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Article

Azoxystrobin and Picoxystrobin Lead to Decreased Fitness of Honey Bee Drones (Apis mellifera ligustica)

by
Wenlong Tong
1,†,
Lizhu Wang
1,†,
Bingfang Tao
1,
Huanjing Yao
1,
Huiping Liu
2,3,
Shaokang Huang
1,4,
Jianghong Li
1,4,
Xiaolan Xu
1,4 and
Xinle Duan
1,2,4,*
1
College of Bee Science and Biomedicine, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
State Key Laboratory of Agricultural and Forestry Biosecurity, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China
4
Fujian Honey Bee Biology Observation Station, Ministry of Agriculture and Rural Affairs, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(15), 1590; https://doi.org/10.3390/agriculture15151590
Submission received: 19 June 2025 / Revised: 13 July 2025 / Accepted: 22 July 2025 / Published: 24 July 2025
(This article belongs to the Special Issue Honey Bees and Wild Pollinators in Agricultural Ecosystems)
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Abstract

Honey bees (Apis mellifera ligustica) are essential pollinators in both ecosystems and agricultural production. However, their populations are declining due to various factors, including pesticide exposure. Despite their importance, the reproductive castes, particularly drones, remain understudied in terms of pesticide effects. To investigate the effects of azoxystrobin and picoxystrobin on honey bee drones, the drones were exposed to different concentrations of azoxystrobin and picoxystrobin for 14 days; the drone survival, body weight, nutrient content, reproductive organs, and sperm concentration were assessed. Results showed that exposure to both fungicides caused a significant reduction in drone survival rates, with survival rates decreasing progressively as the duration of exposure increased. Compared to the control group, the body weights of drones in all treatment groups were significantly lower on days 7 and 14. Nutrient analysis revealed that low concentrations of azoxystrobin and picoxystrobin increased protein levels, while free fatty acid content decreased significantly in all treatment groups. No significant changes were observed in the total carbohydrate content. Morphological examination of reproductive organs showed that the lengths of the mucus glands and seminal vesicles in drones were significantly shorter in the treatment groups compared to the control group. Furthermore, exposure to azoxystrobin and picoxystrobin resulted in a significant decline in sperm concentration in the drones. These findings indicate that azoxystrobin and picoxystrobin have adverse effects on the health and reproductive capacity of honey bee drones. The present study highlights the need to reassess the risks posed by these fungicides to pollinators, particularly given the critical role of drones in maintaining the genetic diversity and resilience of honey bee colonies. Further research is warranted to elucidate the underlying mechanisms of these effects and explore potential mitigation strategies.

