Apis cerana cerana relies on its sensitive olfactory system to perform foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile compounds with ORs in A. cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of A. cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda (Sf9) cells. AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl) thio) acetamide (VUAA1), whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol. Of the compounds tested, AcerOr1 showed the highest sensitivity to these odorants with EC₅₀ values of 10⁻⁷ and 10⁻⁸ M, and AcerOr2 recognized VUAA1 with higher sensitivity [EC₅₀ = (6.621 ± 0.26) × 10⁻⁸]. These results indicate that AcerOr2 is an essential gene for olfactory signaling, and AcerOr1 is a broadly tuned receptor. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them by electroantennography. The response increased with concentration of the odorant. The present study provides insight into the mechanism of olfactory discrimination in A. cerana cerana.

To address the inefficient crushing of fertilizer during the mechanized spreading process caused by the caking of high-humidity organic fertilizer, a fertilizer spreader with a combined rotor and lining plate crushing mechanism was proposed in this paper. With the introduction of the basic structure and working principle of the spreader, a particle group model for an organic fertilizer consisting of both caked and bulk fertilizer was built, based on the Hertz–Mindlin model with bonding and the Hertz–Mindlin model with JKR contact, in EDEM to construct an organic fertilizer-crushing-and-spreading model. With the rotor speed, the axial distance of the hammer, and the number of circumferential hammer groups as the experimental factors and the maximum broken bond rate of the caked organic fertilizer and the minimum coefficient of variation of spreading uniformity as the experimental indices, the Box–Behnken test method was employed to establish regression equations for response surface analysis and multi-objective optimization of the test results. The results indicated that, when the rotor speed was 6.47 Hz, the axial distance of the hammer was 90.30 mm, the number of circumferential hammer groups was five, the broken bond rate reached 90.86%, and the coefficient of variation was 21.45%. Verification tests under these conditions showed a broken bond rate of 90.03% and a coefficient of variation of 22.12%, which were consistent with the optimization results. Therefore, our research provides a reference for the structural design of an organic fertilizer spreader and the optimization of its working parameters.

Temperature is the most significant abiotic factor that affects the growth and behaviour of insects. However, the mechanism by which the olfactory system senses thermal stimulus and combines temperature and chemical signals to trigger certain behavioural outputs is unclear. This study aimed to clarify the mechanism by which environmental temperature affects olfactory perception in Apis cerana cerana (Hymenoptera: Apidae). We used quantitative reverse-transcriptase polymerase chain reaction and western blotting to analyse the expression of AcerOr1 and AcerOr2. We also used electroantennography (EAG) assays to detect bee antennal responses to odorants at different temperatures. The results revealed that the mRNA expression of AcerOr1 and AcerOr2 was significantly influenced by temperature. These genes exhibited both increases and decreases in expression over time, with the most significant differential observed at 25 °C. Protein expression was similarly affected at 2 hours after different temperature treatments. Electroantennography responses from the antennae revealed that six odorant volatiles – N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide (VUAA1), linolenic acid, eugenol, hexyl acetate, 1-nonanol, and lauroleic acid – had the most dramatic effect at 25 °C. The results indicate that environmental factors affecting the expression of AcerOr1 and AcerOr2 modulate olfactory recognition behaviour in A. cerana cerana, suggesting that changes in environmental temperature can affect bees’ olfactory preferences.

Smoothened (Smo) is a critical component regulating the Hedgehog signaling pathway. However, whether Smo is associated with the modulation of olfactory recognition capabilities of bees remains unclear. In this study, we amplified Smo from Apis mellifera. The coding sequence of Smo was 2952 bp long, encoded 983 amino acids. Smo was most highly expressed in the antennae. Cyclopamine (200 μg/mL) significantly reduced but purmorphamine (800 μg/mL) significantly increased Smo expression (p < 0.05). OR152 and OR2 expression in the cyclopamine group significantly decreased, whereas OR152 expression in the purmorphamine group significantly increased (p < 0.05). A significant decrease in the relative values of electroantennography was observed in the cyclopamine group exposed to neral. Behavioral tests indicated a significant decrease in the attractive rates of neral, VUAA1, linalool, and methyl heptenone in the cyclopamine group. Conversely, the selection rates of linalool and methyl heptenone in the purmorphamine group significantly increased. Our findings indicate that Smo may play a role in modulating olfactory receptors in bees.

Olfactory receptors are essential for recognition and detection of odor in honeybees. Although we have cloned and characterized the sequence of olfactory receptor AcerOr1 before, the tissue distribution and location of this gene in the nurse and the forager worker Apis cerana cerana were not very clear. To further investigate this information of AcerOr1 gene, we analyzed its expression and localization. The results showed that AcerOr1 mRNA was predominantly expressed in the antennae of nurse and forager bees, while the AcerOr1 protein was predominant in thorax, and its expression in antennae was higher in forager than in nurse. IHC revealed that AcerOr1 mainly localized in the olfactory neurons of the antennae. In addition, the staining intensity of AcerOr1 protein by IHC was consistent with the results of qRT- PCR and western blotting. The expression of AcerOr1 in non-olfactory tissues implied that, in addition to olfaction, it may involve in other physiological processes. The localization of AcerOr1 in antennae further suggests that it is involved in the olfactory system. Our research suggest that AcerOr1 may participated in perceive pheromone and odours of floral in nurse and forager bees, and also critical for the behavior of collecting, defending, cleaning between the nurse and the forager worker bees.

To systematically investigate how the olfactory system of Bombus terrestris adapts to its social division of labor and reproductive strategies, this study integrated the micromorphology of antennal sensilla and the expression profiles of olfactory receptor (OR) genes from the heads of its three castes (workers, drones, and queens) for a multi-level analysis. Scanning electron microscopy (SEM) revealed that drones possess significantly longer chaetic sensilla (Sch), sensilla trichodea (Str A/B), and sensilla basiconica (Sba A), as well as larger-diameter sensilla coeloconica (Sco) compared to workers and queens, indicating structural and functional specialization for sensitive detection of single key signals (e.g., queen pheromones). In contrast, workers and queens exhibited a more complete composition of sensilla types and a higher sensilla distribution density, suggesting the construction of a perceptual system capable of processing multiple chemical signals simultaneously. RNA-seq combined with qRT-PCR confirmed the significant upregulation of seven OR genes (e.g., BterOR3, BterOR4) in drones, while workers showed upregulation of BterOR3/5/7 accompanied by enrichment of P450 detoxification pathways. Phylogenetic analysis suggested that BterOR5 serves as a conserved co-receptor, and some OR genes may originate from recent duplication events. In summary, distinct differences were observed in the morphological structure and molecular expression of the olfactory system among B. terrestris castes. Drones exhibited structural and gene expression features consistent with specialization in queen pheromone detection, while workers and queens demonstrated sensilla diversity and olfactory receptor expression patterns indicative of a broader response capacity to diverse chemical signals. These findings support the view that the olfactory system has undergone multi-level adaptive evolution driven by social division of labor and reproductive roles.