Sharma, Prajjval ¹ ; Sharma, S. D.² and Sharma, Shubham ³

¹✉ Department of Entomology, College of Agriculture, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, (HP), 176062 India.
²Department of Entomology, College of Agriculture, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, (HP), 176062 India.
³Department of Entomology, College of Agriculture, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, (HP), 176062 India.

2026 - Volume: 66 Issue: 2 pages: 482-496

Original research

Keywords

Abstract

Introduction

The European honey bee, Apis mellifera L. (Hymenoptera: Apidae), is among the most economically valuable and ecologically important pollinators, which play an important role in the pollination of numerous crops and the maintenance of biodiversity (Frazier et al. 2024). Hive products including honey, wax, royal jelly, and propolis are vital in supporting rural and tribal livelihoods and serve as a source of sustainable income through apiculture (Boncristiani et al. 2020). However, various pests and pathogens have been reported to attack bee colonies and cause a significant loss to the beekeepers. The stable microclimate maintained within colonies through thermoregulatory behaviours such as wing fanning, clustering, and evaporative cooling also provides favourable conditions for the development of these pests and pathogens that affect brood and hive products (Peña-Chora et al. 2023). In addition, the widespread movement of A. mellifera colonies for pollination and honey production exposes them to novel parasites and pathogens in environments where they have not co-evolved (Marcelino et al. 2022), which limits their natural resistance and increase susceptibility to new enemies (Nekoei et al. 2023). The ectoparasitic mite Varroa destructor Anderson and Trueman (Acari: Varroidae) is a major pest of A. mellifera, which feeds on the haemolymph and fat bodies of adult and developing bees and cause physiological disorders, reduced longevity, impaired immunity, and serve as a vector for viral pathogens (Muntaabski et al. 2023; Sawadogo et al. 2024). Heavy infestations can substantially decrease colony productivity and, in severe cases, lead to colony collapse. More than 50 per cent of countries globally report varroosis outbreaks in apiaries (Fanelli and Tizzani 2020). Another ectoparasite, Tropilaelaps clareae Delfinado and Baker (Acari: Laelapidae), a native of Asia, has expanded its host range to A. mellifera and other honey bee species (Chantawannakul et al. 2018). This genus coexists with V. destructor in Asia, while remaining absent but under surveillance in North America (Price et al. 2025). T. clareae primarily parasitizes brood in sealed cells, synchronizing its life cycle with that of the host, which makes its management particularly challenging. Infestations can result in partial to complete colony losses, which necessitates the need for effective control measures (Sharma et al. 1994a; Dwarka et al. 2025). Both species are widely distributed across diverse agro-climatic zones of India and are commonly associated with multiple honey bee species (Poonia et al. 2014; Singh et al. 2019; Thakur et al. 2021)

Currently, the control of these mites mainly depends on organic acids such as formic and oxalic acid or synthetic acaricides such as fluvalinate, coumaphos, amitraz, and flumethrin (Sharma et al. 1994b; Qadir et al. 2021) While initially effective, the repeated use of these chemicals has led to the development of resistant mite populations, contamination of hive products, and potential adverse environmental impacts (Sharma and Kashyap 2001; Almecija et al. 2024). In addition to chemical treatments, non-chemical approaches such as sugar dusting, drone brood removal, brood interruption, and other biotechnical methods are also employed to manage mite populations, but their effectiveness is mostly variable and may not provide complete control, particularly under conditions of co-infestation (Odemer et al. 2022). Furthermore, these methods can be labour-intensive, time-consuming, and may not be feasible for large-scale operations, which limit their practical application in commercial apiaries (Lodesani et al. 2019; Vercelli et al. 2023). These limitations have necessitated the search for alternative, environmentally friendly management strategies for the management of parasitic mites in A. mellifera colonies.