1. Introduction

Honey bees play a pivotal role in both ecosystems and agricultural production, functioning as crucial pollinators for global ecological security and agricultural yield [1,2]. Statistics show that nearly 90% of the world's flowering plants are pollinated by animals, and bees account for 30–50% of agroecosystem services [3,4]. Besides pollination, bees are also useful for research in different areas such as learning, memory, behavior, and sex determination [5]. Additionally, bees are often used as an indicator of environmental conditions. Habitat alteration, climate change, invasive species, and environmental pollution are key factors in the decline of wild bee species and the increased mortality of domesticated ones [6,7,8,9,10].
As a typical eusocial insect, honey bees (Apis mellifera ligustica) exhibit distinct caste differentiation, which is fundamental to their reproductive success and division of labor [11]. Each healthy colony comprises three morphologically and functionally distinct castes: the queen, the workers, and the drones [12]. Although drones are only present in the colony for a few months each year, they play a critical role in colony fitness by transferring genetic material to subsequent generations during mating flights with virgin queens [13]. Therefore, the genetic quality and productivity of a colony is largely determined not only by the reproductive capacity of the queen, but also by the genetic contributions of drones from the maternal colony [14]. Meanwhile, subhealthy drones result in queens that are poorly inseminated, and these queens are more likely to be eliminated or superseded, which is partly of the reason for the increase in colony mortality rates [15,16].
Even though drones have a brief life cycle, their reproductive quality and sexual competitiveness, including aspects like semen volume, semen quality, and sperm motility, are influenced by a range of biotic factors including age, haplotype, genetic background, and parasites, as well as environmental factors such as temperature, season, and pesticides [13]. Age affects the viability of drone sperm; the sperm of the drone showed a gradual decline with increasing age [17]. The sperm viability in older than 20-day-old drones was 50% lower than those that survived less than 20 days [18]. The semen volume of drones decreases from 15 days post-emergence, and the increased viscosity of drone semen from drones older than 21 days old leads to blockages of the queen bee's oviduct [19]. Drones are sensitive to temperature fluctuations, and once the temperature deviates from their optimal developmental temperature (33–35 °C), both their growth and development, as well as their reproductive quality, are adversely affected [20]. High temperatures not only affect the size of drones' testes, seminal vesicles, and mucous glands but also significantly reduce semen volume as well as sperm quantity and motility [18,19,21]. Nutrient deficiencies, particularly protein deficiency, delay sexual maturation in drones [22], while simultaneously causing reductions in body weight, semen volume, ejaculation capacity, sperm volume, and motility, ultimately diminishing their reproductive potential [23].
Numerous studies have demonstrated that pesticide exposure constitutes a major driver of honey bee (A. mellifera ligustica) population decline and colony mortality [8,10,24,25,26]. However, current research has predominantly focused on the effects on non-reproductive worker bees, while largely overlooking the critical reproductive castes: queens and drones [7]. Unlike worker bees that frequently encounter direct pesticide exposure during foraging activities, queens and drones experience primarily indirect exposure through the consumption of pesticide-contaminated food resources within the colony [27]. Compared to diploid queens and workers, haploid drones are more sensitive to cold, imidacloprid exposure, and other external stress [28,29]. Neonicotinoid pesticide exposure results in increased forewing vein asymmetry and wing morphology fluctuation in honey bee drones [14]. Imidacloprid and thiamethoxam exert substantial adverse effects on the survival and reproductive function of drones [30,31]. Thiamethoxam exposure (4.5 ppb) notably reduces the lifespan of drones and results in a 39% decline in the number of living sperm [30]. Imidacloprid at 0.02 ppm significantly decreases the sperm motility of drones, although this value significantly varies among different colonies [31]. Fipronil (0.1 mg/L) decreases the fertility in the drones, and the sperm concentration and motility are also significantly decreased [32]. Glyphosate exposure (0.31 mg/mL) causes sperm death, with dead spermatozoa counts showing significant positive correlations with both exposure duration and herbicide concentration [33]. Chlorothalonil, along with its combinatorial effect with chlorpyrifos, impairs drone reproductive quality and sperm viability [34].
Azoxystrobin and picoxystrobin are derived from the natural fungicidal derivatives of β-methoxyacrylic acid and exert their antifungal activity by inhibiting the cytochrome bc1 complex (Complex III) in fungal mitochondria. This inhibition disrupts electron transport, impairing energy production and ultimately leading to pathogen suppression [35]. Owing to this unique mode of action, these strobilurin fungicides have been extensively adopted in modern agriculture for the prophylactic and therapeutic management of phytopathogenic fungi across diverse crops [36], and exhibit ecotoxicological effects on non-target species [37,38,39]. Azoxystrobin exposure induces oxidative stress, lipid peroxidation, and DNA damage in earthworms (Eisenia fetida) [37]. Picoxystrobin inhibits the community and quantity of earthworms, with a notable decrease in the number of juvenile earthworms [38]. Exposure to azoxystrobin and picoxystrobin can cause significant, time- and dose-dependent effects on the mortality, hatching, and teratogenic rates of eggs, and induce significant oxidative stress in adult zebrafish (Danio rerio) [39]. Azoxystrobin and picoxystrobin cause morphological alterations and cytotoxic changes in the midgut, and this may affect the nutrient absorption functions of this organ in the long term [24,40]. Meanwhile, the field-recommended concentrations of azoxystrobin and picoxystrobin have significant toxicity to worker bees of different ages, causing negative impacts on their survival, enzyme activities, nutritional metabolism, and immune ability [10,41].
Drones are vital for the genetic diversity and resilience of honey bee colonies, and their health directly impacts mating success, genetic flow, overall colony vitality, and disease resistance [13,14]. Despite their critical role, drones remain understudied in ecotoxicological research, particularly regarding the sublethal effects of widely used strobilurin fungicides such as azoxystrobin and picoxystrobin. This study aims to fill the gaps in the understanding of the potential adverse effects of azoxystrobin and picoxystrobin on drones. The impact of exposure to these two fungicides on drones’ (A. mellifera) survival, body weight, nutrient content (protein, free fatty acids, and carbohydrates), reproductive organs (mucus glands and seminal vesicles), and sperm concentration was investigated. These findings provide novel insights into the impact of azoxystrobin and picoxystrobin on drone vitality and highlight the need to reassess the agrochemical risks to pollinators.

