Malaria remains a major global health concern, disproportionately affecting impoverished populations and straining national economies (WHO 2023). Thailand has committed to eliminating malaria by 2030 through its National Malaria Elimination Strategy, which incorporates the 1-3-7 surveillance system (Sudathip et al. 2022). Despite this progress, resurgence of cases, particularly along Thailand’s western border, poses a significant challenge, with 15,367 cases reported in 2024 (MOPH 2023). Malaria transmission is transmitted by Plasmodium-infected Anopheles mosquitoes. In Thailand, primary vectors include Anopheles dirus Peyton and Harrison in forested highlands and Anopheles minimus Theobald in village-forest fringes, whereas Anopheles harrisoni Harbach and Manguin is considered as a secondary vector in the west, and Anopheles epiroticus Linton and Harbach is a potential vector along the coastal areas (Sinka et al. 2011, Tainchum et al. 2015, Tananchai et al. 2019). Current vector control efforts rely heavily on chemical insecticides, notably pyrethroids and organophosphates (Chareonviriyaphap et al. 2013). However, their long-term application raises concerns regarding environmental safety, cost, and the emergence of insecticide resistance (Chaumeau et al. 2017). Consequently, there is a growing interest in sustainable and environmentally friendly alternatives. Among these, plant-derived compounds offer promising solutions (Tisgratog et al. 2016). β-caryophyllene, a naturally occurring bicyclic sesquiterpene found in essential oils of clove, basil, hops, and rosemary, is recognized as safe by the U.S. Food and Drug Administration (FDA) and exhibits low mammalian toxicity alongside diverse therapeutic properties (Oliveira et al. 2017). Its volatile, aromatic nature lends itself to potential application as an insect repellent. Studies have reported repellent efficacy of β-caryophyllene and its oxide derivative against Aedes aegypti (L.), Ae. albopictus (Skuse), and An. dirus (Gillij et al. 2008; Nerio et al. 2010). Nevertheless, data on its behavioral effects against major Anopheles vectors in Thailand remain limited.

This study evaluated the contact irritancy and noncontact repellency of β-caryophyllene against four Anopheles species using an excito-repellency assay. Laboratory strains included An. dirus, An. minimus, and An. epiroticus, all insecticide-susceptible. A field population of An. harrisoni was collected as adults from Kanchanaburi Province and reared under semi-field conditions. Mosquito colonies were maintained at Kasetsart University under controlled insectary conditions (25 ± 2 °C, 80 ± 10% RH, 12:12 h L:D). Larvae of An. epiroticus were reared in artificial brackish water, whereas adults were sustained on 10% sucrose and human blood via membrane feeding (Sukkanon et al. 2020). Anopheles dirus required artificial insemination (Yang et al. 1963), An. minimus and An. epiroticus were self-mating. For testing, 3–5-day-old sugar-starved, non-blood-fed females were used. Pure β-caryophyllene was diluted in absolute ethanol to prepare 2.5%, 5%, and 7.5% (v/v) solutions. Whatman No. 1 filter papers (14.7 × 17.5 cm) were treated with 2.8 ml of each solution and air-dried for at least 1 h (Sukkanon et al. 2020). Each paper was used only once. Experiments were conducted using an excito-repellency assay system composed of four stainless steel chambers with escape windows (Choomsang et al. 2018). In noncontact (repellency) trials, treated papers were placed behind mesh screens; for contact (irritancy) trials, papers were laid directly on the mesh. Fifteen mosquitoes were introduced per chamber (60 total per test) after a 3-min acclimation period. Escaping mosquitoes were recorded at 1-min intervals for 30 min. Post-exposure mortality was assessed at 24 h. All experiments were replicated 4 times during daylight hours.

Data were corrected using Abbott’s formula (Abbott 1925). Contact-specific irritancy was isolated using a modification of the Henderson-Tilton method (Sukkanon et al. 2020). Escape responses were analyzed using Kaplan–Meier survival analysis, with escape time (ET) for 25%, 50%, and 75% of mosquitoes (ET25, ET50, ET75) compared, using log-rank tests (Mantel and Haenszel 1959, Roberts et al. 1997). Statistical analyses were performed using SAS software, with P < 0.05 considered significant.

β-caryophyllene significantly enhanced mosquito escape behavior in both contact and noncontact trials, with no knockdown or mortality observed after 24 h (Table 1). In noncontact trials, An. minimus demonstrated 100% escape at 5%, whereas An. harrisoni reached 72.73% at 7.5%. An. epiroticus had the lowest escape rates (12.07–25.42%). In contact trials, An. minimus again exhibited 100% escape at 2.5%, with An. harrisoni reaching 94.34% at 7.5%. In contrast, An. dirus and An. epiroticus had modest responses at 7.5% (64.29% and 20.69%, respectively; Table 2). Escape responses between contact and noncontact trials were largely comparable, except for An. minimus at 2.5% and An. harrisoni at 7.5%, which showed stronger responses in contact trials. After adjusting for spatial repellency, most contact irritancy values showed no significant difference, though numerical escape rates were often higher. Anopheles minimus consistently had the fastest escape times (ET75 within 2–6 minutes), whereas An. harrisoni had ET50 values of 14–21 min in noncontact trials and 2–4 min in contact trials. Anopheles dirus showed an ET50 of 11 minutes at 7.5% in noncontact trials (Table 2). Escape times for An. epiroticus were not calculable because of insufficient responses. Log-rank analysis indicated significant differences in escape rates for An. minimus across concentrations in both test types (Table 1). Anopheles harrisoni exhibited dose-dependent responses in contact trials. Anopheles dirus showed significant differences only at 7.5%, whereas An. epiroticus displayed no significant variation.

Table 1.Percentage escape responses and log-rank statistics for four mosquito species exposed to β-caryophyllene.

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Table 2.Time (min) for 25% (ET₂₅), 50% (ET₅₀), and 75% (ET₇₅) of adult female mosquitoes to escape from treated excito-repellency chambers containing various concentrations of β-caryophyllene.

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This study demonstrated that β-caryophyllene effectively induces both contact irritant and spatial repellent responses in several Anopheles species, particularly An. minimus and An. harrisoni. These findings align with earlier reports on β-caryophyllene oxide’s efficacy against An. minimus (Nararak et al. 2019) and its synergistic effects when combined with essential oils (Nararak et al. 2023). The comparatively lower responses observed in An. dirus and An. epiroticus suggest species-specific sensitivities and possibly underlying genetic factors. The strong repellent responses of field-collected An. harrisoni support the practical applicability of β-caryophyllene in outdoor settings. This is particularly relevant in Southeast Asia, where many malaria vectors exhibit exophilic and exophagic behaviors, limiting the effectiveness of indoor-based interventions (Saeung et al. 2024). Formulations based on β-caryophyllene could offer a safe, plant-based alternative for personal protection and outdoor vector control, contributing to reduced insecticide reliance and resistance development (Deng et al. 2023). β-caryophyllene shows promise as a natural excito-repellent agent against important Anopheles vectors in Thailand, especially An. minimus and An. harrisoni. Its ability to trigger rapid escape responses without inducing mortality underscores its potential as a nonlethal control tool. Further field evaluations are warranted to validate these laboratory findings and explore formulation strategies for effective outdoor use.

This research was funded by the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand (Grant no. FF(KU) 51.68) and the National Research Council of Thailand (NRCT) High-Potential Research Team Grant Program (Contract no. N42A670406).