Larvicidal effect and mechanism of action of the essential oil and major compound from Piper brachypetiolatum (Piperaceae) against Aedes aegypti (Linnaeus, 1762) larvae, with protection of non-target aquatic animals

doi:10.21203/rs.3.rs-5233897/v1

Botanical larvicides like essential oils (EO) and their main compounds extracted from plants, such as those in the Piper species offer eco-friendly approaches to mosquito control on account of promote activity against Culicidae larvae, while demonstrating low toxicity to non-target aquatic animals. This study investigated the mechanism and larvicidal activity of the essential oil from Piper brachypetiolatum and its main compound against Aedes aegypti, as well as the lethal effects on the non-target aquatic organisms Toxorhynchites haemorrhoidalis, Anisops bouvieri, and Diplonychus indicus. The EO was extracted from the leaves of P. brachypetiolatum using the hydrodistillation method, yielding 1.5 ± 0.7%. Gas chromatography analyses revealed the presence of sesquiterpenes (64.70%), oxygenated sesquiterpenes (17.64%), monoterpenes (11.76%), and oxygenated monoterpenes (5.88%). The major compound identified was (E)-nerolidol, comprising 64.32% of the EO. Both EO and (E)-nerolidol exhibited larvicidal activity against A. aegypti, with LC50 values of 15.51 and 9.50 ppm, respectively. They also inhibited AChE activity, with IC50 values of 44.97 and 11.07 ppm, respectively, and induced RONS overproduction (p < 0.05) compared to the positive control, α-cypermethrin. Additionally, EO and (E)-nerolidol showed no lethal effects on T. haemorrhoidalis, A. bouvieri, and D. indicus, with these species exhibiting 100% survival after exposure. In contrast, α-cypermethrin caused 100% mortality in these species. These findings highlight the promising potential of the EO from P. brachypetiolatum and (E)-nerolidol as effective and safe alternatives for controlling A. aegypti larvae.

Botanical larvicides

compounds

oxidative

Aedes

non-target animals

Dengue is a viral disease caused by the etiological agent of the Flavivirus genus (Flaviviridae) majority transmitted by the female mosquito Aedes aegypti (Culicidae), which is also a vector for chikungunya and Zika viruses (WHO, 2024). In 2023, the Americas region reported over 4.5 million cases of dengue, with 2,300 deaths (PAHO, 2024), while in Brazil, during the first 23 epidemiological weeks of 2024, there have been over with 3,643 (82.4%) reported cases of dengue, which have resulted in 1,227 deaths (PAHO, 2024).

Currently, the use of synthetic larvicides like organophosphates and pyrethroids for larvae control is problematic due to widespread resistance (PAHO, 2023). Moreover, the use of these synthetic larvicides faces significant challenges on account of their high toxicity to non-target aquatic animals, which are natural predators of Culicidae larvae (Baranitharan et al. 2017). In fact, studies have demonstrated the high toxicity of temephos against Anisops sp. (Hemiptera), Toxorhynchites sp. (Culicidae), Gambusia sp. (Poeciliidae), and Diplonychus sp. (Heteroptera), with LC₅₀ values ranging from 4.85 to 5.82 ppm (De Oliveira et al. 2022a). Additionally, α-cypermethrin showed significant toxicity against Toxorhynchites sp. and Gambusia sp., with LC₅₀ values of 0.22 and 0.29 ppm, respectively (De Oliveira et al. 2022b). In a study by Lima et al. (2024), α-cypermethrin evaluated at concentrations of 0.13 to 0.65 ppm, proving toxic to T. haemorrhoidalis, A. bouvieri, and D. indicus, resulting in 100% mortality. Emphasizing the importance of developing new mosquito control methods that are effective yet safe for beneficial animals and the environment (Da Costa et al. 2024).

Researchers have turned their attention to plant-derived solutions for mosquito larvae control, particularly essential oils (EOs) and compounds extracted from various Piper species, which can act as larvicides through various mechanisms of action (Huong et al. 2020), including overproduction of Reactive Oxygen and Nitrogen Species (RONS), inhibition of acetylcholinesterase (AChE), and changes in enzymatic activities of catalase (CAT), and glutathione S-transferase (GST) (Janner et al. 2021; Da Costa et al. 2024; Lima et al. 2024). Besides their biological properties, EOs show low toxicity to non-target aquatic animals, making them a viable alternative compared to commercial synthetic insecticides (De Oliveira et al. 2022).

