Larvicidal Activity of Leaf Extract From Mauritiella armata (Aceraceae) on Aedes aegypti and Culex quinquefasciatus (Culicidae)

The mosquitoes Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) are important vectors of several arboviruses, and are relevant public health problems. Conventional control, using chemical larvicides have selected resistant Culicidae populations and caused negative effects on the environment and human health. However, the use of plant extracts has represented a sustainable alternative for insect control. Popularly known as Xiriri, Mauritiella armata (Mart.) Burret (Aceraceae) is an abundant palm tree in Vereda ecosystems and has economic and social importance. In this study, the aim was to evaluate the larvicidal activity of the aqueous extract (AE) leaves of this plant on Ae. aegypti and Cx. quinquefasciatus larvae. The mortalities of larvae were analyzed after treatment with four concentrations of the extract, comparing with a negative control using mineral water. The AE promoted 100% efficacy against Ae. aegypti larvae at 7.9 mg/mL. The lethal concentration to promote 90% mortality of Cx. quinquefasciatus larvae was estimated at 30.57 mg/mL. After chromatographic analyses, flavonoids, catechin and carbohydrates were detected. AE from M. armata leaves presented high larvicidal activity against Ae. aegypti and Cx. quinquefasciatus, and represents a promising alternative to be used in vector control.


Introduction
Vector-borne diseases account for 17% of the estimated global burden of all infectious diseases (WHO, 2018). Aedes aegypti (Linnaeus, 1762) and Culex quinquefasciatus (Say, 1823) are vectors of several arboviruses, which are considered a public health problem due to their increasing territorial dispersion, high capacity to adapt to artificial ecotopes and difficulty to be controlled (Donalisio, Freitas, & Zuben, 2017).
Vector control is the main form of prevention, and the elimination of breeding sites is the most effective method; however, conventional treatments with chemical larvicides select resistant populations of Culicidae (Gray et al., 2018;Atyame et al., 2019;Lopes et al., 2019). As a result, the use of plant extracts appears as a promising alternative, ecologically and environmentally safe, and biodegradable, contributing to the reduction of negative effects on the environment and public health (Hwang et al., 2017;Hari & Mathew, 2018).
Mauritiella armata (Mart.) Burret belongs to the Arecaceae family and is popularly known as Xiriri. It is a palm tree widely distributed in the Brazilian territory, in the Vereda areas and has economic and social importance for the Vereda communities (Andrew Henderson, 1995;Martins, 2012). Although some studies have shown that plants of the Arecaceae family have a larvicidal effect against Culicidae larvae (Tayler et al., 2019, Koc et al., 2016, the phytochemical composition of M. armata extracts and the potential of metabolites in the control of insects however, are not known. In this perspective, the study proposed to evaluate the larvicidal activity of aqueous extract of Mauritiella armata leaves on Aedes aegypti and Culex quinquefasciatus.

Production of Extract
Leaves of M. armata were selected and washed in running water, and damaged or deteriorated were discarded. Afterwards, they were dehydrated in a greenhouse with forced air circulation at 40 °C for 72 hours and ground in a feed mill. The resulting material was placed in paper bags, free from the incidence of light.
The aqueous extract (AE) was prepared by adding 500 mL of distilled water to 50 g of raw powder of the plant species, being heated in a water bath at 40 °C for 60 min. After this period, the extracts were hot filtered in a funnel with gauze and cotton and later sent to a greenhouse with forced air circulation at 40 °C until constant weight was obtained (Nery et al., 2010, Morais-Costa et al., 2015. The subsamples of the extracts were submitted to dry matter determination at 105 °C, to calculate the tested concentrations (Patricia Cunniff, 1995). After determining the dry matter weight, the extracts were adjusted to the concentrations to be tested.