Plant-derived essential oils have emerged as promising natural acaricides due to their broad-spectrum antimicrobial and miticidal properties, low mammalian toxicity, and reduced risk of chemical residues in hive products (Hosamani et al. 2007; Ntalli et al. 2022; Farina et al. 2025). Essential oils contain volatile compounds such as monoterpenes, sesquiterpenes, terpenoids, and phenylpropanoids, which possess fumigant, contact, or repellent actions against mites (Mutinelli 2016; Reyna-Fuentes et al. 2024). Numerous studies have reported the efficacy of essential oils, including thyme, eucalyptus, clove, lemongrass, neem, and citronella, against V. destructor under laboratory and field conditions. Some essential oils, such as thymol-based formulations, have been successfully used in commercial apiaries, and provide substantial mite control with minimal impact on bees (Mutinelli 2016). Essential oils' toxicity can vary among species and developmental stages of honey bees, and comprehensive assessments of their selectivity and safety are limited (Caren et al. 2025). Although mite biology and management have been extensively studied, there is limited information on the concurrent incidence patterns of V. destructor and T. clareae and in areas where both mites co-occur, data on their susceptibility to various hard and soft acaricides remain scarce (Buawangpong et al. 2015). Moreover, very less standardized evaluation exists for their efficacy against co-infestations of both V. destructor and T. clareae.

In this study, we aimed to evaluate the potential of four essential oils viz., thyme, eucalyptus, clove, and lemongrass, for the management of V. destructor and T. clareae in co-infested A. mellifera colonies. The investigation was conducted in two phases: laboratory assays to determine the fumigant toxicity and lethal concentrations (LC₅₀) of the oils against both mite species, followed by field trials to assess their efficacy under hive conditions.

Materials and Methods

Study site and honey bee colonies

Honey bee colonies housed in standard Langstroth hives, located in the A. mellifera apiary of the Department of Entomology, College of Agriculture, CSK HPKV, Palampur, Kangra (HP), India (32°05′ 59.77″ N, 76°32′ 50.07″ E; 1250 m amsl), were used to collect parasitic mites (V. destructor and T. clareae) and worker bees, and to evaluate the essential oils against both mite species. The colonies were kept free from any type of treatment for the last one year and were naturally co-infested with both species of parasitic mites, as evidenced by regular monitoring through mite fall and brood examination.

Collection of parasitic mites and worker bees of A. mellifera for bioassay

Varroa destructor

The hives were opened on a warm sunny day to collect worker bees. A 0.5-litre plastic jar was filled with 15 g of powdered sugar. Worker bees were gently transferred into the jar with the help of a bee brush. The lid of the jar was closed, and the jar was shaken vigorously for one minute to dislodge the mites attached to the bodies of the worker bees. After shaking, the jar was opened and kept tilted on a white plain paper outside the hive, allowing the bees to come out of the jar. After all the bees had moved out, the sugar powder from the jar was collected on plain paper in the laboratory, and the dislodged mites were collected and separated with the help of a fine brush. These steps were repeated until the required number of alive female Varroa mites was obtained.

Tropilaelaps clareae

Due to the shorter phoretic phase of T. clareae, mites were collected from the worker brood of A. mellifera. After uncapping the sealed brood with the help of forceps, the pupae were examined for infestation by adult T. clareae. The reddish-brown mites attached to the pupae were visible to the naked eye. The required number of infested pupae thus collected was transferred to the laboratory, and the attached mites were gently removed with the help of a soft brush and transferred to sterile Petri plates under a stereomicroscope.

Apis mellifera

Worker bees were collected directly from fully covered lateral combs of five honey bee colonies by brushing them into a 500 ml glass jar and transported to the laboratory for immobilization by low-temperature exposure in a refrigerator (at 4 °C for 3 minutes.). To ensure population variability in the experimental groups, an equal number of bees were collected from full-strength bee frames of the selected colonies.