The Piper genus, comprising approximately 2,000 species, holds the distinction of being the largest within the Piperaceae family (Yoshida et al. 2018). Piper species are widely distributed in the northern region of Brazil and have attracted considerable attention due to their potential insecticidal and larvicidal properties against various mosquitos such as Aedes sp. and Anopheles sp. (Machado et al. 2015; De Oliveira et al. 2022b).

Piper brachypetiolatum Yunck is a plant commonly found in Manaus, Amazonas, Brazil, which is a shrub that typically grows to about one meter in height and features small, irregular, light green inflorescences (Garcia, 2005). The EO extracted from its leaves is notably rich in (E)-nerolidol, which constitutes 44.23 ± 2.23% of the EO (Araujo et al. 2021). (E)-Nerolidol is a well-known oxygenated sesquiterpene with various biological activities, such as insecticidal, acaricidal activity (Silva et al. 2020), antifungal properties (Sampaio et al. 2020), antinociceptive and anti-inflammatory effects (Ogunwande et al. 2019), and antioxidant activity (Piekarski-Barchik et al. 2021). However, specific studies on the biological activities of this EO against mosquitos such as A. aegypti, as well as its mechanism of action, remain scarce.

Thus, the aim of this study was to investigate the larvicidal activity and mechanism of action of the EO from P. brachypetiolatum and its major compound (E)-nerolidol, against A. aegypti, as well as to assess its toxicity to non-target aquatic animals.

Chemicals and reagents

The following reagents were obtained from Merck (Brazil): AChE enzyme from Electrophorus electricus (200–1,000 units/mg protein), dimethyl sulfoxide (DMSO) (≥ 99%), phosphate buffer solution (pH 7.3), bovine serum albumin (BSA) (pH 7, ≥ 98%), anhydrous sodium sulfate (Na₂SO₄), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (≥ 99%), acetylthiocholine iodide (AChI) (≥ 99%), n-alkane series (C₈–C₃₀) (Supelco), α-cypermethrin (PESTANAL®, analytical standard), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) (≥ 95%), 2’,7’-dichlorofluorescein (DCF) (≥ 90%), hexane (HPLC) (≥ 99%), ethyl acetate (HPLC) (99.9%), (E)-nerolidol (99%), and methanol (HPLC) (99.9%).

Plant material

The collection of the P. brachypetiolatum was authorized by the Sistema de Autorização e Informação em Biodiversidade (No. 78372/1) and registered in the e Sistema Nacional de Gestão do Patrimonio Genético e do Conhecimento Tradicional Associado (No. AE3F373). The plant was collected in the city of Manaus, in the state of Amazonas, Brazil (latitude 2°92'68"S and longitude 59°97'77"W). The collected material was authenticated and deposited in the Herbarium of the Universidade Federal do Amazonas, under the registration HUAM No. 12102. The leaves were removed from the branches, dried at room temperature (28 ± 6 °C), ground using a knife mill, and stored in a glass recipient until the extraction of the EO (De Oliveira et al. 2020).

Extraction and chromatograph analyses

For the extraction of the EO, 200 g of pulverized leaves of P. brachypetiolatum were subjected to hydrodistillation using a Clevenger apparatus. The collected EO was dried with anhydrous sodium sulfate (Na₂SO₄) and stored at 4 °C until chromatographic analyses. The extraction, conducted in triplicate over 3 hours, had its yield calculated using the equation: EO (%) = (volume of EO / weight of the sample) × 100 (Girard et al. 2007).

For the Gas Chromatography-Mass Spectrometry (GC–MS) and Gas Chromatography-Flame Ionization Detection (GC-FID) analyses, 1 mg of the EO was diluted in 1 mL of ethyl acetate, following the methodology previously reported by Oliveira et al. (2022b). Compound identification was based on mass spectra and retention indices (RI), compared with values from the literature (Adams, 2017). The RI was determined using a series of n-alkanes (C₈-C₃₀) and the Van Den Dool and Kratz equation (1963).

