Apiol-Rich and Caryophyllene-Oxygenated Essential Oils from Amazonian Piper Species as Dual-Action Biopesticides: Broad-Spectrum and Selective Antifeedant

1. Introduction

Resistance to synthetic pesticides in crop pests and disease transmitting arthropods demands alternative control strategies [1]. Key resistant species include the polyphagous lepidopteran Spodoptera littoralis (Boisduval), with documented resistance to pyrethroids, organophosphates, and spinosad [2]; the aphid vectors Myzus persicae (Sulzer), which has developed resistance to more insecticide classes than any other herbivorous arthropod [3], and Rhopalosiphum padi (L.) [4,5]; and the tick Hyalomma lusitanicum Koch principal reservoir/vector of Crimean, Congo hemorrhagic fever virus (CCHFV) in southwestern Europe [6,7,8,9,10]. These species collectively illustrate how multi-class resistance compromises conventional chemical control [1,3,10], highlighting the urgency of identifying new biocidal agents effective against both agricultural pests and arthropod vectors of public health concern.

Essential oils (EOs) from aromatic plants have emerged as promising candidates for sustainable pest management owing to their rapid biodegradability, low mammalian toxicity, and multiple simultaneous modes of action, targeting the octopaminergic system, GABA receptors, and acetylcholinesterase, which reduce selection pressure for resistance [11,12,13,14,15]. Among plant-based strategies, the antifeedant approach is particularly valuable for integrated pest management (IPM) by modulating contact chemoreception rather than exerting direct lethal toxicity, and lowering resistance risk [16,17].

The genus Piper L. (Piperaceae), with approximately 2000 tropical species, produces a remarkable diversity of essential oils rich in terpenoids and phenylpropanoids with pesticidal properties [18,19,20]. Of particular interest are phenylpropanoids bearing the methylenedioxyphenyl (MDP) pharmacophore, the same structural motif present in the synergist piperonyl butoxide (PBO), which inhibits cytochrome P450 monooxygenases involved in insect detoxification [21]. Dillapiole, the dominant MDP, phenylpropanoid in Piper aduncum L., has been extensively studied as an insecticide and acaricide [22], and safrole from P. hispidinervum C. DC. has shown antifeedant activity against S. littoralis, M. persicae, and R. padi with documented synergistic interactions [23]. In contrast, apiol (2,5-dimethoxy-3,4-methylenedioxy-1-allylbenzene), another MDP-bearing phenylpropanoid that dominates the essential oil of several Piper species, has received comparatively little attention despite sharing the same pharmacophore. To the best of our knowledge, neither the antifeedant nor the ixodicidal activities of apiol have been systematically evaluated, and no previous study has compared these activities across apiol-rich Piper chemotypes.

In a previous study, our group characterized the GC-MS composition, antifungal activity against Botrytis cinerea, and herbicidal potential of essential oils from ten Piper species collected in the Peruvian Amazon [24]. The essential oils exhibited diverse chemotypes: two species were dominated by apiol (P. sancti-felicis, 76.1%; P. mituense, 51.6%), while others were characterized by sesquiterpene hydrocarbons (β bisabolene, caryophyllene, germacrene D, bicyclogermacrene) or the monoterpene limonene. Three essential oils (P. casapiense, P. soledadense, and P. mituense) were described for the first time, and dillapiole was not detected in any of the ten essential oils [24], making apiol the sole dominant MDP-bearing compound in this collection. However, the insect antifeedant and ixodicidal potential of these essential oils and their dominant compound apiol was not investigated.

Phenylpropanoid, are a large and structurally diverse class of plant secondary metabolites involved in ecological interactions such as defense against herbivores, pathogens, and environmental stress [25,26]. Apiol, a benzodioxole-type phenylpropanoid, is widely distributed in plant essential oils and has been associated with several biological activities, including specific inhibitory effects on aflatoxin G₁ biosynthesis in Aspergillus parasiticus [27].

