GC-MS Analysis and Evaluation of Essential Oils as Volatile Biopesticides: Assessing Their Acaricidal Potential against Varroa destructor

Abstract

Honey bees are crucial for ecosystem pollination and honey production, yet the Varroa destructor mite hinders their productivity and health. Efforts to manage Varroa mites with synthetic pesticides have had limited success, highlighting the need for naturally derived acaricides as a primary option. However, the acaricidal efficacy of essential oils from Salvia officinalis L. (sage), Cannabis sativa (hemp), and Laurus nobilis (laurel) remains to be fully understood. This study aims to investigate the acaricidal efficacy of these three essential oils at varying concentrations and their impact on honey production, focusing on the efficient reduction in Varroa mites. The sugar roll method was employed to assess Varroa mite infestation levels, while GC-MS analysis was utilized to verify the composition of the essential oils. Honey production measurements were also performed. The efficacy rates (%) at concentrations of 15%, 10%, and 5% for hemp oil were 95.4% ± 0.30%, 85.71% ± 0.85%, and 64.48% ± 0.26%, respectively; for sage oil, they were 81.08% ± 0.57%, 69.42% ± 1.72%, and 50.35% ± 0.70%; and for laurel oil, they were 68.96% ± 0.34%, 54.66% ± 0.37%, and 33.58% ± 0.30%, respectively. Key compounds identified include trans-caryophyllene, α-pinene, and viridiflorol in hemp oil; myrcene, limonene, and β-caryophyllene in sage oil; and phytol, β-myrcene, and n-heneicosane in laurel oil. The overall findings indicate that hemp oil is highly effective in controlling Varroa mites. However, further research is needed to evaluate its potential side effects on bees to ensure its sustainability and safety.

1. Introduction

Apis mellifera L. is a species of crucial environmental, agricultural, and economic importance [1] that exists worldwide except in Antarctica [2]. Honey bees represent a vital component of agricultural systems, primarily due to their pollination activities, encompassing fruits, vegetables, and cash crops. This pollination significantly contributes to approximately one-third of the American food supply, underlining the indispensable role of honey bees in food production and ecosystem health. Moreover, honey bees are prolific producers of various by-products such as honey, royal jelly, beeswax, and propolis [3,4]. Recent declines in global honey bee populations, accompanied by an uptick in colony losses reported by beekeepers, have sparked scientific concern [5]. With the precipitous decline of honey bee populations, specifically A. mellifera, the academic sphere has increasingly recognized this phenomenon, with numerous studies pinpointing various contributing elements [6]. These encompass pathogens, parasites, environmental stressors, and the deployment of agricultural pesticides. Amongst the array of threats to global honey bee populations, such as habitat degradation and pesticide application, the V. destructor mite is identified as a predominant factor exacerbating colony collapses. Varroa mites cause stress to honey bees and can be a vector for viral pathogens [7,8]. Significantly, Rosenkranz et al., 2010 highlight the Varroa mite as a predominant factor exacerbating colony collapses (as reviewed in their article) [9].

The Varroa mite is an ectoparasite afflicting honey bee colonies, leading to significant health detriments. This parasitic mite damages the bees’ immune systems, culminating in adverse effects such as reduced lifespan, impaired metabolic processes, and increased rates of viral infections [10,11,12]. Additionally, Varroa mites indirectly harm honey bees by lowering the number of bees in the colony, affecting their ability to forage and pollinate, reducing honey production as a result of fewer foraging trips and the poor health of worker bees, and making colonies weaker and more vulnerable to other environmental pressures. These mites multiply fast and can take over a bee colony in roughly half a year [13,14,15].

The challenge in controlling these mites is heightened as they have developed resistance to the acaricides traditionally used to combat them [16]. Mechanical and cultural Varroa management methods are widely used, but their efficacy is inconsistent. Global research has identified promising chemical compounds for mite toxicity, but prohibitive costs have hindered further experimentation. Despite this, chemical screenings are helpful, but their effects on Varroa mites and honey bees remain unexamined [17,18,19]. Predominantly, Varroa control methodologies employ synthetic chemical agents. These pyrethroid insecticides, acaricides like tau-fluvalinate and organophosphate insecticides like Coumaphos, are reported to be persistent and tend to accumulate in the hive products, like honey and beeswax, and this is likely to cause sublethal exposure of the Varroa mites for a long period. Such prolonged exposure to the acaricides is expected to contribute to the resistance buildup in the mites [20,21,22]. Additionally, research sheds light on the harmful effects of these substances on bee health and the broader environment, compelling the search for safer alternatives [23,24,25].

