Tobacco is a self-pollinating crop; however, several studies have shown that insect pollination also contributes to its reproduction [1], [2]. Moreover, bee pollination can improve seed quality in tobacco, and the pollen and nectar of tobacco provide bees with abundant food sources [3], [4]. Therefore, bees and tobacco can form a mutually beneficial ecological relationship. During the flowering period, pesticides are frequently used to control different types of pests and diseases, which may pose a serious threat to bees foraging on tobacco pollen and nectar [5], [6]. However, current ecotoxicological research and chemical risk assessments primarily focus on individual pesticides [7], often overlooking the potential interactions and cumulative effects that arise from co-exposure to multiple chemicals [8], [9]. These pesticide combinations may produce toxicological profiles distinct from those of individual pesticides [10]. Therefore, assessing the toxicity of pesticide mixtures under real-world scenarios is crucial [11], [12].

Honeybees (Apis mellifera L.) are essential for pollinating approximately 35% of global crops, making them crucial for global agriculture [13]. Pollination by bees can not only increase tobacco yield, but also ensure the excellent quality of tobacco seeds [2]. However, honeybee populations are experiencing significant declines in many countries, posing a serious threat to crop production [14], [15]. This decline is attributed to multiple factors, including pathogens, habitat loss, and pesticide exposure [16], [17]. Among these stressors, the impact of pesticides on honeybees has become a focal point [12], [18]. Honeybees are exposed to pesticide residues through multiple routes, such as consuming contaminated nectar and pollen [19] or contacting with treated soil and plant surfaces [20]. These exposures can severely affect honeybee health, as evidenced by increased mortality rates and oxidative stress following 16 days of exposure to pesticides like imidacloprid, glyphosate, and difenoconazole [21]. Furthermore, pesticide exposure disrupts the composition of honeybee gut microbiota, leading to immune suppression and increased susceptibility to pathogenic infections [22], [23]. Growing evidence demonstrates that pesticide mixtures can induce more distincted biological effects than single pesticides. Specifically, co-exposure to thiamethoxam (1 µg/L) and picoxystrobin (18 µg/L) induced greater lifespan reduction and cellular damage compared to individual exposures [24]. However, current ecotoxicological assessments primarily focus on the toxicity of individual pesticides [25], [26], neglecting the more ecologically realistic scenario of combined pesticide exposure.

Nitenpyram (NIT), a second-generation neonicotinoid introduced in 1995, exhibits relatively low mammalian toxicity and negligible long-term bioaccumulation [27]. Similar to other neonicotinoids, NIT acts as a potent agonist of nicotinic acetylcholine receptors, disrupting synaptic transmission in the central nervous system [28]. This mechanism makes NIT highly effective against sucking pests such as tobacco aphids (Myzus persicae Sulzer) and tobacco whitefly (Bemisia tabaci Gennadius), the important pest species in tobacco growth [29], [30]. Due to its widespread use, NIT has become globally distributed, with residues detected in hive pollen within 30 km of Saskatoon, Canada [31], and in thirty honey samples from China at concentrations of 13-41 μg/kg (equivalent to 19.24-60.68 μg/L) [32]. Notably, NIT is highly toxic to honeybees, with a 48-hour LC50 of 3.37 mg/L. Chronic exposure (14 days) to sublethal NIT concentrations (3-300 μg/L) disrupts gut microbial composition, impairs metabolic homeostasis, and suppresses immune responses in honeybees [28]. Despite these known risks, the combined effects of NIT with other pesticides remain poorly understood. Spiroxamine (SPX), a spiroketalamine-class fungicide extensively used in tobacco cultivation for gray mold and powdery mildew control [33], [34], also accumulates in the environment; pollen concentrations can reach 18 µg/L, posing exposure risks to honeybees [35]. Although in vitro evidence indicates that SPX (1.1 μM, 7 days) disrupts cardiomyocyte differentiation by perturbing all-trans-retinoic acid signaling homeostasis [36], critical knowledge gaps persist regarding its sublethal effects on honeybees at field-realistic concentrations. Systematic explorations are urgently needed to assess the effect of SPX on honeybee health. During the growth process of tobacco, NIT and SPX are frequently applied either simultaneously or sequentially within the same fields or adjacent areas [37]. A key concern is that honeybees may encounter NIT and SPX while foraging on treated crops, as both pesticides have been detected in honey and pollen [32], [38]. This co-occurrence is further supported by residue monitoring studies reporting the simultaneous presence of NIT and SPX in various environmental matrices. Moreover, the likelihood of co-exposure is increased by the environmental persistence of these compounds. Specifically, NIT exhibits a log Kow of –0.66 and a soil half-life (DT₅₀) of approximately 8 days, whereas SPX has a log Kow of 2.89 and a considerably longer field DT₅₀, ranging from 19.8 to 145.3 days [39], [40]. Given these exposure risks, systematic investigations are essential to assess the toxic effects of NIT and SPX on honeybees, both individually and in combination.

Melatonin (MT), a neurohormone derived from tryptophan metabolism, acts as a potent biological regulator with multifaceted activities, particularly in scavenging reactive oxygen species and attenuating inflammatory responses [41]. MT has been shown to protect against imidacloprid-induced intestinal damage in common carp (Cyprinus carpio) by suppressing the peptidoglycan (PGN)/p38 mitogen-activated protein kinase signaling pathway [42]. Furthermore, in diquat-exposed piglets, MT administration enhanced antioxidant defenses, thereby alleviating growth impairment and restoring intestinal barrier integrity [43]. Despite its established protective effects in vertebrates, the potential of MT to mitigate pesticide-induced midgut damage in honeybees remains largely unexplored. In this study, we hypothesized that co-exposure to NIT and SPX elicits synergistic response on honeybees by triggering oxidative stress and inflammation‐mediated midgut injury, and that MT may attenuate these effects. To test this, we quantified acute mortality in A. mellifera following individual and combined pesticide treatments, then evaluated midgut specific biomarkers including oxidative damage, inflammatory mediators, histopathology, and barrier integrity in 48-hour-old worker bees subchronically exposed to field-relevant residue levels for 10 days. By elucidating the molecular and cellular mechanisms underpinning pesticide mixture toxicity and demonstrating MT’s protective potential, these data not only deepen our understanding of honeybee health hazards posed by agrochemical interactions but also inform targeted strategies to safeguard pollinator colonies.