Different susceptibilities of Aedes aegypti and Aedes albopictus larvae to plant-derived products

Mosquitoes are major vectors of several diseases, transmitting pathogens to more than 700 million people annually¹. Dengue is a mosquito-borne viral infection whose incidence has increased dramatically worldwide in recent decades. The dengue virus is transmitted mainly by Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Aedes albopictus (Skuse, 1894) (Diptera: Culicidae), which are adapted to both tropical and temperate regions².

Difficulties related to the production of effective vaccines against chikungunya, Zika, and dengue viruses have led to more emphasis on vector control. For many years, the control of Ae. aegypti and Ae. albopictus populations has involved the use of synthetic insecticides such as organochlorines, organophosphates, and pyrethroids. However, the indiscriminate and frequent use of these substances has caused insecticide resistance, environmental pollution, destabilization of ecosystems, and toxic hazards to humans and other non-target organisms³.

In the past decade, research using botanical products as pesticides has increased, since these substances are potentially a rich source of bioactive compounds that are also biodegradable. Extracts and essential oils obtained from plants have been suggested as alternative sources of substances for insect control, because some are selective, biodegrade to nontoxic products, and have few effects on non-target organisms and the environment. These products are thought to contain insecticidal phytochemicals that are predominantly secondary metabolites produced by plants to protect themselves against herbivorous insects⁴.

In the present study, essential oils, ethanol extracts, and seed oil methyl esters of nine plants were tested against third and fourth instars of Ae. aegypti and Ae. albopictus larvae. We analyzed their different susceptibilities and discovered a natural product useful for the control of infections transmitted by these mosquitoes.

Leaves of Anadenanthera colubrina (Vell.) Brenan (Fabaceae), Momordica charantia L. (Cucurbitaceae), and Sterculia striata St. Hil. et Naud (Malvaceae) were collected in Piauí state, to prepare the ethanol extracts. We purchased oils of sunflower (Helianthus annuus L., Asteraceae), linseed (Linum usitatissimumL., Linaceae), and rapeseed (Brassica napus L., Brassicaceae) from a local drugstore, to obtain lipids and prepare the fatty acid methyl esters. Finally, we extracted essential oils from the leaves of Cymbopogon citratus (DC) Stapf (Poaceae) and Croton nepetaefolius Baill (Euphorbiaceae), and flower buds of Syzygium aromaticum (L.) Merrill & Perry (Myrtaceae). The above products were tested against the third and fourth instars of Ae. aegypti and Ae. albopictus larvae, to evaluate their potential larvicidal activity. We deposited reference plant specimens in the Graziela Barroso Herbarium and Parnaíba Delta Herbarium-both of which are associated with the Federal University of Piauí-and they were identified by their botanists.

The essential oils were extracted by hydrodistillation in a Clevenger apparatus⁵. Leaves of M. charantia, A. colubrina, and S. striata were dried in the shade and subjected to cold extraction with ethanol for 72 h, repeating the process three times. To obtain the extracts, the solutions were processed in a rotary evaporator for complete elimination of the solvent. The percentage of extraction was calculated by the ratio between weight of the extract / dry weight of the leaves, multiplied by 100.

The oils of H. annuus, L. usitatissimum, and B. napus were methylated: 500 mg of lipid, hexane (5 mL), and 0.1 M KOH in methanol (5 mL) were mixed and then added to 30 cm test tubes. The tubes were placed in a water bath at 50 °C for 1 h. Hexane (5 mL) and 5% HCl solution (15 mL) were added, and the mixture was transferred to a separating funnel, where the hexane phase, containing the methyl esters, was separated and dried with Na₂SO₄ to produce the corresponding fatty acid methyl esters.

Ae. aegypti and Ae. albopictus eggs were collected using ovitraps in the field in March 2016, during activities of the Fortaleza Dengue Control Program. The traps were sent to the Entomology Laboratory at the Federal University of Ceará, where eggs were removed and placed in dishes to hatch. The resulting larvae were maintained under laboratory conditions (25 ±2 °C, 80 ± 10% relative humidity, and 12:12 h light-dark photoperiod) in plastic containers with 1000 mL of distilled water and fed with fish feed, until they reached the third and fourth instars.

The third and fourth instars of Ae. aegypti and Ae. albopictus larvae (20 specimens of each) were exposed to different concentrations (50, 100, 250, and 500 mg/L) of the ethanol extracts, methyl esters, and essential oils, according to standard methods for testing larvicidal activity, per the World Health Organization⁶ with slight modifications. The bioassays were conducted at the Chemistry Laboratory of the Federal Institute of Education, Science and Technology of Ceará (Maracanaú Campus), where the larvae were observed for mortality after 24 h and 48 h. Three replicates were carried out simultaneously for each concentration. During the assays, no food was offered to the larvae.

A generalized linear model with binomial response (logistic regression) was applied, followed by an analysis of variance (ANOVA) and multiple comparison of the means. The level of statistical significance (p-value) was 0.05.