Conventional pesticides pose several challenges, including pest resistance, environmental pollution, public health threats, and food contamination, considering that new and safer products are lacking in the market [26,27]. Botanical pesticides, seen as low-risk and environmentally friendly, are drawing researchers’ eyes. They promise a green twist on fighting pests, blending old wisdom with new science for a healthier farm future [28,29,30]. They are not just for managing pests anymore but are stepping stones towards greener, safer veterinary products. This shift toward sustainability is crucial for protecting our environment while ensuring the health of our valuable insects, crops, and livestock [31,32,33,34]. Botanicals are considered safe because they are low in toxicity to mammals, do not pollute, and steer clear of environmental toxins, making them a popular choice for both people and the planet [35]. Plant chemicals like pyrethrins, alkaloids, and terpenoids boast a range of biological activities. Their diverse biological actions make them versatile [36]; although the compounds have short-term persistence and low non-target toxicity, there are exceptions. Pyrethrin products like PyGanic can also have toxic effects on pollinators, so special care should be taken to protect the beneficial insects [37,38]. Essential oils can be used as an attractor or repellent for pests, which makes them suitable for use in a trap, and they repel queen bumble bees even if the results are non-lethal [39,40].

The interest in plant secondary metabolites can lead to sublethal effects like reduced hatching, shorter lifespans, or abnormal development. Meanwhile, the volatile compounds in honey do not harm bees. Recent studies show that organic acids and essential oils act effectively against Varroa mites in organic beekeeping [41,42,43,44]. In beekeeping, using natural compounds derived from plants is gaining attention. Terpenoids and other secondary metabolites [45] have acaricidal properties that stand out for their ability to fend off pests [46,47], and subsequently deter them from feeding, disrupt their growth, and affect their reproduction [48]. Cannabis sativa L produces essential oils that are noted for their larvicidal and biopesticidal efficacy against pests like the peach–potato aphid and house fly [48,49,50]. Another research study focuses on various application techniques and assesses the efficiency of essential oils, advocating their incorporation within integrated pest management schemes [51]. The essential oil of S. officinalis L. has several constituents in the form of borneol, camphor, and pinene, and its activity on the human nervous system is interesting [52,53]. The findings revealed that sage oil has significant acaricidal properties against Varroa mites [54]. Laurel, an aromatic angiosperm native to the Mediterranean, is cultivated for its medicinal properties, including antifungal, antibacterial, antiviral, insecticidal, and acaricidal properties [55,56,57,58,59,60]. Bearing the above in mind, we evaluated the acaricidal properties of hemp, sage, and laurel essential oils (EOs) and their main constituents against Varroa mites. Significant increases in honey production and the number of fallen mites were also recorded.

2. Materials and Methods

The pictorial representation of the present research experimental protocol was conducted to assess the efficiency of essential oil, GC-MS analysis, sticky board method, sugar roll method, and yield production after treatment, as shown in Figure 1.