Toxicity of essential oils to parasitic mites (V. destructor and T. clareae) and worker bees of A. mellifera

The fumigant toxicity of four essential oils was evaluated against worker bees of A. mellifera and adult female mites of V. destructor and T. clareae under laboratory conditions (32 °C and 70% RH). Four essential oils (clove, eucalyptus, lemongrass, and thyme) with 100 per cent purity (Essentia Extracts) were used in the study. The method used by Ghasemi et al. (2011), with slight modifications, was followed to evaluate the toxicity of essential oils. After a preliminary dose trial for each essential oil against worker bees and mites, a logarithmic series of dilutions was prepared to identify the effective range of concentrations causing mortality from 10 to 90 per cent. The required volume of pure essential oil was diluted with acetone and applied uniformly onto a filter paper (2 cm diameter) with the help of a micropipette and allowed to air dry. After drying, the filter paper was attached to the inner surface of the lid, and the Petri plates (90 mm diameter × 15 mm height) were sealed with the help of parafilm. Concentrations of the applied essential oil were calculated based on the quantity of oil and the volume of the Petri plate (µl/l air).

Each mite assay set-up consisted of twenty female mites per Petri plate along with five pupae of A. mellifera workers to prevent mortality due to starvation. The control mites were kept without any treatment under sealed Petri plates. Each set-up was replicated thrice, totaling 60 adult mites per assay. In the worker bee assay, twenty worker bees were kept in each Petri plate (150 mm diameter × 25 mm height), and the experiment was replicated three times. The essential oil was applied in the same manner as in the mite assay, and the Petri plates were sealed with parafilm. Mortality data of mites as well as worker bees in each experiment were recorded after 4 hours. Mites and bees showing no movement when touched with a fine soft brush were considered dead.

Field evaluation of essential oils against parasitic mites

The field experiments were carried out during March-April 2025. Pure essential oils (the same as those used in the laboratory bioassays) were tested against V. destructor and T. clareae under colony conditions in A. mellifera apiaries co-infested with both mite species. For each essential oil, ten colonies of similar strength and mite infestation were selected. All colonies were equipped with a mite screening mesh (3 × 3 mm) between the brood chamber and the bottom board. A white sticky paper was placed over the bottom board to trap fallen mites along with hive debris and was changed twice a week. Fourteen days before the first oil treatment, a new sticky paper was placed in the colonies, and cumulative mite fall was recorded weekly at 7 days before treatment (DBT), zeroth day (on the day of treatment before oil application), and 7, 14, and 21 days after treatment (DAT).

For field application, essential oils were applied as fumigants using cotton discs as a slow-release carrier. Each cotton disc was impregnated with 1.5 mL of pure essential oil, and two discs per colony were used per application. One disc was placed between the mite screening mesh and the bottom board, while the other was positioned on the top bars of the central brood frames to facilitate uniform volatilization of the oil within the hive. The discs were replaced at weekly intervals for three consecutive applications (0, 7^th, and 14^th day). This method was adopted to ensure sustained fumigant exposure of mites while minimizing direct contact with bees and brood.

On the 21st day after replacing the sticky paper, one fluvalinate strip was placed between the central brood frames as a follow-up treatment to dislodge the remaining mites. Control hives were treated only with fluvalinate strips on the 21st day. Mite fall due to the follow-up treatment was recorded on the 28th day. The total brood area (including eggs, larvae, and sealed brood) was recorded weekly for all experimental colonies. To measure brood area, a wire grid containing one-inch square divisions was mounted on a comb frame. Squares fully covered by brood were counted as one (1), while those partially covered were estimated proportionally as quarters (0.25, 0.5, or 0.75). Measurements were initially taken in square inches and then converted to square centimetres using a conversion factor of 6.45. The relative efficacy of each essential oil, natural grooming behaviour of A. mellifera, and the efficacy of essential oils over control were evaluated using the following formulae.

Relative efficacy (%)= (Sum of essential oil induced mite fall at 7th,14th,and 21st day)/(Total mite fall at 7th,14th,21st,and 28th day) × 100

Efficacy over control (%)= (Relative efficacy of the oil (%) - Relative efficacy in the control (%))/(100 - Relative efficacy of the control (%)) × 100

Data analysis

Concentration–mortality data for both mite species and A. mellifera workers were analyzed by probit regression to estimate LC₅₀ values, 95% fiducial limits, slope ± SE, and chi-square goodness-of-fit (Finney 1964; Finney 1971) using the computer program Probit-MSChart (Chi 2026). Differences among treatments were considered significant when the 95 per cent fiducial limits did not overlap.

