Altitude-Mediated Chemotypic Shifts in Piper aduncum Drive Differential Nano-Bioinsecticidal Activity Against Crocidolomia pavonana Fabricius (Lepidoptera: Crambidae)

Crops from the Brassicaceae family, including cabbage (Brassica oleracea var. capitata), mustard greens (Brassica juncea), and kale (Brassica oleracea var. alboglabra), are a major source of dietary micronutrients and are an important economic commodity for smallholder farmers in tropical and subtropical regions. In Indonesia alone, the cumulative annual production area of Brassicaceae vegetables exceeds 290,000 ha, yet yields remain chronically depressed below genetic potential due to arthropod pest pressure [1]. Among the lepidopteran complex colonizing these crops, Crocidolomia pavonana Fabricius (Lepidoptera: Crambidae), commonly known as the cabbage looper, is recognized as the most economically damaging leaf-destroying pest species throughout the Indo-Pacific region. Early instar larvae congregate in dense clusters and feed on leaf blades in concentric feeding rings, rapidly stripping the crown and, in later instars, hollowing out the developing seeds. Field studies conducted in West Sumatra and East Java have documented crop losses ranging from 30% at moderate infestation densities to total crop failure (>90%) during severe outbreaks, posing a continuing and increasing threat to food security [2]. Conventional management of C. pavonana has historically been dominated by synthetic insecticides from the organophosphate, pyrethroid, and diamide chemical classes, applied at intervals as short as two to three days during peak population pressure [3]. However, this calendar-based spraying strategy has resulted in a series of unintended agronomic and ecological consequences. Repeated sublethal exposure has driven selection for metabolic and target-site resistance mechanisms in field populations, including increased expression of cytochrome P450 monooxygenases (CYPs) and mutations in voltage-gated sodium channel genes, which progressively erode the effectiveness of the most widely used active ingredients [4]. Simultaneous disruption of natural enemy groups, particularly Hymenoptera parasitoids in the genera Cotesia and Diadegma, disrupts key biological regulation, creating favorable conditions for secondary pest outbreaks [5]. Environmental contamination of soil and water matrices through pesticide runoff further exacerbates the sustainability deficits of current management approaches [6].

These cascading consequences have increased global demand for biodegradable, plant-derived pest management alternatives that are compatible with integrated pest management (IPM) principles and meet increasingly stringent maximum residue limit (MRL) standards for export-oriented agricultural commodities. Botanical insecticides derived from crude extracts and plant extracts have attracted renewed scientific and commercial interest as low-risk alternatives in IPM tools [7]. Among the studied species, Piper aduncum L. (Piperaceae) has emerged as one of the most promising bioinsecticide taxa across its distribution range from the neotropics to the paleotropics. Its crude extract is characterized by a series of phenylpropanoid compounds, primarily apiole, dillapiol, and myristicin, that act synergistically on multiple physiological targets in insects. Apiole and dillapiol have been documented to inhibit acetylcholinesterase (AChE) activity and suppress cytochrome P450-dependent monooxygenases and carboxyl esterases, thereby disrupting neurotransmission and xenobiotic detoxification capacity [8]. Myristicin has demonstrated additional mechanisms of action, including disruption of oxidative phosphorylation and genotoxic effects on hemocytes at sublethal concentrations [9]. This multi-target bioactivity profile substantially reduces the likelihood of rapid resistance evolution compared to single-target synthetic insecticides.

Piper aduncum is native to tropical America but was introduced to the Indonesian archipelago through the Bogor Botanical Gardens in 1860 and has since adapted widely across various biogeographic zones, thriving at altitudes up to 2,000 metres above sea level (m a.s.l.) in ecosystems characterized by annual rainfall of 1,500–4,000 mm and temperatures of 22–30 ℃ [10, 11]. Despite this species' broad adaptation, striking phytochemical variability among populations has been documented. Reported apiole content in fruit crude extract ranges from 28.62% in populations sampled from the Brazilian Amazon to as high as 90.7% in specimens from Manaus, Brazil [12]. Similarly, dillapiol concentrations range from below the limit of quantification (<1%) in plants from the Federal District of Brazil to 73.40% in Atlantic Forest populations [13]. Population-level investigations have linked this intraspecific chemotypic differentiation to the interactive effects of genetic background, geographic origin, and prevailing abiotic conditions, but the relative contribution of each factor, especially altitude, remains poorly understood in the Indonesian context.