Accordingly, the aim of the present study was to evaluate, for the first time, the antifeedant effects of these ten Amazonian Piper essential oils and their main components against three model pest species (S. littoralis, M. persicae, and R. padi), together with their ixodicidal activity against H. lusitanicum larvae and additionally, their effects on the plant parasitic nematode Meloidogyne javanica (Treub) Chitwood. This work expands the biocidal profile of these Amazonian essential oils beyond their previously reported antifungal and herbicidal activities [24], contributing to the identification of apiol-rich Piper chemotypes as dual purpose biopesticidal agents for integrated management of crop pests and ticks. Additionally, molecular docking, a widely used computational method that predicts ligand–protein binding by sampling conformations and ranking them using scoring functions [28,29] has been used to compare the interactions of apiol versus known ligands on acetylcholinesterase and CYP450 proteins from insects, ticks and nematodes [30], to better understand the mechanism of action of apiol and the apiol-rich essential oils.

2. Results

2.1. Essential Oil Composition

Table 1 shows the summarized chemical composition of the Piper essential oils studied here determined by GC-MS. Their chemical composition was previously reported [24]. Based on their dominant chemical classes, the essential oils were classified into four chemotypes: (i) a phenylpropanoid chemotype dominated by apiol (P. sanctifelicis, 76.1%; P. mituense, 51.6%); (ii) a predominantly monoterpene chemotype rich in limonene (P. soledadense, 38.5%); (iii) a bisabolene-type sesquiterpene chemotype (P. coruscans, β-bisabolene 33.4%; P. tuberculatum, β-bisabolene 40.2%); and (iv) a mixed sesquiterpene chemotype characterized by varying proportions of caryophyllenes (P. anonifolium), germacrene D, and bicyclogermacrene (P. obliquum, P. dumosum, P. reticulatum, and P. casapiense). In this work, further chemical identification of previously unidentified compounds has been carried out based on retention index and EI mass spectrum (see table S1 in supplementary information).

Three major (>5%) compounds have been identified in P. casapiense. Compounds eluting at rt 19.49 and 21.13 min (RI = 1711 and 1798) were dominated by ions at m/z 93, 91, 105 and 133, characteristic of caryophyllane-type fragmentation. These compounds have been tentatively identified as caryophyllenone and β-caryophyllenol. A third oxygenated caryophyllane derivative (rt 21.55 min) was detected with a retention index of 1821. Its EI mass spectrum was dominated by characteristic caryophyllane ions at m/z 93, 91, 105 and 133, together with additional fragments at m/z 107, 119 and 121, indicative of a higher degree of oxygenation. Based on its elevated retention index and fragmentation pattern, this compound was tentatively identified as a caryophyllane type diol or highly oxygenated caryophyllenol derivative. Its co-occurrence with caryophyllenone and β caryophyllenol supports a sequential oxidative transformation of a common β caryophyllene precursor within the essential oil. Similarly, in P. anonifolium, the EI mass spectrum (m/z 161/105/81/204/119/162/134/91/159/131) of compound eluting at rt 15.32 min and RI 1503 was characteristic of a caryophyllane alcohol. Additionally, in P. obliquum, the EI mass spectrum (m/z 95/121/161/204/43/109/105/81/164/108) of the compound eluting at rt 18.46 min and RI 1658 was characteristic of a highly oxygenated caryophyllane-type sesquiterpene (diol derivative).

2.2. Bioactivity

The insect antifeedant effects of the tested essential oils are shown in Table 2. The most sensitive insects were the aphids M. persicae (7 active of 10), R. padi (6 active of 10) and the lepidopterous S. littoralis (3 active of 10). Regarding insect species selectivity, P. mituense and P. sancti-felicis were active against all three insect species, P. anonifolium, P. casapiense, P. obliquum and P. reticulatum had significant effects on both aphid species, P. coruscans and P. tuberculatum were only active on M. persicae and P. soledadense on R. padi. The most active oil on R. padi was P. casapiense (EC₅₀, 3.8 µg/cm²), followed by P. sancti-felicis (EC₅₀, 8.6 µg/cm²), P. mituense (EC₅₀, 9.7 µg/cm²) and P. obliquum (EC₅₀, 25.6 µg/cm²), while P. soledadense and P. anonifolium had significant effect but did not reach the 70% inhibition threshold to carry out dose-response experiments. On M. persicae, P. reticulatum was the most active (EC₅₀, 16.2 µg/cm²) followed by P. mituense (EC₅₀, 23.2 µg/cm²), P. tuberculatum (EC₅₀, 39.5 µg/cm²) and P. sancti-felicis (EC₅₀, 53.5 µg/cm²), while P. anonifolium, P. casapiense, P. coruscans and P. obliquum had significant effect but did not reach the 70% inhibition threshold to carry out dose-response experiments. On S. littoralis, P. mituense (EC₅₀, 15.6 µg/cm²) was very active, followed by P. sancti-felicis (EC₅₀, 42.0 µg/cm²) and P. dumosum, with a feeding inhibition value below the 70% inhibition threshold.