2.1. Honey Bee Colonies

Prior to the commencement of the research, we meticulously put out 75 colonies of A. mellifera to document the natural drop of Varroa mites. The experimental colonies were all accommodated within conventional Langstroth hives. Through a rigorous screening process, we narrowed our focus to 50 colonies. This selection was based on several critical criteria: the size of the honey bee population was comparable across the chosen colonies, they were queen right, and they exhibited a mite infestation level exceeding the economic threshold of 3 to >10 adult mites per 100 bees [61]. Moreover, the colonies designated for the experiments were in a mature stage of development and demonstrated high productivity levels. Before initiating the study, these colonies were observed to maintain an average of 8 ± 2 brood frames within their brood chambers [62,63]. In our experiment, we used 50 bee hives, kept at a minimum distance of ten meters, which were divided into ten treatment groups with five replications: three essential oils (hemp, sage, laurel) at three concentrations each (5%, 10%, 15%) and a control group (untreated). Those treated included fifteen colonies of each essential oil and five colonies of the control group; every concentration of each oil had five colonies treated, implementing a randomized block design for uniform environmental conditions across all treatments. The present research was conducted during the winter season, from December to January, at the apiary within the Department of Entomology at the University of Agriculture Faisalabad, Islamic Republic of Pakistan. During the study period, December’s meteorological data indicated a maximum temperature of 20.4 °C and a minimum of 8.2 °C. Wind conditions were recorded at an average of 10.56 km/day, with precipitation levels reaching 13 mm and average relative humidity standing at 60%. In the subsequent month of January, the data reflected a slight decrease in thermal readings, with the maximum and minimum temperatures documented at 16.9 °C and 5.5 °C, respectively. The wind speed escalated to an average of 15.88 km/day, while the rainfall increased significantly to 19 mm, accompanied by a rise in average relative humidity.

2.2. Plant Material Collection and Extraction

Three botanicals, namely L. nobilis, C. sativa, and S. officinalis, were utilized in the study. The seeds and leaves of these three plants were purchased from a traditional medicine shop in Lahore, Pakistan. The extraction methodology, as outlined by Moazam Hyder et al. (2022a) and Moazam Hyder et al. (2022b), was employed with minor modifications [64,65]. Leaves of the botanicals were subjected to washing, followed by drying at 45 °C for 48 h. The dried material was then pulverized and sieved through a 40 µm mesh screen, with each botanical powder stored separately.

The powder was made by mixing one gram of initial powder with five milliliters of ethanol in bottles, which were kept at room temperature (18–25 °C) for seven days. The bottles were agitated twice daily to facilitate the dissolution of the powder in ethanol. Subsequently, the mixture was filtered through filter paper, and any residue was further dissolved using 2.5 mL of the same solvent per gram of powder. The combined first and second solvent fractions were concentrated using a rotary evaporator until the liquid evaporated, resulting in oil extraction, which was stored at 4 °C in brown collection containers. To achieve a final concentration of 10 mg/mL (10,000 ppm), we started with 0.01 g of extract dissolved in 0.3 mL of DMSO. This creates a stock solution with a concentration of 33.3 mg/mL. To dilute the stock to the desired 10 mg/mL, add 0.7 mL of double distilled water to 0.3 mL of the 33.3 mg/mL stock solution. A control treatment was prepared by mixing the same solution, without added oil, with 0.3 mL of DMSO, 1% Tween-20, and distilled water to achieve a volume of 5 mL.

2.3. Identification of the Chemical Components of the Essential Oil

Gas chromatography-mass spectrometry (GC-MS) analysis was conducted on the obtained essential oil samples. The analysis was performed using a 50-GC/320-MS instrument (Varian, Inc., Walnut Creek, CA, USA) equipped with a flame ionization detector and an HP-5MS capillary column (film thickness: 30 m length, 0.25 mm inner diameter). For the gas chromatography phase, the injector oven temperature profile was as follows: initially held at 60 °C for 2.5 min, then ramped at a rate of 10 °C/min to 180 °C and maintained for 1 min, and finally ramped at a rate of 20 °C/min to 280 °C and held for 15 min. Sample injection was performed with one microliter of samples diluted in 1% hexane, using a split ratio 1:10. Helium gas was employed as the carrier gas at a flow rate of 1.0 mL/min, while the column pressure was maintained at 100 kPa. Chemical constituents present in the gas chromatogram were identified by comparing them with NIST 23 Gas Chromatography (GC) [66].