Altitude represents one of the strongest and most ecologically coherent environmental gradients known to modulate secondary metabolite biosynthesis in plants. With increasing altitude, plants experience a decrease in average temperature, an increase in UV-B radiation flux, a decrease in CO₂ partial pressure, and a shift in soil nutrient stoichiometry that collectively activate various stress response signaling cascades, thereby increasing the accumulation of phenolic acids, flavonoids, and terpenoids. The phenylpropanoid pathway, which underlies apiole and dillapiol biosynthesis, is limited by the activity of phenylalanine ammonia-lyase (PAL). PAL is transcriptionally regulated at low temperatures through a cold-responsive promoter element (COR), providing a mechanistic link between altitude-associated temperature decreases and increased phenylpropanoid accumulation [14]. Downstream, the enzyme O-methyltransferase (OMT) catalyzes the final methylation step that distinguishes apiole from dillapiol, and its differential expression under varying environmental signals may further explain the observed chemotypic differences along the altitudinal gradient. Supporting evidence from related Piper taxa is particularly informative; highland accessions of Piper nigrum L. at 1,134 m a.s.l. exhibited significantly higher monoterpene concentrations than lowland counterparts at 20 m a.s.l., and hypericin content in Hypericum perforatum L. increased 4.5-fold when plants were cultivated at 15 ℃ compared to 22 ℃ [15]. These findings collectively indicate that altitude-based sampling strategies are an important, yet underexplored, experimental variable in optimizing the bioinsecticidal potential of plant-derived pest control materials.

A persistent obstacle hindering the wider adoption of botanical insecticides in the field is their inherent physicochemical instability; crude extracts are susceptible to photodegradation, oxidation, and rapid evaporation, leading to unpredictable residual activity under environmental conditions [16]. Nanotechnology-based formulation platforms have substantially overcome these limitations. Nanoemulsions, thermodynamically or kinetically stable oil-in-water dispersions with average droplet diameters in the range of 10–200 nm, offer several important advantages over conventional emulsion concentrates: better penetration through the hydrophobic insect cuticle, increased solubility of lipophilic phytochemicals, protection against photodegradation, and improved resistance to rain [17]. The increased surface area-to-volume ratio at the nanoscale also accelerates the release of active ingredients at the target site, improving contact and fumigant toxicity. Importantly, nano-encapsulated P. aduncum crude extract has been shown to exhibit significantly greater acute toxicity to Spodoptera frugiperda larvae than standard emulsion formulations, providing direct proof of concept for nanoemulsion technology as a platform for this botanical active ingredient [18, 19]. Physical characteristics parameters, including droplet size, polydispersity index (PDI), zeta potential, and encapsulation efficiency, have emerged as key quality benchmarks that must be regularly optimized across source populations to ensure a consistent, field-ready product.

Despite growing evidence documenting the phytochemical variability of P. aduncum and the influence of altitude on secondary metabolite biosynthesis in plants, no study has systematically examined how the altitude gradient in West Sumatra a region extending from lowland river valleys (~100 m a.s.l.) to mountainous volcanic zones (~1,500 m a.s.l.) within a single biogeographic unit shapes the chemotypic composition and, indirectly, the bioinsecticidal activity of P. aduncum nanoemulsions at different altitudes against key pests in the region. This knowledge gap is crucial because any altitude-dependent chemotypic variation that results in significant differences in pesticide performance could inform targeted collection strategies to increase botanical biopesticide production in West Sumatra, where P. aduncum has been classified as an invasive species and represents an easily harvested and low-cost biomass resource. The design does not allow separation of altitude from site-specific edaphic and climatic factors; the findings should be interpreted as provenance-dependent variation rather than altitude-controlled effects. Therefore, this study aims to examine and investigate the effect of altitude variation on the phytochemical composition of Piper aduncum fruit crude extract and the bioinsecticide performance of its nanoemulsion against second-instar larvae of Crocidolomia pavonana, including its insecticidal, antifeedant, and sublethal effects, as well as the relationship between chemotype variation and biological efficacy.