For each essential oil-treated and control colony, a one-way analysis of variance (ANOVA) was performed for mite infestation parameters of pre- and post-treatment weeks as well as for the efficacies of essential oils (following arc-sine transformation), and means were compared using Tukey's post-hoc test at p < 0.05 using SPSS v26.0. The brood area of treated and untreated colonies was compared using a paired t-test (p < 0.05) in SPSS to evaluate pre- and post-treatment changes.

Results

Bioassays and relative toxicity

For both the parasitic mites, V. destructor and T. clareae, the four tested essential oils showed non-significant median lethal concentrations (LC₅₀) after a 4 h exposure period (based on overlapping CLs) (Table 1, Fig 1). However, LC₅₀ values of all oils were significantly greater for A. mellifera compared to the mites, although no significant differences were reported across all the tested essential oils for A. mellifera. Thyme oil showed the highest selectivity ratio of 7.37, followed by eucalyptus, clove, and lemongrass oil. A similar trend for the selectivity ratio was also recorded against T. clareae after the 4 h exposure period (Table 1).

Effects of essential oil treatments on the infestation levels of V. destructor in A. mellifera colonies

In the control hives of A. mellifera, the mite infestation of V. destructor in colonies from 7 days before treatment (DBT) to 21 days after the first treatment (DAFT) remained at par in terms of mite fall, per cent brood, and per cent worker bee infestation (Fig 2a). However, after the follow-up treatment on the 21st day, the per cent brood (F_5,54 = 35.944; p < 0.001) and worker bee infestation (F_5,54* = 13.351; p* < 0.001) reduced significantly and declined to zero, while the mite fall increased significantly to 54.20 on the 28th day (F_5,54 = 52.289; p < 0.001). In colonies treated with thyme oil, the Varroa mite mortality recorded at 7 and 14 days after first treatment increased significantly (F_5,54 = 52.552; p < 0.001) compared to the pre-treatment mite fall counts (7 DBT: 26.90; day 0: 23.50), while 21 days after first treatment, the mite fall count (8.70) declined to a minimum (Fig 2b). The per cent brood infestation reduced significantly (2.10%) from pre-treatment levels at 14 days after first treatment (F_5,54 = 15.429; p < 0.001), while the per cent worker bee infestation reduced significantly (0.6%) at 7 days after first treatment compared to the pre-treatment levels (F_5,54 = 23.007; p < 0.001) and declined to zero during 14 and 21 days after first treatment.

In colonies treated with eucalyptus oil, the maximum mite mortality was observed on day 14 (47.8 mites), which was significantly greater than the pre-treatment mortality of V. destructor (F_5,54 = 46.861; p < 0.001) (Fig 2c). However, the brood infestation of A. mellifera by V. destructor remained at par with the pre-treatment levels until the 21st day after the first treatment and significantly reduced to zero per cent after the follow-up treatment (F_5,54 = 8.765; p < 0.001). In contrast, the worker bee infestation significantly reduced (0.5%) after the first eucalyptus oil treatment in the hives and declined to zero immediately after the second treatment (F_5,54 = 14.919; p < 0.001). In the clove oil treatment, after the three applications of the essential oil, the mite fall count of V. destructor remained statistically similar to the pre-treatment counts and only increased significantly after the follow-up treatment (F_5,54 = 3.988; p < 0.001) (Fig 2d). However, the per cent brood (F_5,54 = 7.366; p < 0.001) and worker bee infestation (F_5,54 = 20.940; p < 0.001) reduced significantly after 21 and 14 days of the first treatment, respectively. Similarly, in the lemongrass treatment, Varroa mite mortality did not increase significantly until the 21st day (37.1 on 21 DAFT) but increased after the follow-up treatment (43.70 on 28 DAFT) (F_5,54 = 4.084; p < 0.001) (Fig 2e). Likewise, the worker bee infestation levels remained at par with the pre-treatment infestation levels till one week and only reduced significantly thereafter (F_5,54 = 36.052; p < 0.001). However, the per cent brood infestation by V. destructor reduced significantly from pre-treatment levels at 21 days after first treatment (3.1%) and further decreased significantly to 0 per cent after the follow-up treatment (F_5,54 =15.672; p < 0.001).