2.4. Sugar Roll Method

Before applying essential oils, the sugar roll method was used to quantify the prevalence of V. destructor infestation in 50 test colonies of A. mellifera [67,68]. For the Varroa mite collection, three brood frames from each colony ensured an accurate representation of the colony’s bee population. The procedure entailed the meticulous transfer of bees from brood comb into a clean white tray through the gentle application of a soft brush, a measure taken to avoid injuring the bees and prevent contamination, excluding the queen to avoid any harm. An approximate tally of 300 bees were transferred into this jar. A glass canning jar with a fine mesh screen lid (8 per inch) was utilized. This setup was crucial as it allowed mites to move through the mesh while ensuring the bees were securely contained within the jar. Subsequently, the procedure involved the addition of three tablespoons of powdered sugar through the mesh into the jar. The bees within were then gently agitated by rolling the jar for 2 to 3 min, ensuring an even coating of powdered sugar on all bees. This gentle agitation was pivotal for the mite-removal process from the bee bodies. The jar was left undisturbed for another 2 to 3 min, allowing the bees sufficient time to self-clean, thereby dislodging the mites and the sugar coating from their bodies. After the time for self-cleaning, the jar was carefully inverted and lightly rolled again, facilitating the collection of the powdered sugar, now containing the dislodged mites, onto a white paper chart. The white paper chart was then scanned for the presence of mites that were counted. The bees, now sampled and free of mites, were gently returned to their hive either at the top of the colony or the entrance, ensuring minimal disruption to their natural state. The sugar roll method was applied immediately before and after a 30-day treatment with essential oils. The final step involved the calculation of the mite infestation rate, which was achieved by dividing the total number of mites by the total number of bees sampled. The research established a standard economic threshold for action at a 2% mite presence per 100 bees, translating to a critical limit of 6 Varroa mites per 300 adult bees during periods of low population (dearth periods) and a heightened threshold of 9 Varroa mites per 300 bees during peak population seasons, typically observed from February to March [61]. The acaricidal potential of the oils was quantified using the following formula for percentage reduction. The reduction (%) in mites was counted per 300 bees by using the sugar roll method before the initial count and post-treatment (final count).

$$\mathrm{C}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{m}\mathrm{i}\mathrm{t}\mathrm{e} \mathrm{p}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{\%}=\frac{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}-\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}}\times 100$$

2.5. Essential Oils Treatment

The essential oils used for the experiment were hemp, sage, and laurel. We experimented with three levels of concentration: 5%, 10%, and 15% for the 3 × 3 factorial block design. The preparation of the treatment solutions involved meticulous dilution of sage, hemp, and laurel oils to concentrations of 5%, 10%, and 15% in sunflower oil, ensuring precision in the formulation for application consistency. In this study, we utilized untreated wooden strips measuring 2 cm in width, 0.5 cm in thickness, and 27 cm in length, tailored to fit within standard Langstroth beehive frames. The wooden strips were immersed in oil solutions for 24 h to ensure deep saturation before being strategically placed within the hives. Precisely, in each colony, three such strips were carefully positioned between the brood combs. These strips were routinely exchanged every three days for optimal control and effectiveness. The experimental framework included a total of 50 hives, categorized into four distinct groups: 45 of these hives were allocated into separate clusters, each receiving a unique concentration of essential oils, while the remaining 5 served as control groups, receiving no essential oil treatment. In our study, the effectiveness of essential oils was determined by counting the mites on approximately 300 bees from each colony before and after treatment.

2.6. Mite Collection

This investigation sought to elucidate the efficacy of evaporated essential oils in mitigating Varroa mite infestation levels within apian colonies. The screened bottom board technique was employed [69], incorporating a layer of petroleum jelly as a viscous trap beneath a modified bottom board precisely dimensioned to the specifications of the beehive. This innovative approach not only preserved the inherent behaviors of the bee populations but also maximized the efficiency of mite capture. The bottom board, tailored and balanced concerning the dimensions of the hive, was efficient in maintaining the required efficacy and was effective in trapping mites due to the uniform application of petroleum jelly. To systematically monitor the levels of mite infestation and evaluate the effectiveness of the applied mite treatments, collection trays, safeguarded by a wire mesh to deter the removal of bees, were strategically situated at the posterior section of the hives. These trays, serving the dual objective of monitoring and evaluation, enabled the quantification of mite infestation levels at predetermined intervals of 10, 20, and 30 days post-treatment, providing a comprehensive assessment of the treatment efficacy over time [63,70,71].