3.1 Phytochemical composition of Piper aduncum fruit extracts by Gas Chromatography–Mass Spectrometry

The GC–MS analysis of Piper aduncum fruit extracts from three altitudinal populations in West Sumatra, Indonesia, identified a total of 38 secondary metabolites, and the active compounds were not standardized (Table 1 and Figure 2). Apiole (retention time: 25.786 min) was the dominant bioactive constituent in all accessions, with relative abundances of 53.50%, 49.32%, and 45.18% in the highland (1,071 m a.s.l.), midland (469 m a.s.l.), and lowland (5.72 m a.s.l.) populations, respectively.

Table 1. Relative abundance of selected secondary metabolites identified by Gas Chromatography–Mass Spectrometry (GC–MS) in Piper aduncum fruit extracts from three altitudinal zones in West Sumatra, Indonesia

Note: asterisk (*) denotes the dominant bioactive compound; (—) = not detected; RT = retention time; m a.s.l. = metres above sea level.

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This positive monotonic relationship between altitude and apiole content indicates a strong chemotypic gradient, reflecting a 18.4% increase in apiole abundance from the lowest to the highest altitude. Two additional phenylpropanoids were detected at lower abundances: myristicin (0.97–3.60%) and β-caryophyllene (1.49–1.87%). Interestingly, myristicin showed the opposite altitudinal trend, reaching its highest relative abundance in lowland samples (3.60%), while β-caryophyllene did not show a consistent gradient across populations. Minor compounds included pentadecane (2.82–3.40%), trimethylsilyl (TMS) silane derivatives (10.69–13.27% only in highland and midland populations), 9-cyanophenanthrene (1.82–2.04%), and sterols consisting of γ-sitosterol, stigmasterol, and campesterol (2.11–2.42%). Phenolic derivatives were significantly more abundant in lowland extracts (6.02%) than in highland (0.28%) and midland (3.26%) populations. This study provides the first systematic evidence linking altitudinal gradients to chemotype differentiation and nano-bioinsecticide efficacy on P. aduncum from West Sumatra, Indonesia. Highland accessions (1,071 m a.s.l.) accumulated significantly higher apiole concentrations (53.50%) compared to lowland populations (45.18%), resulting in nanoemulsions that were approximately 37% more potent in terms of LC₅₀. This finding is consistent with the established ecological principle that abiotic stress at high altitudes promotes increased biosynthesis of plant secondary metabolites [22]. Similar altitudinal chemotype gradients have been documented in Piper nigrum from West Sumatra, where highland accessions (1,134 m a.s.l.) showed significantly increased alkaloid and flavonoid accumulation relative to lowland populations, and in Hypericum perforatum, where hypericin content increased 4.5-fold under lower temperature conditions. The observed chemotypic differences cannot be attributed solely to altitude; temperature, UV-B radiation, and precipitation are co-varying factors that likely contribute to the observed patterns. Future studies with controlled environmental conditions are required to disentangle these variables.

The mechanistic basis for altitude-driven apiole enrichment involves convergent regulatory effects on phenylpropanoid metabolism (Figure 3). At higher altitudes, decreasing ambient temperature increases the catalytic activity of phenylalanine ammonia lyase (PAL), the key gateway enzyme of the phenylpropanoid pathway, and promotes downstream methylation reactions mediated by OMT [23], which converts early pathway intermediates to apioles through sequential aromatic ring modifications [24, 25]. Concurrently, increased UV-B radiation at higher altitudes stimulates the formation of reactive oxygen species (ROS), which activate antioxidant enzyme cascades and induce phenolic biosynthesis as a UV-filtering strategy and a general stress response [26]. The inverse altitudinal gradient of myristicin, highest in lowland populations (3.60%) and lowest in highland populations (1.07%), likely reflects competitive metabolic flux at a shared enzymatic branch point in the methylenedioxyphenylpropanoid biosynthesis network, where low-temperature OMT activation favors the intermediate pathway to apiole over myristicin. The complete absence of dillapiol in the three West Sumatran accessions confirms that these populations belong to an apiole-dominant chemotype, distinct from the dillapiol-dominant and mixed chemotypes prevalent in Brazilian and some Southeast Asian populations [27]. The regional consistency of this chemotype suggests that OMT enzyme specificity is governed by the genetic background of P. aduncum West Sumatra and likely reinforced by local environmental selection pressures that reliably direct biosynthetic flux toward apiol, despite quantitative variation induced by altitude. From a biopesticide development perspective, this chemotype predictability provides a stable biochemical basis on which altitude-based origin selection strategies can be reliably implemented.