Among the major components of the oils (Table 1), the following compounds were tested: apiol, β-bisabolene, δ-cadinene, β-caryophyllene, β-caryophyllene oxide, germacrene D, α-humulene, limonene, nerolidol, β-pinene, α-pinene (Table 2). Additionally, myristicin was included because of its potential synergistic action.

Apiol was active against the three insect targets with EC₅₀ values of 22.24, 16.00 and 17.70 µg/cm² against S. littoralis, M. persicae and R. padi respectively. Nerolidol showed significant effects on both aphid species and α-pinene on S. littoralis but did not reach the 70% inhibition threshold to carry out dose-response experiments (Table 2).

The ten essential oils and apiol were also evaluated for nematicidal activity against Meloidogyne javanica second stage juveniles (J2). None of the essential oils nor the pure compound exhibited significant nematicidal effects at the concentrations tested (see Table S2).

Table 3 shows the ixodicidal activity of the ten Piper essential oils against Hyalomma lusitanicum larvae. Five essential oils were effective ixodicidal agents, with P. mituense being the most active (LD₅₀ = 1.46 µg/mg cellulose), followed by P. sanctifelicis (LD₅₀ = 2.14 µg/mg cellulose), P. reticulatum (LD₅₀ = 6.19 µg/mg cellulose), P. dumosum (LD₅₀ = 8.89 µg/mg cellulose), and P. soledadense (LD₅₀ = 10.05 µg/mg cellulose).

The major components of the active oils (apiol, limonene, α- and β-pinene, caryophyllene, β-caryophyllene oxide, nerolidol, copaene and δ-cadinene) were tested on H. lusitanicum larvae (Table 3). Myristicin was also included similarly to the tests on insects. Apiol (LD₅₀ of 1.96 µg/mg cellulose) and β-caryophyllene oxide (LD₅₀ of 4.46 µg/mg cellulose) and myristicin (LD₅₀ of 2.88 µg/mg cellulose) were active (Table 3).

2.3.1. Principal Component Analysis

Principal component analysis was performed on a matrix of 26 active variables (23 compounds with relative abundance ≥5% in at least one species and three antifeedant EC₅₀ values) across the ten Piper species, using autoscaling. The first two principal components accounted for 21.9% and 18.3% of the total variance, respectively (40.2% cumulative), and their biplot is shown in Figure 1.

The score plot revealed three distinct groupings. The apiol-rich species P. mituense (Pm) and P. sancti-felicis (Psf) clustered in the upper-left quadrant, driven by the strong positive loading of apiol on PC2 and its negative contribution to PC1. In contrast, the β-bisabolene-dominated species P. coruscans (Pc) and P. tuberculatum (Pt) were projected into the lower-center region, associated with the loadings of β-bisabolene, δ-cadinene, nerolidol, and β-caryophyllene. The sesquiterpene-rich species P. anonifolium (Pa) and P. dumosum (Pd) occupied the right side of the plot, separated along PC1 by high loadings of germacrene D, α-cubebene, and α-humulene. P. casapiense (Pcs) was clearly isolated in the lower-left quadrant, driven by oxygenated caryophyllane derivatives (caryophyllenone, β-caryophyllenol, caryophyllane oxide, and oxygenated caryophyllane diol), which are exclusive to this species. P. soledadense (Ps) occupied an intermediate position in the left-center region, consistent with its limonene-dominated monoterpene chemotype. P. obliquum (Po) and P. reticulatum (Pr) were positioned near the center, reflecting their mixed sesquiterpene profiles without a single dominant compound.