2.7. Quantification of Honey Production

Following the experimental phase’s conclusion post-treatment and Varroa assessment through the sugar shake method, the honey extraction procedure was initiated utilizing a manually operated honey harvester. The objective was to ascertain the honey yield from the treated colonies, which was achieved by assessing the weight of each hive body designated for honey collection before and after the extraction process. The variance in weight was then quantified as the volume of honey eligible for harvest. This methodological approach ensured a precise measurement of the honey yield, contributing to the comprehensive analysis of the experiment’s outcomes [63,72]. The total number of fallen mites was counted using three concentrations: 5%, 10%, and 15% of hemp, sage, and laurel oils and the control group. The experimental colonies (n-50) were divided into three treatment groups of colonies (n-15), each along with a control group of colonies (n-5). The time and dose-dependent effect on the number of fallen mites after the treatment with hemp, sage, and laurel oils at 5%, 10%, and 15% concentrations was evaluated over intervals of 10 days, 20 days, and 30 days.

2.8. Statistical Analyses

We used ANOVA analysis for comparative efficiency across the different treatment groups (hemp oil, sage oil, laurel oil, control), Levene’s test to perform the equality of variances, and Tukey’s HSD test to find out which treatment groups had significantly different effects on reducing mite populations. A two-factor ANOVA to evaluate the impacts of each treatment of hemp, sage, and laurel oils (3 groups) and their quantities/concentrations of 5%, 10%, and 15% (9 treatments) on Varroa mites was conducted using Graphpad prism Version 9 [73]. The heat maps were formed to visualize the number of fallen mites after 10, 20, and 30 days. In the case of honey yield, ANOVA and Tukey’s HSD test were performed to evaluate the impact of different essentials and their concentrations on honey yield.

3. Results

3.1. Treatment Efficacy among Essential Oils

The study found that the efficacy of the three major treatments of sage F(6) = 11.68, p < 0.001), hemp F(6) = 12.79, p < 0.001), and laurel essential oils F(6) = 10.59, p < 0.01) to the control group significantly differed, as depicted in Figure 2. Hemp oil had the highest efficiency (81.89%), followed by sage and laurel oils at 66.95% and 52.40%. The control group showed an 88.65% increase in the mite population, indicating significant mite proliferation without treatment. Mite counts were based immediately before the first application of the essential oils, and the post-treatment counts were carried out after 30 days post-treatment, which commenced with the sugar shake method.

Tukey’s HSD test revealed significant differences in mite population reduction between treatments with hemp oil, laurel oil, and sage oil to the control group (p > 0.001). Additionally, comparing the essential oils themselves, hemp oil was more effective than both laurel and sage oils, while no significant difference in efficacy was observed between laurel and sage oils (p = 0.391).

3.2. Concentration Effectiveness

The quantitative analysis delineates a discernible increment in the efficacy of hemp oil as its concentration is augmented from 5% to 15%, as illustrated in Figure 3A. In a parallel vein, sage oil displays a similar trajectory of efficiency enhancement in alignment with concentration increments. However, it is imperative to note that the foundational efficiency of sage oil at a 5% concentration markedly trails behind that of hemp oil, indicating a variance in intrinsic efficacy as shown in Figure 3B. By the time the concentration reaches 15%, as shown in Figure 3C, laurel oil’s efficiency aligns closely with sage oil’s, suggesting a convergence of efficacy at higher concentrations.

Results for the colonies treated with hemp oil at 15%, 10%, and 5% were 95.4% ± 0.30, 85.71% ± 0.85, and 64.48% ± 0.26, (95.49% ± 2.5%) respectively; for sage oil, they were 81.08% ± 0.57%, 69.42% ± 1.72%, and 50.35% ± 0.70%; and for laurel oil, they were 68.96% ± 0.34%, 54.66% ± 0.37%, and 33.58% ± 0.30%, respectively. The hemp oil showed higher efficacy at 15% and lowest efficacy with laurel oil at 5%. Hemp oil showed a significant difference with F(2, 12) = 66.78, p < 0.0001, from concentrations of 5% to 15%, followed by sage oil with F(2, 12) = 190.2, p < 0.0001 and laurel oil with F(2, 12) = 326.8, p < 0.00013.