Mosquito rearing

The rearing conditions were controlled at the Laboratório de Controle Biológico e Biotecnologia da Malária e da Dengue of the Instituto Nacional de Pesquisas da Amazônia, under temperature of 28 ± 2 °C, relative humidity of 80 ± 5%, and a photoperiod of 12 hours light to 12 hours dark, following the methodology described by de Oliveira et al. (2020). A. aegypti eggs were deposited on filter paper and immersed in running water for hatching. Larvae were fed a mixture of rodent and feline food in a 1:1 ratio until reaching the fourth instar. Subsequently, pupae were collected and transferred to entomological cages (30 cm x 30 cm x 30 cm). Adults were fed with a 10% sucrose solution, and females were provided with blood meals from Mesocricetus auratus (Cricetidae) hamsters under authorization from the Ethics Committee on Animal Use (No. 011/2022 - SEI-01280.000601/2022-08) (Beserra et al. 2010).

Larvicidal assay

The larvicidal activity assessment of the EO from P. brachypetiolatum and (E)-nerolidol against A. aegypti was conducted according to the World Health Organization Guidelines for Laboratory and Field Testing of Mosquito Larvicides, with some adaptations (WHO, 2005). Groups of 20 third instar larvae (n = 500) were transferred to containers holding 199 mL of distilled water and concentrations ranging from 5 to 30 ppm of EO oil from P. brachypetiolatum and (E)-nerolidol, previously diluted in 1 mL of dimethyl sulfoxide (DMSO). The synthetic insecticide α-cypermethrin was used as a positive control at concentrations ranging from 0.13 to 0.65 ppm, while DMSO was tested at concentrations ranging from 5 to 30 ppm was used as a negative control. The tests were conducted in quintuplicate with three replicates under controlled conditions of relative humidity (80 ± 5%) and temperature (28 ± 2 °C). The percentage of mortality at each concentration was calculated using the equation: Total dead larvae / total treated larvae x 100.

Acetylcholinesterase (AChE) inhibition assay

The AChE inhibition test was conducted following the colorimetric method described by Ellman et al. (1961), with adaptations by de Oliveira et al. (2020). Neostigmine (1 mg) and AChE (10 μL) were prepared in 1 mL of 0.1 M phosphate buffer at pH 8, while the EO from P. brachypetiolatum and (E)-nerolidol, both at 1 mg, were dissolved in MeOH at 1 mL and evaluated at concentrations ranging from 10 to 100 ppm. The experiment was performed in triplicate using a 96-well microplate, incubated in a light-protected environment. Neostigmine (0.07 to 10 ppm) was used as a positive control and MeOH as a negative control. Absorbance readings were taken over 30 minutes with 5-minute intervals, using a wavelength of 405 nm on a microplate reader (ELx800, Biotek, USA). The percentage inhibition at each concentration was calculated using the formula: Inhibition (%) = A² – (A¹ – A³) x 100 / A², where A¹ is the absorbance of samples with enzyme, A² is the absorbance of enzyme without sample, and A³ is the absorbance of sample without enzyme.

Preparation of the supernatant for oxidative stress indicator assay

After 24 hours of exposure, A. aegypti larvae treated with EO from P. brachypetiolatum (30 ppm), (E)-nerolidol (30 ppm), α-cypermethrin (0.65 ppm), and DMSO (130 ppm) (De Oliveira et al. 2024) were homogenized in 0.1 M phosphate buffer (pH 7.3) at a ratio of 1 mg of larvae per 10 mL of buffer. The homogenates were centrifuged at 4000 rpm for 5 minutes, and the resulting supernatants were stored in eppendorf tubes at – 4 °C for oxidative stress analysis, assessing reactive RONS levels (Janner et al. 2021; Johnson et al. 2021). Protein content was quantified using bovine serum albumin (BSA, 1 mg/mL) as a standard (Lowry et al. 1951).

Measurement of reactive oxygen and nitrogen species (RONS)

To measure RONS, 2′,7′-dichlorofluorescein (DCFH) was employed as an oxidative stress marker, following the methodology outlined by Perez-Severiano et al. (2004). Each supernatant (diluted 1:10) was combined with 5 µL DCFH, 40 µL distilled water, and 150 µL potassium buffer (0.1 M, pH 7.4) in a 96-well microplate, then incubated for 60 minutes at 37 °C. Fluorescence intensity was monitored for 10 minutes at 30-second intervals using a SpectraMax plate reader (Molecular Devices, USA), with excitation set at 488 nm and emission at 525 nm. DMSO and α-cypermethrin served as control groups. Each treatment was tested in triplicate, and the rate of DCF formation was expressed as a percentage relative to the control groups.