2. Materials and Methods

2.1. Fungicides and Reagents

Azoxystrobin (25% suspension) was obtained from Syngenta Crop Protection Co., Ltd. (Nantong, China). Picoxystrobin (22.5% suspension) was purchased from Du Pont China Holding Co., Ltd. (Shanghai, China). The protein (Cat# KMSP-2-W) and carbohydrate (Cat# ZT-1-Y) assay kits were provided by Comin Biotechnology Co., Ltd. (Suzhou, China), while the free fatty acid kit (Cat# G0901W) was supplied by Grace Biotechnology Co., Ltd. (Suzhou, China).

2.2. Maintenance of Drones

A total of five queen-right and healthy honey bee (A. mellifera ligustica.) colonies from the apiary of the College of Bee Science and Biomedicine at Fujian Agriculture and Forestry University (Fuzhou, China) were used in this experiment. These colonies were maintained according to standard beekeeping practices to keep them free from clinical symptoms of adult bee or brood diseases and parasites, and they were not exposed to pesticides or other agrochemicals during the experiment [4]. During the spring breeding season for honey bees in 2024, these healthy egg-laying queens were confined to a defined area on the drone comb with empty cells for laying eggs for 24 h. Then, the combs with drone eggs were isolated to prevent further oviposition by the queen until natural capping was completed. Drone pupae nearing emergence (approximately 23 days post-oviposition) were transferred to artificial incubators (Saifu Experimental Instrument Co., Ltd., Ningbo, China) 24 h before eclosion (34.5 ± 0.5 °C, relative humidity 60 ± 10%, darkness). The newly emerged drones were randomly assigned to different cuboid cages (14 × 9 × 8 cm), and each cage contained 20 adult drones and 40 worker bees, which could provide caretaking duties for the drones [27,42]. To facilitate subsequent fungicide exposure experiments, all these bees in cages were reared with 50% sucrose solution (w/v) and enough fresh pollen in the incubator to enable autonomous drone feeding and to promote the development and maturation of male reproductive organs.

2.3. Fungicide Exposure

Based on the recommended application concentrations in the product instructions, the azoxystrobin and picoxystrobin were diluted 2000- and 3000-fold with sucrose solution (50%), respectively, and the prepared fungicide-containing sucrose solution was stored at 4 °C and used within 2 days. The concentrations of the two fungicides were as follows: azoxystrobin 125 mg/L (AZ-H), azoxystrobin 83 mg/L (AZ-L), picoxystrobin 113 mg/L (PI-H), picoxystrobin 75 mg/L (PI-L), and the control group (CK). Before the fungicide exposure experiment, the aforementioned drones were starved for 8 h, and each concentration treatment group included six cages (replicates), with a total of 120 drones per group. Drones in the CK group were fed with sucrose solution without fungicide, and those in different exposure groups received fungicide-contaminated sucrose solution. These drones were provided with ad libitum access to food resources, and the fresh sucrose solution was replenished daily to ensure consistency of dosage. Considering that the developmental period from germination to sexual maturity of the drones was approximately 14 days, the exposure of drones to azoxystrobin and picoxystrobin was also maintained for 14 days. These drones were checked daily, and dead drones were removed and counted to determine survival statistics. On the seventh and fourteenth days of fungicide exposure, ten drones were randomly selected from each treatment group, and weighed individually to determine their average body weight.

2.4. Detection of Nutrients in Drones

The protein, carbohydrate, and free fatty acid contents in drones exposed to azoxystrobin and picoxystrobin for 14 days were quantitatively analyzed using commercially available assay kits. For each treatment group, five living drones from each treatment group were individually mixed with the specific buffer provided in the kit, followed by rapid homogenization and centrifugation at 8000 rpm for 10 min at 4 °C. The clear supernatant was then mixed with the appropriate buffer from the kit and homogenized again. The resulting mixture was combined with the specific assay reagents provided in the kit and transferred to a 96-well microtiter plate for spectrophotometric analysis. Absorbance was measured at specific wavelengths (620 nm for proteins, 540 nm for carbohydrates, and 715 nm for free fatty acids) using a Thermo Scientific Varioskan™ LUX (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was analyzed in triplicate to ensure consistency of results. The content of different nutrients in the drones was calculated according to the instructions of the assay kits.