The projection of the antifeedant EC₅₀ variables (red arrows, Figure 1) provided insight into the relationship between chemical composition and biological activity. The EC₅₀ vectors for S. littoralis (Sl) and M. persicae (Mp) pointed toward the center-right of the plot, while the EC₅₀ vector for R. padi (Rp) was directed toward the lower-left quadrant. Since lower EC₅₀ values indicate greater antifeedant potency, the most active species are those projected in the direction opposite to the EC₅₀ vectors. Accordingly, Pm and Psf, located opposite to all three EC₅₀ vectors, were associated with the strongest broad-spectrum antifeedant activity, consistent with their apiol-rich chemotype. The proximity of the apiol loading vector to these species and its opposition to the EC₅₀ vectors confirmed that apiol content is the primary chemical determinant of broad-spectrum antifeedant efficacy. Conversely, the sesquiterpene-dominated species (Pa, Pc, Pt) were projected near or along the EC₅₀ vectors, consistent with their weak or absent antifeedant effects. P. casapiense (Pcs), despite its position opposite to the Rp vector, showed strong selective activity against R. padi (EC₅₀ = 3.8 µg/cm²), suggesting that its oxygenated caryophyllane derivatives may contribute to taxon-selective antifeedant effects.

A second PCA (Figure 2) was performed on a matrix of 24 active variables (23 compounds with relative abundance ≥5% in at least one species and the LD₅₀ against H. lusitanicum larvae) across the ten Piper species, using autoscaling. PC1 and PC2 explained 22.0% and 19.1% of the total variance, respectively (41.1% cumulative). The LD₅₀ vector (red arrow) pointed toward the upper-left quadrant, where the five ixodicidally active species were clustered: P. mituense (Pm, LD₅₀ = 1.46 µg/mg), P. sancti-felicis (Psf, LD₅₀ = 2.14 µg/mg), P. reticulatum (Pr, LD₅₀ = 6.19 µg/mg), P. dumosum (Pd, LD₅₀ = 8.89 µg/mg), and P. soledadense (Ps, LD₅₀ = 10.06 µg/mg). The apiol loading vector was co-directional with the LD₅₀ vector and closely aligned with Pm and Psf, the two most potent species, confirming that apiol is the primary driver of ixodicidal activity, in agreement with the high potency of pure apiol (LD₅₀ = 1.97 µg/mg, Table 3). Notably, Pd was projected in the upper quadrant alongside the dillapiol, bicyclogermacrene, and β-pinene vectors, suggesting that its ixodicidal activity (100% mortality) may be attributable to dillapiol (8.9%) and myristicin (3.7%), both MDP-bearing compounds, rather than to its sesquiterpene background. This contrasted with the antifeedant PCA (Figure 1), where Pd failed to achieve broad-spectrum activity, indicating that the MDP-phenylpropanoid threshold for ixodicidal effects may be lower than that required for antifeedant efficacy.

In contrast, the inactive species (LD₅₀ > 40 µg/mg) were projected in the opposite region of the biplot. P. anonifolium (Pa) was isolated in the far right, driven by α-humulene, β-caryophyllene, caryophyllane alcohol isomer II, and neoalloocimene loadings, while P. coruscans (Pc) and P. tuberculatum (Pt) clustered in the lower-center region along the β-bisabolene and δ-cadinene vectors. Their position opposite to the LD₅₀ vector was consistent with the absence of ixodicidal activity for pure β-caryophyllene, limonene, α-pinene, β-pinene, and copaene (all with LD₅₀ > 20 µg/mg, Table 3). P. casapiense (Pcs) was again isolated in the lower-left quadrant by its exclusive oxygenated caryophyllane derivatives (caryophyllenone, β-caryophyllenol, caryophyllene oxide); despite its moderate mortality (53 ± 7%), it did not achieve a calculable LD₅₀, indicating that the oxygenated caryophyllane chemotype that conferred potent selective antifeedant activity against R. padi (Section 2.3.1) did not translate into equivalent ixodicidal effects. The only active non-apiol compound was β-caryophyllene oxide (LD₅₀ = 4.46 µg/mg) and myristicin (LD₅₀ = 2.89 µg/mg), whose individual contributions may partially explain the ixodicidal activity of Ps (caryophyllene oxide 13.3%) and Pr (apiol 15.0% combined with a mixed sesquiterpene profile).