Effects of essential oil treatments on the infestation levels of T. clareae in A. mellifera colonies

In the control hives of A. mellifera, all three mite infestation parameters studied for T. clareae remained at par across different time periods before the follow-up treatment (Fig 3a). However, after the follow-up treatment, the mite fall increased significantly to 32.9 mites per hive per week (F_5,54 = 43.648; p < 0.001), while the per cent brood (F_5,54 = 13.153; p < 0.001) and worker bee infestation (F_5,54 = 4.092; p = 0.003) reduced significantly and declined to zero per cent. In the thyme oil treatment, the mite fall of T. clareae increased significantly from pre-treatment levels 7 days after the first treatment (37.6 dead mites/hive/week), which was also greater than the mite mortality after the follow-up treatment (12.22 mites/hive/week) (F_5,54 = 78.341; p < 0.001) (Fig 3b). The per cent brood infestation by T. clareae reduced significantly to 0.60% after the first treatment and remained at par until the follow-up treatment (0%) (F_5,54 = 19.559; p < 0.001). Similarly, the per cent worker bee infestation followed the same trend, showing complete absence and significant reduction only after the first treatment and remaining absent until the follow-up treatment (F_5,54 = 31.607; p < 0.001).

In the eucalyptus oil treatment, the mite mortality increased significantly after the first treatment (32.10 mites/hive/week) compared to the pre-treatment fall (F_5,54 = 16.807; p < 0.001) (Fig 3c). The brood infestation reduced significantly and declined to 0 per cent after 21 days of the first treatment (F_5,54 = 10.490; p < 0.001), whereas worker bee infestation declined significantly to 0 per cent after 7 days of the first treatment (F_5,54 = 19.150; p < 0.001). The application of clove oil significantly increased T. clareae mortality after 7 (27.40 mites/hive/week) and 14 days (27.70 mites/hive/week) of the first treatment (F_5,54 = 14.627; p < 0.001) (Fig 3d). The brood infestation remained at par with the pre-treatment levels until 14 days after first treatment (2.30%), then reduced significantly after 21 days of the first treatment (0.4%) (F_5,54 = 7.911; p < 0.001). The worker bee infestation also reduced significantly after 21 days of the first treatment (F_5,54 = 8.773; p < 0.001). The lemongrass oil treatment had no significant effect on mite mortality and brood infestation until 14 days after the first treatment (Fig 3e). Only the follow-up treatment significantly reduced the mite population on the 28th day after the first treatment (mite fall: F_5,54 = 9.170; p < 0.001; brood infestation: F_5,54 = 8.321; p < 0.001; worker bee infestation: F_5,54 = 8.084; p < 0.001).

Treatment effectiveness of essential oils against V. destructor and *T. clareae

The relative efficacy of each essential oil and natural grooming behaviour (control = 53.06%) of A. mellifera was evaluated in comparison to the mortality of Varroa and Tropilaelaps mites caused after the follow-up treatment with fluvalinate (Fig 4a). All tested essential oils exhibited a significantly greater relative efficacy against V. destructor compared to the control (F_4,45 = 39.123; p < 0.001). Thyme (84.13%) and eucalyptus oil (82.32%) possessed significantly greater relative efficacy than clove (69.87%) and lemongrass (70.95%) oils against Varroa mites. Similarly, all tested essential oils were significantly more effective against T. clareae than the control (F_4,45 = 28.824; p < 0.001). Colonies treated with thyme oil exhibited the highest relative efficacy of 81.70% against T. clareae, which was at par with eucalyptus (74.53%) and clove oil (76.95%), while lemongrass oil showed the lowest relative efficacy (69.74%).