2.5. Length of Mucus Glands and Seminal Vesicles

Following 14 days of fungicide exposure, five surviving drones from each treatment group were randomly selected and immobilized on wax plates for dissection. Their mucus glands and seminal vesicles were meticulously excised from the abdomen by using a dissecting microscope and transferred into a 1.5 mL tube containing 600 μL Kiev+ buffer [7]. For the measurement process, the excised mucus glands and seminal vesicles were placed on a clean glass slide and gently flattened using fine forceps to ensure they lay flat. The length measurements were conducted using the Leica Stereozoom S9i insect stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany), equipped with standardized illumination settings and an attached camera for image capture. The microscope was calibrated before each use to ensure accurate measurements. Images were captured at consistent magnification and lighting conditions. Each sample was measured three times to account for technical variation. After imaging, the seminal vesicles were carefully dissected from the mucus glands for subsequent sperm concentration analysis. The acquired images were then analyzed using ImageJ software v1.8.0 [43] to determine the lengths of the mucus glands and seminal vesicles. The average length of the seminal vesicles along the central axis and the average length of the mucus glands along the central axis were measured following the methods described by Metz and Tarpy [44].

2.6. Sperm Concentration Detection

Semen was collected from drones by manual eversion, and then the pooled semen from each drone was transferred to pre-labeled microcentrifuge tubes and diluted 100-fold in pre-cooled 100-fold Kiev+ buffer for subsequent sperm concentration detection, as described by Ciereszko et al. [31]. This process is summarized below: 10 μL of the diluted semen was further diluted in Kiev+ buffer to a series of predetermined multiples to ensure that the absorbance readings fell within the optimal range of the spectrophotometer. Then, 50 μL of each diluted suspension was added to a UV cuvette, and the samples were gently vortexed for three seconds to ensure homogeneity without causing damage to sperm cells. Absorbance was then measured at 600 nm using a Thermo Scientific Varioskan™ LUX (Thermo Fisher Scientific Inc., Waltham, MA, USA). In parallel, the spermatozoa in different diluted suspensions were counted using a hemocytometer under a Leica DM2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany). The average sperm concentration from the three replicates was recorded for each sample, and the calculation formula of sperm concentration was referred to Straub et al. [7]. According to the counting results and absorbance values, a regression equation of absorbance versus concentration was plotted. Five drones were randomly selected from each treatment group for the sperm concentration determination. The semen samples from each treatment group were diluted 2000-fold with Kiev+ buffer, and the absorbance of the diluted samples was measured at a wavelength of 600 nm. Finally, the measured absorbance values were substituted into the pre-established regression equation to calculate the sperm concentration.

2.7. Data Analysis

The survival of drones in each fungicide treatment and control group was analyzed using the Kaplan–Meier survival function in GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Data on body weight, nutrient content, the length of mucus glands and seminal vesicles, and sperm concentration were organized using Excel 2016. One-way ANOVA was performed via SPSS Statistics 17 (IBM, Armonk, NY, USA). Tukey’s HSD was used for multiple comparisons between groups, with significance marked at p < 0.05. All data are presented as mean ± standard deviation and were plotted using GraphPad Prism 8 for graphing.

3. Results

3.1. Effect of Azoxystrobin and Picoxystrobin Exposure on the Survival Rate of Drones

Exposure to azoxystrobin and picoxystrobin caused a reduction in drone survival, with survival rates decreasing progressively as the duration of fungicide treatment increased (Figure 1, Log-rank (Mantel-Cox) test, x2 = 36.12, df = 1, p < 0.0001). After 14 days of continuous exposure, the survival rates of drones in the AZ-L, AZ-H, PI-L, and PI-H treatment groups decreased to 77.5%, 51.67%, 62.50%, and 41.67%, respectively. All treatment groups except the AZ-L treatment group showed significantly lower survival compared to the control group (80.00%).