Chromatographic Analysis of the Extract
Quotas (1.0 mg) of the plant extract was measured in a conical glass and then dissolved in 60 μL of pyriride and 100 μL of BSTFA (N,O-bis(trimethylsilyl) = triflouroacetamide) containing 1% of chloratrimethylsilane. The reaction mixture was heated at 60 °C for 30 min. Of the solution obtained, only 1 μL was injected into the CG-MS, and the procedure was performed in triplicate.
Chromatographic analyses were performed in an Agilent Technologies gas chromatograph (GC 7890A) equipped with an electron impact ionization detector (CG-MS) and a DB-5MS capillary column (Agilent Technologies, 30 m long × 0.25 mm internal diameter × 0.25 µm film thickness). Helium (99.9999% purity) was used as the trailing gas at a rate of 1 mL min-1. Using an autoinjector (CTC combiPaL), 1 μL of the sample was injected into the chromatograph at a split ratio of 1:10. The split/splitless injector was kept at 290 °C. The chromatographic column, initially at 80 o C, isothermal for 5 min, was heated at a rate of 4 °C min-1 to 260 °C for 10 min. After separating the compounds, the temperature was raised to 300 °C and maintained for 2 min (after running). The interface temperature was maintained at 280 °C and the ionization performed by impact of 70 eV. The m/z sweep range was from 30 to 600 Da.

Origin of Insects
Native larvae of Culex quinquefasciatus were provided by the Montes Claros Zoonoses Control Center (Centro de Controle de Zoonoses-CCZ) and identified in accordance with the Cx. quinquefasciatus surveillance guide (Ministério da Saúde, 2011). The eggs of Aedes aegypti came from the F4 and F5 generations of the already established closed colony, provided by the insectarium of the Insect Behavior Laboratory of the Federal Institute of Northern Minas-Campus Salinas.

Bioassays and Data Analysis
The bioassays were carried out according to the methodology described by the WHO (1981). Final third instar or early fourth instar larvae were transferred to disposable plastic containers containing 30 mL of AE or negative control containing mineral water. Tests were performed in triplicate; 20 larvae of Ae. aegypti and 15 Cx. quinquefasciatus larvae were added to each container, and the final concentrations evaluated were 10.5; 7.875; jas.ccsenet.

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The results obtained demonstrate that the AE of M. armata leaves presented larvicidal potential at the evaluated concentrations. In 48 h, there was a higher mortality of larvae in both insects. This fact corroborates the results of Hari and Mathew (2018), who in the same period, observed higher larval mortality of Ae. aegypti and Cx. quinquefasciatus through a combination of plant extracts. According to Santos et al. (2015), the longer the exposure time of larvae to the AE, the higher the mortality percentages, due to the absorption of toxic substances.
The control larvae of both insects showed high mobility and quick reaction to any touch. In contrast, larvae submitted to AE showed loss of mobility and morphological changes after 24 h of treatment. According to Barreto et al. (2007), the first sign of the extract's larvicidal action is the reduction of larval mobility.
Additionally, the AE from the leaves of M. armata caused deterioration and induction of elimination of the intestinal contents of the larvae of both vectors (Figure 2). Procopio et al. (2015), observed a similar effect on A. aegypti larvae exposed to the leaf extract of Schinus terebinthifolius (Anacardiaceae). This behavior has been reported as a defense mechanism of mosquito larvae in order to expel substances that are toxic to them (Gusmão et al., 2002). Although the larvae present this defense mechanism, our results showed that this was not enough to avoid the harmful effects of the extract, since the larvae survival rate decreased after the larvicide test.
Assessing the chemical composition of M. armata, Royo et al. (2019) detected significant flavonoid content in its leaves, petiole and root. Flavonoids are a diverse and abundant group among secondary metabolites in different plants (Filho, Antonio Carlos Pereira;Castro, 2019). In the study by Tayler et al. (2019), EA from leaves of Cocos nucifera (Arecaceae) showed antiparasitic activity against the malaria protozoan, due to the synergistic action of flavonoids with other compounds.
Diterpene extracted from Copaifera reticulata, and a fraction rich in catechetical tannins, extracted from Magonia pubescens caused the death of Ae. aegypti through cell destruction in the midgut (Volotto et al., 2011). In the work by Elumalai et al. (2016), the catechin isolated from Leucas aspera showed 100% mortality in Ae aegypti, Anopheles stephensi and Cx. quinquefasciatus at a concentration of 20 ppm. The catechin, which has insecticidal properties, was identified in the phytochemical analysis of the xiriri leaf.
The aqueous extract of the leaves of Mauritiella armata represents a promising alternative for the control of Aedes aegypti and Culex quinquefasciatus, due to its high larvicidal effect. In addition, this study presents a simple, clear, and low-cost methodology that contributes to the valuation of ecosystem services and the promotion of unique health.