2.3.2. Docking

Apiol demonstrated binding affinities comparable to or better than those of reference ligands across several targets (Figure 3, Supplementary Table S3). In AChE enzymes, improved binding was observed in S. littoralis and Ixodes scapularis, whereas slightly reduced affinity was found in M. persicae and M. javanica. In CYP450 enzymes, apiol showed improved binding in Drosophila melanogaster relative to α-pinene but slightly reduced affinity in H. asiaticum compared to terpinolene.

Apiol maintained interactions with catalytic residues in AChE. The interactions with the Ser and His of the catalytic triad were consistent across the generated poses. Also, the same interactions of acetylcholine with non-catalytic residues were detected in the case of apiol, indicating proper positioning within the active site (Supplementary Table S1). Additional interactions not observed in control ligands were identified, suggesting alternative stabilization mechanisms within the binding pocket. In the case of D. melanogaster’s CYP450, only the interaction with the catalytic Ile was detected, nonetheless, other non-catalytic interactions were shared between the reference ligand and apiol, suggesting the compatibility of the molecule with the binding position.

3. Discussion

The present study demonstrates that apiol-rich essential oils from Amazonian Piper species exhibit strong and consistent biocidal activity across both insect pests and ticks, supporting their potential as dual-purpose agents for integrated pest management.

The broad-spectrum antifeedant activity observed for P. mituense and P. sancti-felicis, together with their high ixodicidal efficacy and the strong correlation between apiol content and biological activity revealed by PCA highlights the central role of apiol as a key determinant of bioactivity. This finding is consistent with the ecological function of phenylpropanoids in plant defense, where these metabolites contribute to herbivore deterrence and protection against biotic stress [25,26].

A notable exception to the apiol-driven pattern was P. casapiense, which exhibited the strongest selective antifeedant activity against R. padi despite lacking high apiol content. This species, characterized by a chemotype rich in oxygenated caryophyllene derivatives, particularly β-caryophyllenol, caryophyllenone, and related highly oxygenated caryophyllane-type compounds, was clearly separated in the PCA and positioned opposite to the R. padi EC₅₀ vector. This indicates that its activity arises from a distinct chemical mechanism compared to apiol-rich oils. Oxygenated caryophyllane sesquiterpenes, particularly caryophyllene oxide, have been widely reported to exhibit multi-target bioactivity against insects, including larvicidal effects, oviposition deterrence, developmental disruption, and strong repellency, often exceeding the activity of the non-oxygenated parent compound β-caryophyllene [32,33,34,35]. A similar pattern was observed for P. obliquum, characterized by bicyclogermacrene, oxygenated caryophyllene derivatives, and other caryophyllene-related sesquiterpenes. Bicyclogermacrene exhibited potent larvicidal activity against mosquito larvae, including Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus [36]. Sesquiterpene-rich essential oils have been shown to interfere with insect feeding behavior and sensory perception, particularly in aphids, which rely heavily on contact chemoreception [17,37]. Therefore, in addition to apiol-mediated broad-spectrum activity, alternative chemotypes based on oxygenated caryophyllenes and related sesquiterpenes can confer potent but selective antifeedant effects.

The observed differences between antifeedant and ixodicidal responses further suggest that distinct biological targets and thresholds may be involved. While high apiol content was required to achieve broad-spectrum antifeedant activity, ixodicidal effects were observed at lower concentrations, as evidenced by the activity of P. dumosum, which contains moderate amounts of MDP-bearing compounds such as dillapiol and myristicin.