3.2. Effect of Azoxystrobin and Picoxystrobin Exposure on the Body Weight of Drones

Compared with the control group, the body weights of drones in all picoxystrobin and picoxystrobin treatment groups were significantly lower on days 7 and 14 (one-way ANOVA, for 7 d: F (4, 85) = 11.69, p < 0.0001; for 14 d: F (4, 85) = 7.569, p < 0.0001, Figure 2). On the 7th day of fungicide exposure, the body weights of drones in the AZ-L, AZ-H, PI-L, and PI-H treatment groups were 0.2207 g, 0.2167 g, 0.2127 g, and 0.2161 g, which were significantly lower than those of the control group (0.2476 g, for AZ-L group, p < 0.0001; for AZ-H group, p < 0.0001; for PI-L group, p < 0.0001; for PI-H group, p < 0.0001), but there was no significant difference between the fungicide treatment groups. On the 14th day of fungicide exposure, the body weights of drones in each treatment group were all significantly lower than those in the control group (0.2430 g, for AZ-L group, p = 0.763; for AZ-H group, p = 0.0028; for PI-L group, p = 0.0007; for PI-H group, p < 0.0001), with decreases by 8.18%, 12.39%, 13.83%, and 18.19%, respectively, and the drones in the PI-H treatment group had the lowest average weight of 0.1988 g.

3.3. Effect of Azoxystrobin and Picoxystrobin Exposure on the Nutrient Content in Drones

Exposure to azoxystrobin and picoxystrobin can affect the levels of proteins, free fatty acids, and carbohydrates in drones. However, the trends of the effects on these nutrients are different (Figure 3). Both azoxystrobin and picoxystrobin treatments induced alterations in protein content within drones (Figure 3A). In particular, the low concentration treatment groups (AZ-L and PI-L) exhibited a significant increase in protein levels compared to the control group (for AZ-L group, p = 0.0051; for PI-L group, p = 0.0238). However, no statistically significant differences were observed between the high concentration treatment groups (AZ-H and PI-H) and the control group (for AZ-H group, p = 0.9995; for PI-H group, p = 0.4679), suggesting a concentration-dependent response pattern in the regulation of protein metabolism. In contrast to the protein content alterations, the free fatty acid content in drones showed a significant decrease in both azoxystrobin and picoxystrobin exposure groups, with the free fatty acid content decreasing by 29.83%, 14.71%, 21.89%, and 22.39% in each treatment group, respectively (Figure 3B, for AZ-L group, p = 0.0001; for AZ-H group, p = 0.0051; for PI-L group, p = 0.0004; for PI-H group, p = 0.0005). Although exposure to azoxystrobin and picoxystrobin caused fluctuations in the total carbohydrate content in the drones, there were no statistically significant differences between the fungicide treatment and control groups (Figure 3C, for AZ-L group, p = 0.9950; for AZ-H group, p = 0.7909; for PI-L group, p = 0.9635; for PI-H group, p = 0.7586).

3.4. Effect of Azoxystrobin and Picoxystrobin Exposure on the Mucus Glands and Seminal Vesicles in Drones

Azoxystrobin and picoxystrobin exerted certain effects on the lengths of the mucus glands and seminal vesicles in drones (Figure 4). The lengths of the mucus glands in drones from all azoxystrobin and picoxystrobin treatment groups were shorter compared to those in the control group (Figure 4A). In particular, the mucus gland lengths in the AZ-H, PI-L, and PI-H treatment groups were significantly reduced, with decreases by 10.69%, 9.54%, and 18.21%, respectively (for AZ-H group, p = 0.0372; for PI-L group, p = 0.0056; for PI-H group, p = 0.0252). Similarly, the lengths of the seminal vesicles in male drones from all treatment groups exposed to different concentrations of azoxystrobin and picoxystrobin were shorter than those in the control group (Figure 4B). However, a statistically significant difference in seminal vesicle length was only observed in the PI-L treatment group compared to the control group (p = 0.0079).