3.3. Number of Fallen Mites

The level of effectiveness of different essential oils (hemp, sage, and laurel) after 10, 20, and 30 days of treatment regarding the number of fallen mites compared to the control through the application of heatmaps for enhanced visual interpretation is depicted in Figure 4A. The acaricidal efficiency of essential oils derived from hemp, sage, and laurel was evaluated using concentrations of 5%, 10%, and 15% over intervals of 10, 20, and 30 days as shown in Figure 4B–D. The results demonstrated notable differences in the rates of mite fall, which were dependent on the essential oil used, its concentration, and the length of exposure. Among the oils tested, hemp oil exhibited superior effectiveness across all tested concentrations and durations, particularly at lower concentrations and for brief exposure periods.

3.4. Honey Yield

Hemp oil reflected a higher yield than sage and laurel, and when compared to the control group, it shows that hemp oil has a significantly strong positive correlation with honey production; comparison was made between essential oils, F (3, 6) = 28.68, p = 0.0006, as illustrated in Figure 5A.

The highest average honey yield was recorded in treated honey bee colonies with hemp oil at different concentrations (5%, 10%, and 15%) at 11.778 kg, 13.26 kg, and 16.98 kg, respectively, as shown in Figure 5B. The lowest average honey yield was recorded and treated with laurel oil (5%, 10%, and 15%) at 5.64 kg, 6.6 kg, and 8.06 kg, respectively, as shown in Figure 5B. The results of Tukey’s multiple comparisons tests were as follows: for 5% hemp vs. sage, F(12) = 81.47, p < 0.0001; for hemp vs. laurel, F(12) = 83.93, p < 0.0001; and for sage vs. laurel, F(12) = 2.461, p = 0.2308: for 10% hemp vs. sage, F(8) = 30.75, p < 0.0001; for hemp vs. laurel, F(8) = 50.68, p < 0.0001; and for sage vs. laurel, F(8) = 19.94, p < 0.0001. Lastly, in the case of 15% concentration, the results showed hemp vs. sage, F(8) = 62.27, p < 0.0001; hemp vs. laurel, F(8) = 87.89, p < 0.0001; and sage vs. laurel, F(8) = 25.62, p < 0.0001.

3.5. GC-MS Analysis

The components of hemp oil, sage oil, and laurel oil were analyzed using GC-MS analysis to study their acaricidal potential against Varroa mites. The components and their percentages in the essential oils are illustrated in Figure 6, Figure 7 and Figure 8.

In the GC-MS analysis of S. officinalis, three compounds stood out as the most abundant constituents: trans-b-caryophyllene (8.56%), α-pinene (8.14%), and viridiflorol (7.34%). These compounds represent the major components contributing significantly to the chemical profile of S. officinalis. On the other end of the spectrum, the three compounds with the lowest percentages were p-Cymen-8-ol (0.01%), (+)-trans-caryophyllene (0.05%), and isobutyl acetate (0.04%). While present in smaller quantities, these compounds add to the overall diversity of chemical constituents identified in S. officinalis, as depicted in Figure 6.

In the chemical analysis of C. sativa by GC-MS, the three compounds with the highest percentages were myrcene (23.45%), limonene (16.24%), and β-caryophyllene (16.64%). These constituents represent the major components contributing significantly to the volatile profile of C. sativa. Conversely, the three compounds with the lowest percentages were camphene (0.5%), (E)-β-ocimene epoxide (0.1%), and linalool (0.3%). While present in smaller quantities, these compounds still contribute to the overall chemical diversity of C. sativa, as shown in Figure 7.

In the GC-MS analysis of L. nobilis essential oil, phytol, β-myrcene, and n-heneicosane emerged as the compounds with the highest area percentages, at 10.65%, 10.12% and 4.65%, respectively. Conversely, the compounds with the lowest detected area percentages were eugenol, thymol, and β-caryophyllene, each constituting less than 0.3% of the oil’s composition, with exact values of 0.24%, 0.26%, and 0.26%, respectively, as illustrated in Figure 8. These findings highlight the dominant and trace components contributing to the oil’s unique chemical profile.