Assessment of toxicity in non-target animals

The study assessed the toxicity of the EO from P. brachypetiolatum and (E)-nerolidol on non-target aquatic animals T. haemorrhoidalis, A. bouvieri and D. indicus, following the methodology described by Sivagnaname and Kalyanasundaram (2004). The animals were collected at the Instituto Nacional de Pesquisas da Amazônia and identified using the taxonomic key by Hamada et al. (2014). After 48 hours of acclimatization, the animals were transferred to containers containing 499 mL of natural habitat water and exposed to concentrations of 228 and 239 ppm of the EO from P. brachypetiolatum and (E)-nerolidol, prepared in 1 mL of DMSO. These concentrations were calculated by multiplying the CL₉₀ values of the EO from P. brachypetiolatum and (E)-nerolidol by 10. DMSO at 239 ppm was used as a negative control, while the positive control α-cypermethrin was tested at 0.39 ppm. The assays were conducted in five replicates with n = 25 for each non-target animals, under controlled conditions of relative humidity (80 ± 5%) and temperature (28 ± 2 °C), over a 30-day treatment period.

Statistical analyses

The LC₅₀ and LC₉₀ values from the larvicidal test, along with Chi-square, slope ± standard error, and degrees of freedom, were determined using Probit analysis with IBM® SPSS® Statistics software. Kaplan-Meier analysis (p ≤ 0.05) was used to calculate the survival curve of non-target animals (Kishore et al. 2010). For the AChE assay, absorbances were logarithmically transformed, normalized, and analysed using nonlinear regression to determine the IC₅₀. Statistical analyses were performed using GraphPad Prism® 9 software. Furthermore, one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test (p ≤ 0.05), was conducted to compare treatments in the oxidative stress assay.

Chemical composition of the EO

The EO from P. brachypetiolatum, obtained from the extraction of 200 g of fresh leaves, yielded 1.5 ± 0.7%. The chemical composition analysis, performed using GC-MS and GC-FID, revealed that the oil is predominantly composed of sesquiterpenes (64.70%), followed by oxygenated sesquiterpenes (17.64%), monoterpenes (11.76%) and oxygenated monoterpenes (5.88%). The major constituent identified was (E)-nerolidol, which accounts for 64.32% of the total oil composition. Other relevant components include hinesol (12.42%), α-terpinene (8.10%), and β-caryophyllene (5.72%), as detailed in Table 1.

Table 1. Compounds identified in the EO extracted from P. brachypetiolatum leaves.

Substances	RI^calc	RI^lit	(%)^a	Chemical Class
α-Terpinene	1417	1417	8.10	Monoterpene
Limonene	1023	1024	0.26	Monoterpene
Borneol acetate	1275	1278	0.23	Oxygenated monoterpene
α-Cubebene	1348	1351	0.53	Sesquiterpene
α-Copaene	1374	1376	1.22	Sesquiterpene
β-Caryophyllene	1417	1419	5.72	Sesquiterpene
γ-Gurjunene	1475	1477	0.42	Sesquiterpene
γ-Muurolene	1478	1479	0.76	Sesquiterpene
α-Zingiberene	1493	1493	0.71	Sesquiterpene
Valencene	1496	1496	0.51	Sesquiterpene
α-Muurolene	1500	1500	1.17	Sesquiterpene
γ-Cadiene	1513	1513	0.46	Sesquiterpene
δ-Cadinene	1522	1523	1.33	Sesquiterpene
α-Cadinene	1537	1538	0.30	Sesquiterpene
(E)-Nerolidol	1561	1563	64.32	Oxygenated sesquiterpene
Guaiol	1600	1600	1.54	Oxygenated sesquiterpene
Hinesol	1632	1638	12.42	Oxygenated sesquiterpene
Total identification			100
Monoterpene			11.76
Oxygenated monoterpene			5.88
Sesquiterpene			64.70
Oxygenated sesquiterpene			17.64

RI^calc - Calculated retention index using n-alkanes (C₈-C₃₀) on the TR5 column. RI^lit - Literature retention index (ADAMS 2007; NIST 2020; Internal library). ^a – Relative area calculated from the peak area in relation to the total peak area on the GC-FID chromatograph of three replicates. ^b - Mass spectra compared to the literature (ADAMS 2007; NIST 2020; Internal Library). Retention index calculated using n-alkanes.