At the molecular level, docking results provide mechanistic support for the experimental observations. Apiol exhibited similar or better binding affinities than endogenous ligands in acetylcholinesterase (AChE), suggesting that it may act as a competitive inhibitor. Since AChE plays a central role in neurotransmission by hydrolyzing acetylcholine, its inhibition leads to accumulation of the neurotransmitter and disruption of neural signaling, ultimately causing paralysis and death in insects [38,39]). The preservation of interactions with catalytic and non-catalytic residues supports the hypothesis that apiol binds within the active site and may interfere with enzymatic function.

In addition to AChE inhibition, apiol also showed relevant interactions with cytochrome P450 enzymes. Inhibition of cytochrome P450 monooxygenases is a mechanism well documented for methylenedioxyphenyl (MDP) phenylpropanoids such as apiol [40]. The variability observed across CYP450 targets reflects the structural diversity and functional adaptability of these enzymes, which are known to metabolize a wide range of xenobiotics [41]. Enhanced CYP450 activity is a major mechanism of insecticide resistance, allowing insects to degrade toxic compounds efficiently [42,43]. This structural motif is also present in piperonyl butoxide, a well-known synergist that inhibits cytochrome P450-mediated detoxification pathways in insects [21,44].

The dual interaction of apiol with both AChE and CYP450 enzymes is particularly relevant from an applied perspective. Multi-target compounds are less prone to resistance development because they simultaneously affect multiple physiological pathways [11,12]. In this context, apiol and apiol-rich essential oils may offer an advantage over single-target synthetic insecticides, aligning with current strategies aimed at reducing resistance pressure and improving sustainability in pest management.

The integration of chemical composition, bioactivity, and molecular docking data allows the identification of clear structure–activity relationships (SAR) within the essential oils of Piper species. Two major chemotypes emerge as biologically relevant: (i) apiol-rich phenylpropanoid systems, associated with broad-spectrum activity, and (ii) sesquiterpene-rich systems, particularly those dominated by oxygenated caryophyllenes, associated with selective antifeedant effects. Taken together, these findings support a dual SAR framework in Piper essential oils: (i) MDP-phenylpropanoids (apiol, dillapiol-like systems) confer broad-spectrum activity through combined AChE inhibition and CYP450 modulation; (ii) oxygenated sesquiterpenes (caryophyllene derivatives) confer selective antifeedant activity through behavioral and sensory interference mechanisms. This duality provides a mechanistic explanation for the diversity of biological responses observed and highlights the potential of combining different chemotypes to achieve both broad-spectrum and target-specific pest control strategies.

Overall, the combination of chemical, biological, and molecular docking data provides a comprehensive understanding of the activity of apiol-rich essential oils. The consistency between PCA, bioassays, and in silico results supports a unified mechanism in which apiol acts as a central bioactive compound, modulated by synergistic interactions and capable of targeting multiple physiological systems. The integration of chemical, biological, and computational data provides a comprehensive understanding of the activity of apiol-rich essential oils. Consistent results from PCA, bioassays, and molecular docking indicate that apiol is the main active compound, with its effects enhanced by synergistic interactions and its ability to target multiple physiological systems.

4. Materials and Methods

4.1. Plant Material and Essential Oil Extraction

The aerial parts (leaves, stems, and flowers) of ten Piper species were collected in three districts of the Loreto Department, Peruvian Amazon (Figure X): Mazán District (P. coruscans, voucher 039849; P. casapiense, 041044; P. obliquum, 027690; P. anonifolium, 042381; P. tuberculatum, 020115), Punchana District (P. sancti,,felicis, 036367), and San Juan Bautista District (P. dumosum, 040311; P. reticulatum, 042127; P. soledadense, 033308; P. mituense, 041473). Voucher specimens were deposited at the Herbarium AMAZ, Universidad Nacional de la Amazonia Peruana (UNAP), Iquitos, Peru. All plant material was collected under Regional Management Resolution No. 035, 2021, GRL, GGR, GRDFFS. Essential oils were obtained by hydrodistillation of the dried aerial parts, separated by decantation, and dried over anhydrous Na₂SO₄, yielding between 0.078% and 1.26% (dry weight basis). Collection coordinates, dry weights, and detailed extraction procedures have been previously reported [24].