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Figure 3. Chemical structure of apiole (chemical formula = C₁₂H₁₄O₄; molar mass = 222.23 g mol^-1, and density = 1.15 g mL^-1) in Piper aduncum fruit extracts from three altitudinal zones in West Sumatra, Indonesia

3.2 Physicochemical characterization of nanoemulsions

Physicochemical characterization confirmed successful nanoemulsion formation for all three P. aduncum accessions (Table 2). Average droplet diameters ranged from 98.4 to 112.3 nm, consistent with operationally defined nanoemulsion size criteria (<200 nm). PDI values were below 0.25 for all formulations (0.18–0.24), indicating narrow size distribution and monodispersity. Zeta potential values ranged from -31.4 to -35.2 mV, universally exceeding the -30 mV threshold, indicating adequate electrostatic repulsion. All formulations exhibited near-neutral pH (6.8–7.0), reflecting compatibility with the surface chemistry of cruciferous leaf waxes. Nanoemulsions derived from the highland region exhibited the smallest average droplet diameter (98.4 ± 3.2 nm) and the most negative zeta potential (-35.2 ± 1.4 mV), suggesting that the high phenylpropanoid content in the highland extract, particularly apiole, modulates the interfacial layer properties during high-energy emulsification. The amphiphilic character of apiole, caused by its methylenedioxy and methoxy functionalities, likely enhances surfactant packing at the oil-water interface, thereby reducing droplet size and increasing surface charge density. These physicochemical differences, although modest, have practical implications for formulation stability, bioavailability, and cuticle penetration efficiency in field applications.

Table 2. Physicochemical properties of Piper aduncum nanoemulsions prepared from plant materials collected at three altitudinal zones in West Sumatra, Indonesia

Note: PDI = Polydispersity index; m a.s.l. = metres above sea level, and values represent means ± standard deviation (SD) (n = 3 samples).

The superior insecticidal potency of highland nanoemulsions against C. pavonana larvae is mechanistically consistent with the co-existing apiole and phenylpropanoid mechanisms of action. Apiole functions primarily as an inhibitor of CYP-mediated detoxification enzymes, thereby impairing the metabolic clearance of co-existing minor bioactives and increasing the net toxicity of extract-based nanoemulsion, an effect analogous to the synergistic interaction documented between dillapiol and pyrethroids in other Piper-derived formulations. The minor component myristicin exerts complementary neurotoxic effects through inhibition of AChE and carboxylesterase (CarE), inducing neuronal hyperexcitation and motor dysfunction, while β-caryophyllene contributes to contact toxicity and insect cannabinoid-like receptor agonism [9].

The steeper probit regression slope for the highland formulation (b = 7.42 ± 1.69) compared to the lowland formulation (b = 5.52 ± 1.59) indicates a narrower effective concentration range and a more uniform physiological response at the population level, a pattern characteristic of formulations where a single dominant mechanism of action governs mortality, consistent with the higher apiole content focusing toxicological activity on the CYP inhibitor target. The current LC₅₀ value of 0.18% for the highland nanoemulsion compares favorably with the 0.24% reported for an extract-based nanoemulsion of P. aduncum against Plutella xylostella and with recent findings on nanoemulsion-enhanced biopesticides. The advantages of nanoemulsion formulations that work through increased droplet surface area, enhanced cuticular dissolution, and enhanced transcutaneous bioavailability substantially reduce the minimum effective dose relative to contact-applied bulk or powder emulsions, directly resulting in lower field application rates and reduced environmental burden.