Larvicidal assay

The EO evaluated at concentrations ranging from 5 to 30 ppm, exhibited significant larvicidal activity against A. aegypti, with LC₅₀and LC₉₀ values of 15.51 ppm and 22.79 ppm, respectively, and a relative potency of 0.013. The major compound, (E)-nerolidol, tested at the same concentrations as the EO, demonstrated even more pronounced larvicidal activity, with LC₅₀ and LC₉₀ values of 9.50 ppm and 23.89 ppm, respectively, and a relative potency of 0.022 (Table 2). No mortality was observed in the negative control group (DMSO). In contrast, the synthetic insecticide α-cypermethrin exhibited the highest larvicidal activity among the tested products, with LC₅₀ and LC₉₀ values of 0.21 ppm and 0.39 ppm, indicating extreme toxicity compared to the EO and (E)-nerolidol (p < 0.05).

Table 2. Lethal concentrations of the EO from P. brachypetiolatum and (E)-nerolidol against A. aegypti larvae.

	Concentration (ppm)	Mortality (%)	LC₅₀ (ppm) (LLC-ULC)	LC₉₀ (ppm) (LLC-ULC)	χ2 (DF)	Slope ± SE	RP
P. brachypetiolatum	10	0 ± 0	15.51^b (13.743 -17.141)	22.798^b (20.311– 27.450)	5.69 (3)^*	7.666 ± 0.570	0.013
	15	23 ± 4
	20	66 ± 5
	25	80 ± 3
	30	100 ± 0
(E)-Nerolidol	5	1 ± 0	9.50^c (7.714 -11.301)	23.899^a (19.433- 32.194)	3.22 (3)^*	3.201 ± 0.246	0.022
	10	19 ± 2
	20	54 ± 4
	30	79 ± 3
	40	92 ± 5
α-Cypermethrin	0.13	21 ± 4	0.21^a (0.172-0.264)	0.39^a (0.323-0.554)	7.52 (3)^*	5.022 ± 0.378	1
	0.26	53 ± 5
	0.39	79 ± 3
	0.52	95 ± 4
	0.65	100 ± 0

LC₅₀ and LC₉₀ - Lethal concentrations to kill 50 and 90% of larvae. LLC - Lower confidence limit of 95%. ULC - Upper confidence limit of 95%. ^* Non-significant Chi-square (p > 0.05). Df - Degree of freedom. Letters in same column indicate statistical difference (ANOVA One-way and Tukey's test p < 0.05). RP - relative potency (LC₅₀ standard/LC₅₀ of natural larvicidal) (Cheng et al. 2009).

Acetylcholinesterase inhibition assay

The essential oil of P. brachypetiolatum and its major compound, (E)-nerolidol, demonstrated significant inhibition of the enzyme AChE, with IC₅₀ values of 44.97 ppm and 11.07 ppm, respectively. In comparison, the reference standard neostigmine exhibited an IC₅₀ of 6.796 ppm (p < 0.05) (Figure 1).

Measurement of reactive oxygen and nitrogen species (RONS)

The EO and the compound (E)-nerolidol, evaluated at a concentration of 30 ppm, induced the oxidation of DCFH, resulting in an increase in the production of RONS, indicating the presence of oxidative stress in A. aegypti larvae after exposure to these products. Additionally, a significant elevation in RONS production was observed following exposure of the larvae to the positive control, α-cypermethrin, at a concentration of 0.65 ppm (p < 0.05). In contrast, the negative control, DMSO at 30 ppm, exhibited a lower production of RONS (Figure 2).

Toxicity against non-target animals

The essential oil from P. brachypetiolatum and (E)-nerolidol did not exhibit lethal effects on the non-target aquatic organisms T. haemorrhoidalis, A. bouvieri and D. indicus, resulting in 100% survival after 30 days of exposure. A similar outcome was observed in the negative control group (DMSO) (Figure 3). In contrast, the synthetic insecticide α-cypermethrin, at a concentration of 0.13 ppm, demonstrated extreme toxicity to these organisms, leading to 100% mortality immediately after exposure. These findings highlight the safety of the EO and (E)-nerolidol concerning these species, in contrast to the harmful effects of the synthetic insecticide.