3.5. Effect of Azoxystrobin and Picoxystrobin Exposure on Sperm Concentration in Drones

The regression curve equation depicting the relationship between sperm concentration and absorbance values is presented in Figure S1. There is a significant regression between sperm concentration and the absorbance at 600 nm, with a correlation coefficient determined to be 0.9953. Based on this regression curve equation, the sperm concentrations in the drones from each treatment group were calculated (Figure 5). The results reveal a decline in sperm concentration in drones exposed to two fungicides, particularly in AZ-L, PI-L, and PI-H treatment groups. The average sperm concentration of drones in these three treatment groups decreased by 26.34%, 25.4%, and 29.4%, which were significantly lower than those of the control group (for AZ-L group, p = 0.0009; for PI-L group, p = 0.0013; for PI-H group, p = 0.0008).

4. Discussion

Drones contribute half of the genetic diversity of a honey bee colony, and their fitness directly influences mating efficiency, population health, and the sustainability of the colony [13,14]. Our study demonstrates that exposure to azoxystrobin and picoxystrobin harms the health of honey bee drones. Both fungicides significantly reduced survival rates and caused progressive body weight loss over time. The fungicide treatments also induced complex alterations in nutrient metabolism, including protein increase at low concentrations and fatty acid decrease at all concentrations. Furthermore, exposure resulted in morphological changes to the reproductive organs and potential impairment of fertility, indicating adverse effects on the reproductive capacity of drones.
Azoxystrobin and picoxystrobin exposure significantly reduced drone survival rates, with survival rates decreasing progressively as the duration of fungicide treatment increased (Figure 1). These results are consistent with previous studies that have documented the detrimental effects of fungicides on bee survival [10,45]. Azoxystrobin is harmful to worker bees, negatively influencing their survival [41]. Similarly, exposure to picoxystrobin led to a chronic substantial reduction in the lifetime of Africanized workers [10]. Drones typically reach sexual maturity around 14 days after emerging from their cells, at which point they are capable of mating with the queen [13]. However, on day 14 of exposure to the fungicide, the average mortality rate was 35.42% for drones treated with azoxystrobin, while those treated with picoxystrobin showed a higher rate of 47.92%, both of which were higher than the control group. The progressive decrease in survival rates observed in our study underscores the cumulative and potentially chronic effects of azoxystrobin and picoxystrobin exposure on drones. This is particularly concerning given the critical role drones play in maintaining the genetic diversity and resilience of honey bee colonies [13,14]. Reduced drone survival rates could lead to smaller drone populations, which in turn may limit the genetic pool available for mating, potentially compromising colony health and adaptability [7,27,28].
Drone body weight serves as a critical indicator that directly determines both individual survival fitness and reproductive potential in honey bee colonies [23]. Empirical evidence demonstrate that body mass correlates positively with copulation duration, mating success, and overall reproductive output, which ultimately affects the quality of the progeny colony [13,14]. Drones exposed to azoxystrobin and picoxystrobin exhibited significantly lower body weight than the control group on the 7th and 14th day of fungicide exposure (Figure 2). These results are consistent with prior studies suggesting that both azoxystrobin and picoxystrobin can adversely affect bee development and body weight [10,41]. Mating success in drones is influenced by their physical condition and vitality, and drones with light body weights demonstrate compromised flight endurance and mating proficiency [23]. This physiological disadvantage manifests as a reduced capacity to maintain the prolonged aerial pursuit of virgin queens during mating flights, and diminished competitive viability with other normal drones in nuptial flight contests, ultimately decreasing their reproductive contributions to colony genetic diversity [7,13,23]; this disadvantage appears also in a short copulation duration with the queen that could delay the establishment of colonies [14,46].