The strongest larvicidal effects against Ae. aegypti larvae, among the tested natural products, were shown by the essential oils of S. aromaticum and C. nepetaefolius, for which the concentrations that caused 50% mortality (LC₅₀) were 32.7 ppm and 81.7 ppm, respectively (Table 1). Regarding Ae. albopictus, the essential oil of C. nepetaefolius had greater larvicidal effect (LC₅₀ = 76.1 ppm) than did S. aromaticumessential oil (LC₅₀ = 138.1 ppm) (Table 2).

The fatty acid methyl esters from the three oils showed differences in larvicidal activity. The most active was from B. napus (LC₅₀ = 342.8 ppm against Ae. aegypti, Table 1; and LC₅₀ = 181.4 ppm against Ae. albopictus, Table 2), showing medium activity.

Increases in the concentrations of the ethanol extract of M. charantia, methyl esters of H. annuus, L. usitatissimum, and B. napus, and essential oils of C. nepetaefolius and S. aromaticum increased Ae. aegypti larval mortality (Table 1). However, only methyl esters of H. annuus and B. napus and the essential oils of C. nepetaefolius and S. aromaticum had increasing larvicidal effect against Ae. albopictus, with rising concentrations (Table 2). However, increased exposure, from 24 h to 48 h, was significant only for Ae. albopictus (Table 3) (Ae. aegypti, p = 0.869).

The damage caused to humans and the environment by the extensive use of synthetic insecticides beginning in the second half of the 20^th century to combat the transmission of numerous mosquito-borne pathogens has prompted the need to find new strategies to control these vectors⁷. Thus, integrated mosquito management is an eco-friendly way to control immature forms of the mosquito vectors, based on the use of natural products, such as phytochemicals. Unlike conventional insecticides, which are based on a single active ingredient, plant-derived insecticides typically contain botanical blends of chemical compounds that act synergistically on both behavioral and physiological processes¹.

The LC₅₀ values of the essential oils of S. aromaticum and C. nepetaefolius against Ae. aegypti larvae in this study contrast with those obtained by previous studies⁸, which showed that their LC₅₀ values against Ae. aegypti larvae were 92.5 ppm and 66.0 ppm, respectively. In another study⁹, the main chemical constituents of C. nepetaefolius were methyleugenol and alpha-copaene, with an LC₅₀ value of 84 ppm against Ae. aegypti, similar to our results. The larvicidal activity of S. aromaticum against Ae. aegypti was previously reported¹⁰ (LC₅₀ = 93.56 ppm), similar to our findings, and the chemical analysis revealed the presence of eugenol (65.99%) and caryophyllene (28.32%). These results may have been influenced by site-specific differences in the tested larval strains and essential oil composition.

Ae. aegypti and Ae. albopictus larvae showed different levels of sensitivity to the natural products tested. These differences might be related to factors such as the intraspecific and interspecific differences that naturally occur in these mosquitoes, which are subject to different environmental selection pressures, thus influencing their resistance/susceptibility.

The fatty acid compositions of several edible oils were analyzed by Kamal-Eldin and Andersson¹¹. They found that sunflower oil’s main constituents were linoleic (56.78%) and oleic acid (33.7%), while linseed oil contained linolenic (57.8%), linoleic (15.7%), and oleic (17.7%) acids; and rapeseed oil contained oleic (60.1%), linoleic (21.4%), and linolenic (11.4%) acids. To investigate these differences, Du et al.¹²compared the fatty acid composition of these oils. Several fatty acid methyl esters were isolated via chromatography-mass spectrometry using Pharbitis purpurea seed oil, and these were evaluated for acaricidal activity. Methyl oleate was the most active. Thus, enhanced biocidal activity could explain the higher insecticidal activity of B. napus, which contains more oleic acid.

The larvicidal activity of a product is usually improved by increasing its concentration and exposure time. Exploration of plant extracts with significant larvicidal activity should be based on using different plant parts, associated with the extraction of various solvents¹³. In our study, this variability may have contributed to the reduced larvicidal effect of A. colubrina, M. charantia, and S. striata, since only their ethanol extracts were tested.

Generally, plant essential oils are recognized as important natural sources of insecticides and can be composed of complex mixtures of substances at different concentrations. In this study, the essential oils of C. nepetaefolius and S. aromaticum showed significant larvicidal activity against Ae. aegypti, while Ae. albopictus larvae were very susceptible to the essential oil of C. nepetaefolius, as shown by LC₅₀ values < 100 ppm.

Methyl esters, essential oils, and other lipophilic compounds are more active larvicides than polar extracts of plants, based on facilitated transport through insect cell walls and cytoplasmic membranes¹⁴. Despite the low larvicidal effect of the methyl esters of H. annuus, L. usitatissimum, and B. napus observed in this study, oils with high oleic acid content should be preferably evaluated. Among the essential oils, S. aromaticum was the most active and Ae. aegypti showed higher susceptibility than Ae. albopictus to this oil.