The presence of (E)-nerolidol in the EO from P. brachypetiolatum was documented for the first time by Araujo et al. (2021), with 44.23 ± 2.23%. This sesquiterpene is widely found in the EOs of various species in the Piperaceae family, as observed in P. bellidifolium Yunk. (20.3 ± 0.4%) (Araujo et al. 2018), P. longum L. (19.56%) (Dash et al. 2021), P. lolot C. DC (18.43%) (Phong et al. 2022), and P. aduncum (26.75%) (Alonso-Hernández et al. 2023). Additionally, (E)-nerolidol has also been reported in species from different botanical families, such as Stevia rebaudiana (Bert.) Bertoni (Asteraceae) with 8.0% (Benelli et al. 2020), Zingiber montanum (J. Koenig) Link ex A. Dietr (Zingiberaceae) with 14.3% (Huong et al. 2020), and Melaleuca leucadendra L. (Myrtaceae) with 90.8% (Padalia et al. 2015), highlighting its widespread distribution in nature.

Regarding larvicidal activity, the EO from P. brachypetiolatum demonstrated greater efficacy against A. aegypti larvae compared to the EOs of other species within the Piper genus. For instance, P. purusanum C.DC exhibited an LC₅₀ of 62.33 ppm (de Oliveira et al. 2022), P. tuberculatum (Jacq.) had an LC₅₀ of 48.61 ppm (Lima et al. 2024), P. alatipetiolatum Yuncker an LC₅₀ of 33.74 ppm (de Oliveira et al. 2020), and P. corcovadense (Miq.) C.DC an LC₅₀ of 30.52 ppm (da Silva et al. 2016).

The species Magnolia denudata Desr. (Magnoliaceae) also demonstrated significant larvicidal activity against Culex pipiens pallens (LC₅₀ of 9.84 ppm) and Aedes albopictus (LC₅₀ of 16.34 ppm) (Wang et al. 2016). This study, although involving different plant species and insect larvae, presented similar larvicidal activity to that of the EO from P. brachypetiolatum against A. aegypti larvae.

The major compound (E)-nerolidol also demonstrated larvicidal activity, as corroborated by the study conducted by Benelli et al. (2020), which indicated that the EO from Stevia rebaudiana (Asteraceae), containing 37% (E)-nerolidol, exhibited activity against Metopolophium dirhodum (Aphididae) with an LC₅₀ of 3.5 ppm. In addition to its insecticidal properties, (_E)-nerolidol is widely recognized for its various biological activities, including antileishmanial, antimalarial, antibacterial, and antifungal effects (Pasquali 2022; De Moura et al. 2020; Assenço 2022).

AChE serves a fundamental role in the hydrolysis of acetylcholine, an essential neurotransmitter in the central nervous system of animals (Moldogazieva et al. 2020). The inhibition of AChE by insecticides leads to the accumulation of acetylcholine at synapses, resulting in significant toxic effects (Chao et al. 2017). These changes also impact the levels and activities of antioxidant enzymes, increasing the susceptibility of insects to the toxic effects of chemical agents (Janner et al. 2021).

The larvicidal mechanism involving AChE inhibition has been extensively documented in various studies with essential oils from different plant species. For example, the EO from Salvia officinalis (Lamiaceae), tested on A. aegypti larvae, demonstrated AChE inhibition with an IC₅₀ value of 37 ± 2.6 ppm, with the main components being 1,8-cineole and α-thujone (Castillo-Morales et al. 2019). Additionally, the EO from Piper baccans (Piperaceae) exhibited neurotoxic activity through AChE inhibition, showing an IC₅₀value of 38.37 ppm against A. aegypti larvae, where δ-cadinene was the predominant compound (Souza, 2023). This mechanism was also observed in the EO of Piper tuberculatum (Piperaceae), where its main compound, β-caryophyllene, demonstrated significant AChE inhibition, with IC₅₀ values of 57.78 ppm and 71.97 ppm, respectively (Lima et al. 2024).

Reactive Oxygen and Nitrogen Species (RONS) are inherent byproducts of aerobic metabolism, playing crucial roles in regulating cellular processes such as survival, growth, proliferation, and apoptosis (Aranda-Rivera et al. 2022). However, the overproduction of these molecules promotes the uncontrolled generation of free radicals, which can cause severe damage to essential biomolecules, including lipids, proteins, polysaccharides, and nucleic acids (Abolaji et al. 2015). Following the significant increase in RONS generation observed in A. aegypti larvae exposed to the EO from P. brachypetiolatum and the compound (E)-nerolidol, it was determined that this excess of reactive species leads to a significant imbalance in the organism antioxidant system. This imbalance compromises the effectiveness of endogenous antioxidant defenses, making the organism more susceptible to the deleterious effects of oxidative stress (Chang et al. 2020).