Insect macronutrients (protein, free fatty acids, and carbohydrates) not only provide energy for growth, development, and metabolism [47], but also serve as pivotal regulators in xenobiotic detoxification, thereby enhancing organismal resilience to environmental stressors [48,49,50]. Insect proteins serve as structural elements for growth and development, enzymatic catalysts for metabolic processes, and key mediators of immune defense and nutrient storage during critical life stages [48,51]. Low concentrations of azoxystrobin and picoxystrobin (AZ-L and PI-L) significantly increased protein levels in insects. This indicates that exposure to low-concentration fungicides can trigger a rapid stress response in drones, which alters protein metabolism and affects the activity of related detoxification and protective enzymes [52,53]. These metabolic shifts demonstrate the capacity of drones to mobilize biochemical defenses against fungicide exposure, although prolonged stimulation may incur fitness costs as evidenced by reduced pupation rates and body weight [4]. Free fatty acids are important metabolites in insects, and are involved in oxidative energy supply, lipid synthesis, and signal transduction [54,55]. In this study, the free fatty acid content in drones was significantly decreased in all azoxystrobin and picoxystrobin treatment groups (Figure 3B). This decline indicates impaired lipid metabolism in the treated drones and is consistent with previous studies [56]. This could lead to drone malnutrition, which would negatively impact the development of the reproductive organs, the quality and vitality of the sperm, and, therefore, their reproductive capacity and behavior [57,58]. Carbohydrates in honey bees are often required as an energy source to sustain high-energy behaviors such as daily activities and flight, and to contribute to physiological homeostasis by supporting developmental processes, growth, and reproductive functions [59,60]. Under the stress of azoxystrobin and picoxystrobin, the carbohydrate content in drones showed fluctuations but no statistically significant differences. Perhaps, due to the complex metabolic regulation mechanisms in insects, these drones might adjust other metabolic pathways to compensate for changes in carbohydrate metabolism under azoxystrobin and picoxystrobin exposure [50,61]; further investigation is warranted to evaluate this hypothesis. In summary, these alterations in nutrient levels could affect drone survival, vitality, and reproductive capacity, as adequate nutrient reserves are essential for optimal sperm production and overall health.
The reproductive success of drones is fundamentally dependent on the effective transfer of semen to a queen during mating flights, wherein high-quality semen (comprising sperm and mucus) is ejaculated and stored in the queen's oviducts [62,63,64]. In honey bee drones, organs such as the testes, seminal vesicles, and mucus glands play a vital role in the formation of seminal fluid and sperm preservation [65,66]. The accessory gland secretions are the primary source of seminal fluid in insects, and are significant for sperm survival, transport, and storage [67,68]. In the present study, the length of the mucus glands in drones was significantly reduced following exposure to azoxystrobin and picoxystrobin (Figure 4A). A large number of proteins are secreted by the mucus glands during the sexual maturation of drones, which is important for maximizing sperm survival [64]. However, the reduction in the size of mucus glands may directly influence the amount of secretion protein, thereby exerting detrimental effects on sperm viability and motility [69,70]. The seminal vesicle, a principal tissue for sperm storage in drones, and its development could influence sperm vitality [71]. The length of the seminal vesicle has a significant positive correlation to sperm viability [72], and the short length of seminal vesicles in azoxystrobin- and picoxystrobin-exposed drones impairs sperm production and quality, potentially leading to reduced fertility.
The reproductive success of drones is fundamentally dependent on the effective transfer of semen to a queen during mating flights, wherein high-quality semen (comprising sperm and mucus) is ejaculated and stored in the queen's oviducts [62]. Meanwhile, exposure to azoxystrobin and picoxystrobin significantly decreased sperm concentrations in drones (Figure 5). A series of research studies reported similar phenomena and showed that many pesticides, such as oxalic acid, acetamiprid, deltamethrin, fipronil, and clothianidin, could reduce the sperm concentration of drones [32,73,74]. Reduced sperm concentration could directly impact drone fertility and the genetic health of subsequent generations. This is particularly concerning as the genetic diversity of honey bee colonies is crucial for their resilience to environmental stressors and diseases. Also, the quality of insemination is crucial for the survival of both the queen and the entire colony [75] as a low quality of semen leads to the insufficient fertilization of the queen bee. Poorly inseminated queens are easily expelled or replaced because they are less attractive to workers [7,15,16].

