Plant materials

Leaves, stems, and inflorescences of P. marginatum and P. tuberculatum were collected in Cumanda, Chimborazo province (2°12'12" S, 79°06'36" W) and on the El Triunfo - Bucay road, Guayas province (2°18'9" S, 79°14'16" W) from Ecuador. The identification of the botanical species was carried out in the GUAY Herbarium of the Faculty of Natural Sciences of the University of Guayaquil, Ecuador, where the vouchers of the specimens were deposited under the codes MER06 (P. marginatum) and MER07 (P. tuberculatum).

Hydrodistillation

The leaves, stems, and inflorescences of the species were subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus. The oils, once obtained, were meticulously dried over anhydrous sodium sulfate and then stored in sealed vials. These vials were then placed in a controlled environment at 4°C in the dark to ensure the oils' stability and integrity until they were ready for analysis and testing.

Acaricidal activity

Acaricidal activity was analyzed using the contact toxicity technique described by ^{Tabari et al. (2020)} with minor modifications. Larvae between 14 and 21 d old of the tick R. microplus obtained from gravid females collected from infested cattle were used. The tick species was identified by Prof. Antonio Ascencão of the Laboratory of Biodiversity of Arthropods, Department of Biology, Faculty of Sciences, University of Los Andes, Venezuela. Serials of essential oil dilutions were prepared from 20.0 to 0.3 µL mL^-1, in 2% Tween^® 80 solution. A Tween^® 80 solution was used as a negative control and a 20 µL mL^-1 cypermethrin solution dissolved in Tween^® 80 (2%), prepared from the commercial product CIPER-VEEX^® (10% cypermethrin), was used as a positive control. Whatman^® N°1 filter paper discs were impregnated with 1 mL of the test solutions. Tick larvae were transferred to two of the impregnated paper disks, placed inside a Petri dish. The plates were sealed and stored in a glass chamber at 27±2°C and 85±2% humidity. Assays were performed in duplicate. Mortality records were made after 48 h under a stereomicroscope.

Larvicidal activity

Larvicidal activity was analyzed following the guidelines of the World Health Organization (^{WHO, 1981}) for third and fourth instar larvae of wild A. aegypti, collected at the Faculty of Natural Sciences of the University of Guayaquil. The larvae were identified by the author using taxonomic guides. An aliquot of essential oils was solubilized in absolute ethanol until a stock solution was obtained, to prepare test solutions in serial dilutions of 3 to 1,000 µL mL^-1. The final volume of the solutions was 50 mL. A total of 10 larvae were exposed to the test solutions. The negative control consisted of a 2% ethanol solution; the positive control consisted of a solution of the organophosphate insecticide malathion (1 µL mL^-1). Each concentration was evaluated in triplicate. The assays were stored at 25±2ºC and relative humidity of 70±2%. Larval mortality readings were taken at 24 and 48 h.

Statistical analyses

The concentrations producing 50 and 95% mortality in the mite and larvae population (LC₅₀) and (LC₉₅) and their respective confidence limits (95 %) were determined by Probit analysis. To establish statistically significant differences between the assays, a one-way ANOVA analysis of variance was performed using the Fisher (LSD) method. Values of P<0.05 were considered significant. All the statistical analyses were carried out using Statgraphics Centurion XVI statistical software.

Acaricidal activity

The mortality percentages of R. microplus larvae after 48 h of exposure to the essential oils of P. marginatum and P. tuberculatum are shown in tables 1 and 2. No mortality was reported in the negative control, while mortality with the positive control was 100%. The results indicate that the essential oil of P. tuberculatum was the most toxic, causing 100% mortality of R. microplus larvae at a concentration of 1 µL mL^-1. The ANOVA comparison analysis found statistically significant differences between the lethal concentrations of the essential oils at 95 % significance level.