3.3 Insecticidal activity and probit analysis against Crocidolomia pavonana larvae

The C. pavonana larval mortality increased proportionally with nanoemulsion concentration and was significantly affected by P. aduncum altitude (Table 3). Probit analysis confirmed statistically significant and altitude-dependent differences in insecticidal potency, as evidenced by non-overlapping 95% confidence intervals for LC₅₀ values. The highland nanoemulsion showed the lowest LC₅₀ of 0.18% (95% CI: 0.12–0.21), reflecting the highest insecticidal activity. In comparison, the midland and lowland formulations showed 44% and 58% lower potency, with LC₅₀ values of 0.26% (95% CI: 0.23–0.31) and 0.29% (95% CI: 0.25–0.36), respectively. LC₉₅ values followed the same altitude ranking: 0.58% (highland), 0.73% (midland), and 0.82% (lowland). The probit regression slope (b) also decreased with altitude: 7.42 ± 1.69 (highland), 6.38 ± 1.61 (midland), and 5.52 ± 1.59 (lowland), indicating that the dose-response curve was steepest for the most bioactive formulation, a pattern consistent with greater homogeneity of pharmacological targets at the higher apiole concentrations found in the highland extract. At a supra-LC₅₀ concentration of 0.35%, 96-hour cumulative mortality reached 80.0%, 62.7%, and 56.0% for the highland, midland, and lowland nanoemulsions, respectively. Further Pearson correlation analysis revealed a strong inverse relationship between apiole content and LC₅₀ (r = −0.94, p < 0.05), which confirms that phenylpropanoid enrichment at higher altitudes is conducive to insecticidal potential.

Table 3. Probit analysis parameters and lethal concentration (LC) values for Piper aduncum nanoemulsions from three altitudinal populations against second-instar Crocidolomia pavonana larvae

Note: CI = confidence interval; Apiole (%) = relative abundance by GC–MS; b = regression slope; SE = standard error; m a.s.l. = metres above sea level.

The concentration-dependent extension of larval development duration (up to +2.30 days at 0.35%; p < 0.001) and reduction in pupal biomass (up to 48.9%) across all populations are consistent with the established sublethal mechanism of phenylpropanoid action on insect endocrinology and intermediary metabolism. At sublethal doses, phenylpropanoids disrupt the hormonal cascade that regulates ecdysis and larval metamorphosis by suppressing ecdysone and juvenile hormone titers and by disrupting the activity of chitin synthase, an enzyme responsible for depositing the cuticular matrix during each larval molt [28]. This hormonal disruption prolongs the inter-molt interval and creates a developmental delay disproportionate to the concentration-mediated reduction in feeding.

The energetic cost of xenobiotic detoxification is a secondary mechanism for pupal biomass reduction. Larvae exposed to sublethal concentrations of phenylpropanoids divert metabolic resources from somatic growth toward Phase I and Phase II detoxification pathways, depleting lipid and protein reserves that would otherwise have been accumulated during the final larval instar for adult development [29, 30]. The convergence of reduced nutrient intake mediated by antifeedant activity with increased metabolic expenditure creates a dual biomass depletion mechanism culminating in energetically compromised pupae. Importantly, both larval development duration and pupal biomass were concentration-dependent but not altitude-dependent (p > 0.05), suggesting that this sublethal endpoint operates through a mechanism activated by a minimum threshold concentration attainable across all altitude populations, unlike the graded lethal response driven by apiole quantity.

The ecological implications of these sublethal effects extend beyond their direct impact on individual larvae. Smaller, energetically compromised pupae are associated with reduced adult emergence rates, reduced lifespan to adulthood, reduced fecundity, and altered sex ratios in lepidopteran populations [31]. These fitness penalties propagate across generations, reinforcing the pest management benefits of sublethal nanoemulsion exposure beyond the direct mortality measured in laboratory bioassays. From an IPM perspective, the combination of direct larval toxicity, developmental disruption, and antifeedant activity represents a multimodal suppression profile that reduces the likelihood of resistance development through any single mechanism.

3.4 Effects on Crocidolomia pavonana larval development duration

Exposure to P. aduncum nanoemulsion at all tested concentrations significantly prolonged the larval development period (2nd–4th instar) of C. pavonana compared to the untreated control (F₅,₂₄ = 47.3–52.1, p < 0.001). The control group showed an average larval duration of 4.02–4.05 days. At the highest sublethal concentration evaluated (0.35%), larval development was extended to 6.22 ± 0.44 days (highland), 6.17 ± 0.52 days (midland), and 6.35 ± 0.48 days (lowland), indicating an increase of 2.20, 2.12, and 2.30 days, respectively, compared to the control. The 0.29% and 0.35% concentrations were not significantly different from each other within each population group (same letter designations in Table 4), indicating an asymptotic threshold effect of bioactive compound exposure on larval developmental delay. Importantly, P. aduncum altitude had no statistically significant effect on larval developmental duration (p > 0.05), despite having a substantial effect on insecticidal potency. This separation suggests that developmental disruption operates through a concentration-threshold pathway activated even by the lower apiole content of the lowland formulation, in contrast to the lethal response that is graded by altitude [32]. This mechanistic separation between sublethal growth disruption and acute toxicity has important implications for understanding the pharmacodynamics of phenylpropanoids delivered via nanoemulsion [33].