4.2. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

The chemical composition of the essential oils was determined by GC,,MS using a Shimadzu GCMS, QP2010, Ultra system equipped with a Teknokroma TRB, 5 column (30 m × 0.25 mm ID, 0.25 µm film thickness) under the conditions previously described [24]. Compound identification was based on comparison of mass spectra with the Wiley 275 Mass Spectral Database and retention indices from the literature. Quantification was performed using relative peak area percentages. The complete chemical profiles of the ten essential oils, including three species described for the first time (P. soledadense, P. casapiense, and P. mituense), are detailed in [24] and summarized in Table 1 of the present work.

4.3. Antifeedant Bioassays

The colonies of S. littoralis, M. persicae, and R. padi were maintained at ICA, CSIC (Madrid, Spain) on artificial diet, bell pepper (Capsicum annuum), and barley (Hordeum vulgare), respectively (22 ± 1 °C, >70% RH, 16:8 h L:D). Antifeedant activity was evaluated using choice tests as previously described [45,46]. Feeding inhibition (%FI) against S. littoralis sixth, instar larvae (6 replicates, 2 larvae each) was calculated as %FI = [1 − (T/C)] × 100, where T and C are the consumption of treated and control leaf disks. Settling inhibition (%SI) against apterous adults of M. persicae and R. padi (22 replicates, 10 aphids each) was calculated as %SI = [1 − (%T/%C)] × 100, where %T and %C are the percentages of aphids on treated and control surfaces. The essential oils were tested at an initial concentration of 100 µg/cm². The pure compound apiol (Sigma, Aldrich, St. Louis, MO, USA) was tested under identical conditions.

4.4. Nematicidal Bioassays

The population of M. javanica was maintained at ICA, CSIC (Madrid, Spain) [47]. Egg masses of M. javanica were manually collected from infected tomato roots. Second-stage juveniles (J2) were obtained from hatched eggs by incubating handpicked egg masses in a water suspension at 25ºC for 24 h. The oils tested were dissolved in distilled water containing 5% of a DMSO-Tween solution (0.5% Tween 20 in DMSO) and evaluated at initial concentration of 1 μg/μL [47]. The control solution was distilled water/DMSO/ Tween 20. Each treatment was performed in quadruplicate. The plates were sealed and incubated in a growth chamber under the same conditions used for egg mass incubation. Dead J2s were counted at 72 h using a binocular microscope and mortality rate (M%) was calculated and corrected with Schneider-Orelli’s formula [13].

4.5. Ixodicidal Bioassays

The engorged females of H. lusitanicum were collected from red deer in Ciudad Real (Central Spain) and maintained at 22–24 °C, 80% RH until larval hatching. Larvicidal bioassays were performed using the impregnated substrate method [46,48]. Test solutions (50 µL) were applied to 25 mg of powdered cellulose at initial concentrations of 20 µg/mg (essential oils) and 10 µg/mg (apiol). Three replicates of 20 larvae (>6 weeks old) were used per treatment. Negative (cellulose) and positive (thymol, 20 µg/mg) controls were included. Mortality was assessed after 24 h and corrected by the Schneider–Orelli formula [13].

4.6. Statistical Analysis

Antifeedant effects (%FI and %SI) were analyzed for significance using the non-parametric Wilcoxon signed rank test. Effective concentrations (EC₅₀) were determined by linear regression analysis (%FI or %SI vs. log dose) for essential oils exhibiting inhibition values ≥75% (Table 2). LD₅₀ and LD₉₀ values for the ixodicidal bioassays were calculated by Probit analysis using 1:2 serial dilutions covering a range between 100% and <50% mortality, with a minimum of three doses (Table 3). For P. mituense, the Probit regression yielded a negative LD₅₀ estimate; a surrogate value (LOD/2) was assigned following established substitution methods for left ensored data [49,50]. EC₅₀ calculations and Probit analyses were performed using STATGRAPHICS Centurion XVI (version 16.1.02).

Principal component analyses (PCA) were performed to explore the relationships between essential oil composition and biological activity. The antifeedant PCA included the 18 identified compounds and three EC₅₀ variables as active variables. The ixodicidal PCA used the 18 identified compounds as active variables and the corrected mortality against H. lusitanicum as a supplementary variable. Statistical analyses and PCA visualizations were performed using Python (version 3.11.6). All computations were executed using the Julius computational platform (https://julius.ai/), which ensured reproducibility and standardization of the workflow.