5. Conclusions

This study provides novel insights into the adverse effects of azoxystrobin and picoxystrobin on honey bee drones. The significant impact on survival, body weight, nutrient content, reproductive organs, and sperm concentration emphasizes the need to re-evaluate the risks posed by these fungicides to pollinators. Given the crucial function of drones in preserving the genetic diversity and resilience of honey bee colonies, these results have significant implications for beekeeping and agricultural practices. Future research should focus on elucidating the underlying mechanisms of these effects and exploring potential strategies to mitigate the impact of fungicides on pollinator health. Furthermore, long-term studies are required to evaluate the cumulative impact of fungicide exposure on bee populations and colony dynamics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15151590/s1, Figure S1. The regression between sperm concentration and absorbance at 600 nm.

Author Contributions

Conceptualization: X.D., X.X. and J.L.; formal analysis, L.W. and W.T.; funding acquisition: X.D., S.H. and J.L.; investigation, L.W. and W.T.; methodology, X.D., L.W., W.T. and S.H.; software, L.W., W.T. and B.T.; Writing—original draft preparation, X.D., W.T., L.W. and H.Y.; Writing—review and editing, X.D., H.L., J.L. and S.H.; visualization, X.X. and W.T.; validation, L.W. and W.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Visiting Scholar Research Program of State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops (KFXZ23007), the Science and Technology Innovation Special Fund Project of Fujian Agriculture and Forestry University (KFB23097, KFB23106), the Natural Science Foundation of Fujian Province of China (2022J01585), and the Special Foundation of Investigation on Basic Resources of Ministry of Science and Technology of China (2018FY100402).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We are grateful for the assistance of staff in Bee Health Group, Fujian Agriculture and Forestry University, Fujian, China.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Agriculture 15 01590 g001
Figure 1. Effect of azoxystrobin and picoxystrobin exposure on the survival rate of drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = 120 drones per treatment group.
Figure 1. Effect of azoxystrobin and picoxystrobin exposure on the survival rate of drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = 120 drones per treatment group.
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Figure 2. Effect of azoxystrobin and picoxystrobin exposure on the body weight of drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = 10 drones per treatment group.
Figure 2. Effect of azoxystrobin and picoxystrobin exposure on the body weight of drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = 10 drones per treatment group.
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Figure 3. Effect of azoxystrobin and picoxystrobin exposure on the nutrient content of drones. (A): protein, (B): free fatty acid, and (C): carbohydrate. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
Figure 3. Effect of azoxystrobin and picoxystrobin exposure on the nutrient content of drones. (A): protein, (B): free fatty acid, and (C): carbohydrate. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
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Figure 4. Effect of azoxystrobin and picoxystrobin exposure on the length of mucus glands and seminal vesicles in drones. (A): mucus glands, and (B): seminal vesicles. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
Figure 4. Effect of azoxystrobin and picoxystrobin exposure on the length of mucus glands and seminal vesicles in drones. (A): mucus glands, and (B): seminal vesicles. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
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Figure 5. Effect of azoxystrobin and picoxystrobin exposure on the sperm concentration in drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
Figure 5. Effect of azoxystrobin and picoxystrobin exposure on the sperm concentration in drones. Data in the figure are mean ± standard deviation. Different letters indicate significant differences between the different exposure treatment groups (p < 0.05, Tukey’s HSD test). Treatments included a control (CK), azoxystrobin at 125 mg/L (AZ-H), azoxystrobin at 83 mg/L (AZ-L), picoxystrobin at 113 mg/L (PI-H), and picoxystrobin at 75 mg/L (PI-L), with N = five drones per treatment group.
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Tong, W.; Wang, L.; Tao, B.; Yao, H.; Liu, H.; Huang, S.; Li, J.; Xu, X.; Duan, X. Azoxystrobin and Picoxystrobin Lead to Decreased Fitness of Honey Bee Drones (Apis mellifera ligustica). Agriculture 2025, 15, 1590. https://doi.org/10.3390/agriculture15151590

AMA Style

Tong W, Wang L, Tao B, Yao H, Liu H, Huang S, Li J, Xu X, Duan X. Azoxystrobin and Picoxystrobin Lead to Decreased Fitness of Honey Bee Drones (Apis mellifera ligustica). Agriculture. 2025; 15(15):1590. https://doi.org/10.3390/agriculture15151590

Chicago/Turabian Style

Tong, Wenlong, Lizhu Wang, Bingfang Tao, Huanjing Yao, Huiping Liu, Shaokang Huang, Jianghong Li, Xiaolan Xu, and Xinle Duan. 2025. "Azoxystrobin and Picoxystrobin Lead to Decreased Fitness of Honey Bee Drones (Apis mellifera ligustica)" Agriculture 15, no. 15: 1590. https://doi.org/10.3390/agriculture15151590

APA Style

Tong, W., Wang, L., Tao, B., Yao, H., Liu, H., Huang, S., Li, J., Xu, X., & Duan, X. (2025). Azoxystrobin and Picoxystrobin Lead to Decreased Fitness of Honey Bee Drones (Apis mellifera ligustica). Agriculture, 15(15), 1590. https://doi.org/10.3390/agriculture15151590

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