Table 4. Duration of larval development of Crocidolomia pavonana following exposure to Piper aduncum nanoemulsions from three altitudinal populations

Note: Different letters within each column indicate significant differences (Least Significant Difference (LSD) test, p < 0.05); () = live larvae, m a.s.l. = metres above sea level, and values represent means ± standard deviation (SD).

3.5 Effects on Crocidolomia pavonana pupal biomass

Pupal weight generally declined at the higher nanoemulsion concentrations, although the response was not strictly monotonic at the lower and intermediate concentrations (Table 5). Control pupae had an average weight of 0.045 ± 0.002 g. At the maximum concentration tested (0.35%), pupal weight was reduced to 0.025 ± 0.006 g (highland), 0.023 ± 0.006 g (midland), and 0.024 ± 0.006 g (lowland), representing reductions of 44.4%, 48.9%, and 46.7%, respectively, compared to the control. Post hoc comparisons identified that the 0.29% and 0.35% differed significantly from the lower concentration and the control, while differences in concentrations among the altitude populations were not significant. This pattern of concentration thresholds, which mirrors the pattern observed for larval duration, suggests that both developmental endpoints are governed by equivalent bioactive compound thresholds despite subtle differences in apiole concentrations among altitude populations [32].

Table 5. Pupal weight of Crocidolomia pavonana reared on broccoli leaves treated with Piper aduncum nanoemulsions from three altitudinal populations

Note: Different letters within each column indicate significant differences (Least Significant Difference (LSD) test, p < 0.05); () = live larvae, m a.s.l. = metres above sea level, and values represent means ± standard deviation (SD).

The magnitude of the reduction in pupal weight (~45–49%) carries significant ecological implications, as reduced pupal reserves are associated with reduced adult emergence, reduced reproductive output, and suppressed growth rates of C. pavonana populations in field environments.

3.6 Effects on Crocidolomia pavonana antifeedant activity

The potent antifeedant activity elicited by P. aduncum nanoemulsions (up to 76.99% at 0.35%) acts through the disruption of gustatory receptor neurons in the insect's peripheral nervous system, specifically repellent receptor cells that, upon activation by phenylpropanoid ligands, suppress the feeding control center via inhibitory interneurons. The steep concentration response in antifeedant activity from approximately 25–30% at 0.16–0.20% to 67–77% at 0.29–0.35% reflects the observed concentration threshold for lethal activity, suggesting that the apiole-rich matrix responsible for the increased toxicity also provides high repellent power through direct sensory stimulation at the larval mouthparts–leaf surface interface.

Antifeedant activity increased with concentration in all three formulations. At 0.35%, the numerical AI values were 73.65%, 75.21%, and 76.99% for the highland, midland, and lowland formulations, respectively (Table 6). Comparable antifeedant indices have been reported for the apiole-rich ethyl acetate extract of Piper dillatatum against Myzus persicae (AI = 84.4%) and Rhopalosiphum padi (AI = 98.9%), confirming the broad antifeedant spectrum of apiole-type phenylpropanoids in various insect orders. This antifeedant mechanism is agriculturally significant because it reduces the volume of bioactive compounds required for effective plant protection and minimizes the possibility of accumulation of phytotoxic residues, thereby expanding the window of selectivity for application in systems with the goal of natural enemy conservation [34, 35].

From an applied perspective, the current findings establish a provenance-based selection strategy for botanical biopesticide development: plant material should be sourced from upland populations, specifically those above 1,000 m a.s.l. in the West Sumatra highlands, to maximize apiole content, insecticidal efficacy, and antifeedant potential. The 37.90% increase in LC₅₀ from lowland to upland accessions directly translates into proportionally lower field application rates, lower formulation costs, and a smaller ecological footprint per treatment cycle.

Table 6. Antifeedant index (AI) and mean leaf area consumed by Crocidolomia pavonana second-instar larvae exposed to Piper aduncum nanoemulsions from three altitudinal populations

Note: Different letters within each column indicate significant differences (Least Significant Difference (LSD) test, p < 0.05); AI = antifeedant index; (—) = not calculated for control; m a.s.l. = metres above sea level, and values represent means ± standard deviation (SD).