4.7. Molecular Docking

The protein sequences of acetylcholinesterase were obtained from the UniProt database. The entries chosen were B7QEQ1 (I. scapularis) Q8T7U9 (M. persicae), A0A915MGH7 (M. javanica) and A0A9P0N5I4 (S. littoralis) ( https://doi.org/10.1093/nar/gkae1010) . The protein CYP450 3A8 of H. asiaticum was obtained from Genbank (https://doi.org/10.1093/nar/gks1195) (entry: PQ058615.1) and the protein CYP450 6A2 of D. melanogaster was obtained from UniProt (entry: P33270). All the 3D models were generated using the SWISS-MODEL tool (https://doi.org/10.1093/nar/gky427).

Ligands were prepared using the OPLS4 force field, which provides enhanced accuracy in modeling molecular interactions and conformational energetics (Schrödinger, 2026). Ionization states were assigned using Epik, and stereochemistry was preserved. Protein structures were prepared at physiological pH (7.4). Hydrogen atoms were added and protonation states optimized to ensure proper representation of electrostatic interactions.

Docking grids were centered on active-site residues with a grid size of 20 Å. Fifty poses were generated for each ligand–protein pair and ranked based on docking score. Docking predicts the preferred binding orientation and affinity of ligands to protein targets through scoring functions (Sharmila et al., 2025). The best (lowest) docking score per system was selected. Interaction patterns were analyzed across all generated poses to identify conserved residues and unique contacts. Interaction types were assigned based on residue chemical properties and ligand orientation, considering standard criteria (hydrogen bonding, π–π stacking, hydrophobic and polar interactions) derived from docking poses.

4.7. Use of Generative Artificial Intelligence

In preparing this manuscript, the authors utilized AI, assisted tools (Claude Opus 4.6, Anthropic) to enhance the grammatical structure and linguistic clarity of the English writing, and Python, based PCA visualizations were generated with the assistance of Julius AI. All scientific content, experimental design, data collection, analysis, and interpretation represent the original intellectual contribution of the research team. No AI, assisted tools were used for data generation, scientific reasoning, or interpretation of results. The authors reviewed and take full responsibility for the accuracy and integrity of all content presented in this publication.

5. Conclusions

This study demonstrated, for the first time, that apiol-rich essential oils from Amazonian Piper species possess dual biopesticidal activity: broad-spectrum antifeedant effects against S. littoralis, M. persicae, and R. padi (EC₅₀ 0.38–5.35 µg/cm²), and potent ixodicidal activity against H. lusitanicum larvae (LD₅₀ 0.046–0.267 µg/mg cellulose). Among the ten species evaluated, P. mituense and P. sancti-felicis—both dominated by the phenylpropanoid apiol—were the only essential oils that exceeded the 75% inhibition threshold against all three pest species while simultaneously achieving 100% tick larval mortality. The consistently superior potency of P. mituense over both P. sancti-felicis and pure apiol across all bioassays provides evidence for synergistic contributions from minor constituents, particularly myristicin, bicyclogermacrene, and germacrene D. Principal component analysis confirmed that the chemical class of the dominant constituent determines biocidal selectivity: apiol-rich chemotypes exhibited the strongest broad-spectrum activity, while sesquiterpene-dominated oils conferred taxon-selective effects of lower magnitude. The absence of nematicidal activity against M. javanica defines a useful selectivity window for IPM applications. Apiol exhibits favorable binding interactions with acetylcholinesterase and cytochrome P450 enzymes across multiple arthropod species. Its interaction with catalytic residues and its ability to form additional stabilizing contacts support its potential as a bioactive compound.

These findings fill a significant knowledge gap for apiol, previously unexplored as an antifeedant or ixodicidal agent, and extend the biocidal profile of these Amazonian Piper chemotypes beyond their previously reported antifungal and herbicidal activities, positioning apiol-rich essential oils as promising dual-purpose candidates for integrated management of crop pests and arthropod vectors of public health concern.