Previous studies, such as those conducted by Martelli et al. (2020), demonstrated an overproduction of RONS in Drosophila melanogaster (Drosophilidae) following exposure to low doses of the insecticide imidacloprid, which was associated with notable changes in antioxidant enzyme levels. This imbalance in the antioxidant defense system is exacerbated by the interaction of these reactive species with various biomolecules, resulting in extensive oxidative damage (Phaniendra et al. 2015; Ndonwi et al. 2019). Similarly, a study by de Castro Oliveira et al. (2022) identified that EOs extracted from Eugenia uniflora L. (Myrtaceae), Melaleuca armillaris (Myrtaceae), and Schinus molle (Anacardiaceae) induced oxidative stress in Culex quinquefasciatus (Culicidae) larvae, causing structural alterations in lipids, proteins, and DNA, ultimately leading to the mortality of these invertebrates. Similar results were observed by Shahriari et al. (2018) when investigating the effects of compounds such as α-pinene, trans-anethole, and thymol on Ephestia kuehniella (Pyralidae) larvae, where the excessive production of free radicals caused profound structural damage to the cells.Parte inferior do formulárioParte inferior do formulário

Non-target aquatic animals, such as T. haemorrhoidalis, A. bouvieri, and D. indicus, are frequently studied to assess the environmental safety of insecticide products (Da Costa et al. 2024). This analysis highlights concerns regarding synthetic insecticides, which can cause significant harm to non-target organisms, compromising biodiversity and ecosystem health (De Oliveira et al. 2022).

Similar studies were conducted with the EO from P. tuberculatum and its main compound, β-caryophyllene, which also showed no toxicity against A. bouvieri, D. indicus, and T. haemorrhoidalis after 30 days of exposure to the products (Lima et al. 2024). Additionally, the EO from Syzygium lanceolatum (Myrtaceae), tested on non-target organisms such as A. bouvieri, D. indicus, G. affinis, and P. reticulata, exhibited low toxicity, with LC₅₀ values ranging from 4.148 to 15.762 ppm (Benelli et al. 2018). Analyses conducted with the same organisms by Govindarajan et al. (2018) reported LC₅₀ values ranging from 3.123 to 9.104 ppm for the toxicity of the EO from Amomum subulatum Roxb. (Zingiberaceae).

This information is essential for assessing the ecological safety of natural products and emphasizes the importance of further investigating the properties of EOs. Furthermore, the findings reinforce the need to consider toxicity at different trophic levels and the potential of natural products as sources of bioactive insecticidal compounds that preserve ecosystem integrity.

This study highlighted the significant larvicidal potential of the EO from P. brachypetiolatum leaves and its major compound, (E)-nerolidol, against A. aegypti larvae. It further elucidated their mechanisms of action and confirmed the absence of lethal effects on non-target organisms. These findings emphasize the potential of this EO and (E)-nerolidol as eco-friendly and safe alternatives to conventional insecticides.

Funding

This project was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), process (140522/2021-2). It was also supported by the project Technological Innovations for Monitoring, Vector Control, and Etiological Agents of Malaria and Dengue in the Amazon, process (01.02.016301.04682/2022-87) from Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM - POSGRAD).

Competing Interest

The authors declare no conflict of interest.

Author Contributions

Suelen C. Lima, André C. de Oliveira, Hergem V. de Souza, Maria Luiza L. da Costa, and Aylane Tamara dos S. Andrade made substantial contributions to the conception of the study, as well as to the analysis and interpretation of the data. Rosemary A. Roque, Suelen C. Lima, Dayane D. Abensour, and Cláudia P. S. Tavares were responsible for drafting and critically revising the work, ensuring its intellectual quality. All authors reviewed and approved the final version of the manuscript.

Data Availability

The data will be available upon request.

Ethical Approval

The authors are grateful to Central Analítica of the Universidade Federal do Amazonas, Laboratório de Etnoepidemiologia and the Laboratório de Princípios Ativos da Amazônia, both from Instituto Nacional de Pesquisas da Amazônia. As also to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM – POSGRAD).

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No competing interests reported.

Larvicidal effect and mechanism of action of the essential oil and major compound from Piper brachypetiolatum (Piperaceae) against Aedes aegypti (Linnaeus, 1762) larvae, with protection of non-target aquatic animals

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Abstract

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Introduction

Materials and methods

Results

Discussion

Conclusions

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References

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