4. Discussion

Hemp plays a promising role in integrated pest management thanks to its widespread availability, affordability, and eco-friendliness. In 2018, Benelli and colleagues demonstrated the environmental safety of C. sativa essential oil residues, specifically their non-toxicity to non-target organisms [47]. Past research has validated the effectiveness of C. sativa against mites. Its traditional use in Pakistan for treating livestock ticks with its leaves underscores its acaricidal potential [74]. C. sativa L. is a useful and environmentally benign plant distinguished for its natural resilience against parasites and pests. The formation of bioactive secondary metabolites, such as terpenes and cannabinoids, ensures the presence of this feature. Hemp essential oil (EO), known for its terpenoid compounds with pesticide properties, was evaluated for its efficacy against mites Dermanyssus gallinae and ticks Hyalomma dromedarii [75]. Our analysis revealed that C. sativa (EO) demonstrated acaricidal activity, presenting a potential treatment method against the mite. Further, the study expanded to compare the effectiveness of various essential oils under winter conditions, including hemp, sage, and laurel, with hemp EO showing notable efficacy. These findings contribute to the growing body of evidence supporting essential oils as viable alternatives to chemical pesticides in managing mite infestations in beekeeping.

Essential oil derived from S. officinalis’ aerial parts is rich in over 120 identified components, including borneol, camphor, caryophyllene, cineole, elemene, humulene, ledene, pinene, and thujone [57,76,77]. Significantly, this oil impacts the nervous system [78], with compounds like camphor, thujone, and terpene ketones noted for their potent toxicity. Research by Veličković et al. [79] highlights bornyl acetate, camphene, camphor, humulene, limonene, and thujone among the most prevalent phytochemicals in the leaves. However, environmental factors such as climate, water availability, and altitude influence S. officinalis’s chemical composition, echoing the variability observed in other herbs [80]. In our study on treating bee mites, we found that the essential oil of sage (S. officinalis L.) is effective against the V. destructor mite, a common parasite in honey bees (A. mellifera L.). The effectiveness of the treatment changes depending on how much oil is used and how long the mites are exposed to it. Specifically, we discovered that using a 5% oil concentration led to a 50.35% success rate, while a 15% concentration increased the success rate to 81.07%. These findings are significantly better than those of previous studies, like Bendifallah, Leila, et al., who reported a 21.07% effectiveness [54], and Ghomari et al. [81], who saw a 48.7% success rate; our results even surpassed the effectiveness of Apivar. This commonly used mite treatment has a 3.13% success rate and was much more effective than the disappointing results seen with Thymus vulgaris oil, as noted by Giovenazzol et al. [82]. Additionally, Moussaoui et al. [83] have indicated that the eucalyptus bioproduct shows toxicity at an early stage. It is important to note that the chemical makeup of essential oils, and thus their biological activity, can vary based on the part of the plant used, the extraction method, and where the plant was grown [84].