Table 3 summarizes the LC₅₀ and LC₉₀ values of P. marginatum and P. tuberculatum oils against R. microplus tick larvae. The most potent oil was that of P. tuberculatum, with a LC₅₀ of 0.73 µL mL^-1 while the oil of P. marginatum showed an LC₅₀ of 0.90 µL mL^-1.

This groundbreaking report unveils the acaricidal activity of essential oils from Piper species against R. microplus larvae, a discovery of utmost significance as larvae are pivotal in tick control systems. ^{Da Silva et al. (2017)} suggest that essential oils from plants of the genus Piper, rich in phenylpropanoids and hydrocarbonated sesquiterpenes, as in this study, exhibit potent insecticidal and acaricidal activity.

The practical implications of this research are evident in the evaluation of the essential oil of P. marginatum and P. tuberculatum against the mite Tetranychus urticae, a crop pest from the family Tetranychidae. The obtained LC₅₀ values of 0.90 and 0.50 µL mL^-1, respectively (^{Ribeiro et al., 2016}; ^{Araújo et al., 2020)} underscore the potential of these essential oils in pest control.

According to ^{Selles et al. (2021)}, the acaricidal activity of essential oils is related to their hydrophobic nature, which is responsible for several mechanical effects, causing death by water stress or asphyxia due to alteration of cuticular waxes and blockage of respiratory stigmas. Essential oils have been shown to act on the motor function of R. microplus larvae, increasing catecholamine levels within the central nervous system (^{Salman et al., 2020}) and inhibiting the action of acetylcholinesterase in this tick species (^{Cardoso et al., 2020}).

The acaricidal capacity observed in the extracts supports the ethnobotanical use of P. tuberculatum in Ecuador as a lice and tick repellent (^{De la Torre et al., 2008}).

Larvicidal activity

The concentrations of the essential oils and their respective percentages of mortality after 24 and 48 h of exposure are shown in tables 4 and 5. A. aegypti larvae's mortality percentage was 100% from the concentration of 50 µL mL^-1 in both oils evaluated. No mortality was reported in the negative control, while mortality with the positive control was 100%.

The lethal concentrations of the essential oils are shown in table 6. P. tuberculatum obtained the highest activity with LC₅₀ of 8.42 µL mL^-1 and LC₉₅ of 51.38 µL mL^-1 while P. marginatum obtained LC₅₀ of 11.87 µL mL^-1 and LC₉₅ of 28.29 µL mL^-1 at 48 h of exposure. The ANOVA comparison analysis found statistically significant differences between the lethal concentrations of the essential oils at 95% significance level.

The volatile compounds of Piper species are characterized by their great potential for pest control and the low environmental impact (^{Pereira et al., 2021}). Previous larvicidal evaluations on the oils of P. marginatum and P. tuberculatum (^{Lavor et al., 2012}; ^{Pereira et al., 2021}), reported LC₅₀ of 39 and 106 µL mL^-1, respectively. Essential oils from Ecuadorian species showed higher larvicidal activity, probably due to their different chemical composition and synergistic interactions between their compounds. The plant extracts used in the present study were previously characterized to identify their phytochemical composition by our research team. Compounds such as sesquiterpenes curzerene (22.44%) and atractylone (18.15%) were identified in P. marginatum, while in P. tuberculatum, the phenylpropanoid dilapiol (49.15%) was identified (^{Moncayo et al., 2021}). Species with essential oils predominantly composed of these compounds have shown insecticidal properties against both adult and larvae stages of A. aegypti and Culex quinquefasciatus mosquitoes (^{Benelli et al., 2017}; ^{Hung et al., 2020}; ^{Pereira et al., 2021}). Dilapiol, a potent inhibitor of cytochrome activity (P450 and CYP34A), reduces the ability to excrete xenobiotics causing the insect's death by accumulation of toxic substances in the digestive tract (^{Santos et al., 2021}). In addition, its structure can amplify the effects of other chemicals (^{Durofil et al., 2021}).