The efficacy over control of thyme oil (65.91%) and eucalyptus oil (62.36%) against V. destructor was significantly greater than that of clove oil (35.71%) and lemongrass oil (37.97%) (F_3,36 = 13.352; p < 0.001) (Fig 4b). Similarly, against Tropilaelaps mites, thyme oil had the highest efficacy over control (63.12%) and was at par with eucalyptus (48.68%) and clove oil (53.32%). Lemongrass oil showed the lowest efficacy over control (39.59%) and was significantly lower than thyme oil (F_3,36 = 3.490; p = 0.025).

Brood area of the colonies

In untreated colonies of A. mellifera co-infested with V. destructor and T. clareae, a significant reduction in brood area was observed over a 21-day period (0 and 21) (t = 3.943; df = 9; p = 0.003) (Fig 5). However, in the eucalyptus (t = -1.843; df = 9; p = 0.098), clove (t = -2.015; df = 9; p = 0.075), and lemongrass oil (t = -1.865; df = 9; p = 0.095) treated colonies, a non-significant increase in brood area was observed, while the increase was significant in thyme oil treated colonies (t = -3.279; df = 9; p = 0.010).

Discussion

The present study evaluated the acaricidal efficacy and selectivity of essential oils against the major parasitic mites of Apis mellifera viz., V. destructor and T. clareae under laboratory and field conditions. The findings highlight differences in mite mortality and effects of treatments on the colony-level dynamics of mite infestation. In most managed Apis mellifera apiaries, colonies are frequently co-infested with these two parasitic mite species, yet control strategies are often evaluated against a single mite in isolation. Therefore, an integrated evaluation is particularly important for identifying control methods that are broadly effective, selective to honey bees, and suitable for practical adoption in apiary-level mite management programmes (Lee et al. 2005; Thongsawang et al. 2021).

In the present study, the laboratory bioassays demonstrated that all tested essential oils exhibited comparable toxicity against V. destructor and T. clareae, as reflected by non-significant differences in LC₅₀ values between the two mite species. In contrast, the LC₅₀ values for A. mellifera were consistently higher than those for the mites, which indicates a clear margin of safety for the host. The higher selectivity ratios observed for thyme oil, followed by eucalyptus, clove, and lemongrass oils, suggest that these oils possess differential toxicity favouring mite control over bee mortality. Such selectivity is a critical attribute for the practical application of any acaricidal agent within honey bee colonies. Selectivity ratios for 11 essential oils were evaluated by Hýbl et al. (2021), with the highest value recorded for thyme oil (6.848) after 4 h of exposure, which was comparable to that observed in the present study (7.37). This similarity may be attributed to the presence of common bioactive constituents such as thymol and carvacrol, which exhibit selective toxicity towards mites while being relatively less harmful to honey bees (Brasesco et al. 2017; Begna et al. 2023). However, the selectivity ratios of thyme and eucalyptus oils for V. destructor were reported to be 4.76 and 1.75. respectively with LC₅₀ of 1.07 and 1.74 µl/l, respectively by Ghasemi et al. (2011), which were notably lower than those of the present study. These variation in mite mortality and infestation reduction among the essential oils indicates differences in their biological activity and persistence within the hive environment.

In our study, field evaluations revealed that essential oil treatments effectively reduced V. destructor infestation levels in brood and adult bees, with variable timing of reduction among oils. Thyme oil proved to be the most effective under field conditions, causing significantly greater mortality of both V. destructor and T. clareae within 7 days of the first treatment as evidenced by the mite fall data. In addition, thyme and eucalyptus oils induced a more rapid and pronounced reduction in mite populations, whereas clove and lemongrass oils showed delayed effects, particularly prior to the follow-up treatment. These differences suggest that essential oils may vary in their mode of action, volatility, and residual efficacy, which can influence their performance under colony conditions. Earlier studies have shown that thyme volatiles are toxic to parasitic mites of honey bees, while bees are attracted to these volatiles, indicating their adaptability to this oil (Ghasemi et al. 2016). GC–MS analyses have identified bioactive constituents such as carvacrol, thymol, and ρ-cymene, responsible for its acaricidal activity (Safaei-Ghomi et al. 2009). The decline in brood and worker bee infestation following treatment indicates that these oils were capable of suppressing both phoretic and reproductive stages of the mite. The pronounced effect observed after the follow-up treatment highlights the importance of repeated applications to achieve sustained control, particularly in colonies with established infestations. The response of T. clareae to essential oil treatments differed from that of V. destructor, which reflects species-specific differences in biology and behaviour. Thyme and eucalyptus oils resulted in rapid reductions in brood and worker bee infestation, while clove and lemongrass oils required additional applications to achieve comparable effects. These findings indicate that essential oils can be effective against T. clareae, but optimal control may depend on oil type and treatment schedule.