Recent studies on the composition of laurel oil revealed a mix of key components like 1,8-cineole, α-terpinyl acetate, sabinene, linalool, and methyl-eugenol [85]. Linalool, in particular, stands out due to its abundance in L. nobilis, L. officinalis, and L. hybrida oils. Field research shows that its effectiveness varies significantly between bee colonies [86]. Moreover, laboratory tests have indicated that high doses of linalool can negatively affect brood survival and mite reproduction [87]. Our findings have shown that, at a 15% concentration, laurel oil is notably effective against Varroa mites, with a success rate of approximately 69%. This was in line with studies by Al-Ghzawi and his team in 2008, which tested the impact of various essential oils on Varroa mites. They found L. nobilis oil to be particularly potent [88]. Likewise, research by El-Zemity and colleagues in 2006 discovered similar efficacy with other oils. For example, T. vulgaris oil was capable of eliminating 50% of mites with the lowest dosage after just 24 h. However, the most effective results over longer exposures, 48 h and 72 h, were observed with L. officinalis oil, which showed the best balance of effectiveness and safety for bees [89]. In 1999, Colin et al. posited a hypothesis regarding long-term repellency’s impact on female mites’ fecundity. They suggested that minimal concentrations of specific natural compounds, notably monoterpenes and phenolic compounds, could significantly diminish mite fecundity, thereby mitigating the risk of severe parasitism within bee colonies [90]. Further investigations into the composition of essential oils and their acaricidal effects underscored the significance of 1,8-cineole, a major constituent of L. nobilis essential oil. Imdorf et al. (2006) and Ruffinengo et al. (2007) corroborated the high mortality rates among mites attributed to 1,8-cineole exposure [91,92]. Lee et al. (2008) expanded on these findings, associating the efficacy of various essential oils against pests with their molecular structures and emphasizing the role of functional groups over properties like hydrophobicity or vapor pressure in determining toxicity levels [93]. The comprehensive analysis by Calderone and Spivak (1995) illuminated the multitude of factors influencing the effectiveness of acaricide treatments. They delineated how the compound’s concentration, treatment duration, application methodology, intra-colony conditions, apiary environment, and ambient temperature collectively determine a treatment’s success [94]. The study conducted in 2014 showed that trans-β-caryophyllene showed acaricidal activity in two different species of dust mite [95]. The research conducted by Sarri in 2023 showed that essential oil was found to be rich in terpinolene, myristicin, myrcene, limonene, γ-terpinene, and (Z)-caryophyllene, which have acaricidal effects and anti-inflammatory potential [96]. α-pinene, the active component of essential oils of Trachyspermum ammi and Thymus schimperi, exerted toxicity against Varroa mites, as evidenced by antennectomized mites showing signs of restlessness and then moving very fast shortly before death [97]. Ghasemi et al. (2011, 2016) described a large research project in Iran to investigate the potential of essential oils from the medicinal plant flora of Iran to control the Varroa mite. To determine the oil yield percentage, in vitro mites were screened on seeds from many species from different plant families for the safety of the honey bee. The test results revealed a few oils, including Thymus kotschyanus Bioss & Hohen, (47.99% carvacrol, 30.61% thymol), Mentha longifolia L. (36.86% piperitenone, 27.53% piperitenone oxide, 22.21% cis-piperitone epoxide, 8.38% pulegone), and Eucalyptus camaldulensis Dehnh. However, in laboratory studies, these essential oils (74.7% 1,8-cineol and 8.35% α-pinene) effectively killed Varroa mites without causing high mortalities of honey bees [98,99].

Addressing the challenge of controlling Varroa mite populations in honey bee colonies necessitates treatments that exhibit potent acaricidal activity without adversely affecting the bees. Moreover, these treatments should ensure minimal residual presence in honey and wax to safeguard consumer health. Adamczyk et al. (2005) concluded that residual components of essential oils in honey do not pose a health risk, albeit potentially altering its flavor [100]. Isman (2000) attributed the selective efficacy of essential oils as insecticides or acaricides to the absence of octopamine receptors in mammals, a feature exploited by these natural compounds [101]. This body of research collectively underscores the nuanced relationship between acaricide composition, application, and environmental factors and their collective impact on bee colony health. It also highlights the potential of essential oils as a mite control strategy, balancing efficacy with safety for both bees and consumers. But there is much we still need to learn. Critical areas for future studies include figuring out how these oils affect mite numbers and bee health over the long haul, their effectiveness when breeding bees, and how different weather conditions might change things. We also need to see how these oils impact bee behavior and how the colony gets along. Plus, it is essential to know how these treatments can fit into the bigger picture of pest management and whether they are cost-effective for beekeepers worldwide. Diving deeper into these questions will help create better, more sustainable mite control methods that beekeepers can rely on in the future.

5. Conclusions

Our study investigates the acaricidal activity of essential oils extracted from S. officinalis, C. sativa, and L. nobilis on V. destructor mites, a major threat to honey bee colonies. The most effective oil tested was hemp oil, showing rates of effectiveness of 95.4%, 85.71%, and 64.48% at concentrations of 15%, 10%, and 5%, respectively. Sage oil was effective to some extent, while laurel oil was the least effective. Essential oils are a promising and sustainable primary option for controlling V. destructor mites. Major compounds identified in the EOs responsible for their acaricidal properties were trans-caryophyllene, α-pinene, and viridiflorol in hemp oil; myrcene, limonene, and β-caryophyllene in sage oil; and phytol, β-myrcene, and n-heneicosane in laurel oil. Long-term research under field conditions is necessary to confirm the results, with a focus on the effects of these EOs on honey production and colony health. The integrated pest management strategy employing these EOs will allow beekeepers to control their colonies and avoid damage by V. destructor sustainably.