 61.16 minutes in larvae of Ae. aegypti at 100mg.L -1 (Huang et al., 2019).
Synthetic insecticides are widely used in the control of urban vectors; however, they have as a disadvantage their high toxicity to the environment. Assays with temephos have shown that the insecticide can reach LT50 and LT90 in 2,088.6 and 3,432 minutes respectively (Fatimah et al., 2020). Studies using DDT (dichlorodiphenyltrichloroethane), a currently banned insecticide due to high environmental toxicity, showed LT50 and LT90 in 177.73 and 493.54 minutes (Nazni et al., 2009). Thus, S. terebinthifolius has a smaller lethal time than the temephos, the neurotoxic insecticide indicated by WHO as standard for testing and for field use in places where the mosquito population is not yet resistant. Its lethal time is similar to that of DDT; however, it has the advantage of a low toxicity to the environment and may be a possible candidate for the replacement of synthetic larvicides.
The determination of the bioavailability of the EO was performed using LC90. Since EOs are volatile substances, this parameter is essential to verify their bioactivity period. Therefore, it was possible to calculate that Bt90 was nine minutes and Bt50 approximately 134 minutes as shown in Figure 2. The bioassay showed that with time there was a reduction in the bioactivity of the EO, probably caused by its volatilization to the environment. Consequently, the development of formulations that increase the permanence of EO in an aqueous medium is fundamental for the use of S. terebinthifolius with larvicidal purposes. This was the first study that sought to evaluate the bioavailability time of essential oils guided by the larval mortality event.

Conclusion
Thus, it is possible to determine that the most promising essential oil of S. terebinthifolius is that of the fruits since it has a satisfactory yield and potent larvicidal activity against larvae of Ae. aegypti. However, the result of the bioavailability test allowed us to observe that this oil remains bioavailable for a short time in an aqueous solution, which suggests that it is necessary to develop dispersion systems that favor the permanence of the oil dispensed in the liquid medium for a longer time, thus avoiding the loss of volatile compounds to the environment. Therefore, it is a plant with the potential for the development of low environmental impact and effective larvicidal products against Ae. aegypti.
Furthermore, the bioavailability assessment methodology guided by larval mortality was an efficient and relatively low-cost way to measure the persistence time of essential oils in solution, working as an indicator for cases where residual effect tests would not generate accurate information.