The consistent improvement in mite control following the follow-up treatment across all essential oils highlights the necessity of repeated applications when managing co-infestations of V. destructor and T. clareae. Co-infested colonies present a complex management challenge due to overlapping mite populations (Buawangpong et al. 2015). When evaluated relative to natural grooming behaviour and chemical control benchmarks, all tested essential oils demonstrated substantial efficacy against both mite species. Thyme and eucalyptus oils consistently exhibited higher relative efficacy and efficacy over control compared to clove and lemongrass oils. Similarly, Khajehali et al. (2023) reported thyme and eucalyptus oil to be as effective as fluvalinate strips when applied in combination with thymol. In addition, Mahmood et al. (2014) reported the clove oil in combination with tobacco extract to be 96.483 per cent effective against V. destructor suggesting that certain essential oils may be more suitable as primary control agents, while others may serve as supplementary tools within an integrated mite management programme. In contrast to our results, Islam et al. (2016) reported lemongrass oil to be more effective than thyme oil against V. destructor in A. mellifera colonies with a post-treatment cumulative mite fall of 306 compared to 263 mites in thymol treated colonies. This discrepancy may be attributed to differences in environmental conditions, colony strength, or formulation and method of application of essential oils, which are known to influence their efficacy under field conditions (Calderone 1999; Jack and Ellis 2021).

The changes observed in brood area provide an important indicator of colony health following essential oil application (Pătruică et al. 2023). In the present study, the significant decline in brood area in untreated colonies highlights the detrimental impact of the ignorance of the mite infestation. In contrast, the maintenance or increase in brood area in treated colonies, particularly those receiving thyme oil, suggests that effective mite suppression can favourably influence colony growth and brood development. The absence of brood suppression in treated colonies also indicates that the tested essential oils were not detrimental to brood rearing under the conditions of this study. Similar results were also obtained by Masry et al. (2020) for jatropha plant extracts who reported that the two tested concentrations (1 and 2%) were effective in controlling the Varroa mite with higher brood area and colony strength after the treatment. In addition, Lazăr et al. (2021) also reported that application of juniper, thyme, basil, oregano, and cinnamon essential oils were associated with an increase in honey bee health and honey production in A. mellifera colonies.

Overall, the findings suggest that essential oils, particularly thyme and eucalyptus oils, have considerable potential as alternative or complementary tools for managing V. destructor and T. clareae in co-infested A. mellifera colonies. Their selective toxicity, effectiveness under field conditions, and apparent compatibility with colony development support their inclusion in integrated mite management programs aimed at reducing reliance on synthetic acaricides. Despite the promising results, the efficacy of essential oils may be influenced by colony strength, environmental conditions, and application protocols. Further studies evaluating long-term effects, seasonal variability, and integration with other non-chemical control measures would help in understanding their effectiveness for practical use. Standardization of application strategies and assessment across multiple management scenarios will be essential for maximizing their effectiveness in beekeeping systems.

Acknowledgements

We gratefully acknowledge the resources and facilities provided by our university, CSK Himachal Pradesh Krishi Vishvavidyalaya (CSK HPKV), Palampur, India, during the study.

Author Contributions

Prajjval Sharma: Investigation, methodology, writing, and data analysis; S. D. Sharma: Supervision, methodology, writing-review, and formal analysis; Shubham Sharma: Writing and formal analysis.

Declarations

Competing interests: The authors declare no competing interests.

Funding: Not applicable.

Ethics approval: Not applicable.

Consent to participate: Not applicable.

Consent for publication: Not applicable.

Data availability statement: The datasets created during and/or examined during the study are available from the corresponding author on reasonable request.

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