*2.3. Acini Diameters of Hypopharyngeal Glands*

The smallest acini diameter on average was found in the *Nosema*-infected workers (NO) without any treatment, with a distance of 111.05 ± 0.4 μm. The mean diameters of acini of the hypopharyngeal glands were largest in the untreated control bees (CO) (134.55 ± 5.22 μm) and the COS treated control bees (CO-0.5COS) (137.13 ± 8.73 μm), followed by the control bees treated with propolis extract (CO-50P) (128.75 ± 2.9 μm), the infected bees treated with the propolis extract (NO-50P) (125.34 ± 2.9 μm), and the infected bees treated with COS (NO-0.5COS) (120.44 ± 6.8 μm). When we compare between *Nosema*-infected bees and the ones treated with propolis extract and COS, we see a significant increase in the acini diameter on average (*χ*<sup>2</sup> = 33.09, df = 5, *p* < 0.0001, Figure 5). However, the COS treated bees have significantly lower acini distances than the control bees (*χ*<sup>2</sup> = 33.09, df = 1, *p =* 0.0022), but the propolis extract treated bees do not have a significant difference in acini distance in comparison to the control bees treated with propolis (*χ*<sup>2</sup> = 33.09, df = 1, *p =* 0.0553).

**Figure 5.** A box plot showing the median acini diameters across the treatments for the propolis extract and COS experiments. Each box plot represents a treatment: *A. dorsata* infected with *N. ceranae* dosages 106 spores per bee without any treatment (NO) (red), *N. ceranae*-infected bees treated with propolis extract (NO-50P) (light green), *N. ceranae*-infected bees treated with 0.5 ppm COS (NO-0.5COS) (light blue), control bees without any treatment (CO) (grey), control bees treated with 0.5 ppm COS (CO-0.5COS) (purple), and control bees treated with 50% propolis extract (CO-50P) (green). The boxes indicate interquartile ranges, while the vertical bars represent the range of the data. The different letters above each box plot represents significant differences (Kruskal–Wallis test: *χ*<sup>2</sup> = 33.09, df = 5, *p <* 0.0001).

The histological structure of the hypopharyngeal glands of CO bees showed fully developed and contained with several secretory units or acini (oval to rounded shape), each unit composed of 5–8 secretory cells surrounded a central secretory duct. The secretory cells contained with numerous secretory granules stained red-pink with PAS that surround the large cell nuclei, stained greenish with light green (Figure 6), while the secretory units of the hypopharyngeal glands of *Nosema*-infected bees (NO) were incomplete developed in structure indicated by different irregular in shaped and sizes. Therefore, each cell cytoplasm consisted of numerous small secretory vesicles stained pink with PAS (Figure 7). Interestingly, the glands of NO-50P and NO-0.5COS showed fully developed acini. The secretory cell contains several vesicles giving both positive and negative staining with PAS, this indicated the cell storage both carbohydrate and non-carbohydrate molecules. In addition, the large extracellular space between adjacent acinar cells were found indicated by white gap between adjacent cells separating them from each other (Figures 8 and 9).

**Figure 6.** Histology cross sections of the hypopharyngeal gland of *A. dorsata* worker: (**a**) the completely developed secretory units of the glands on 14 dpi of control bees (CO). The secretory cells contain secretory granules surrounded the large nuclei of the secretory cells; (**b**) a section of the hypopharyngeal gland of CO bees with the high magnification of light microscope, the cytoplasm of the secretory cell is seen to contain variable numbers of secretory vesicles (stained red-pink with PAS). The oval nuclei are stained greenish with light green. Abbreviations: ac, acinus; hp, hypopharyngeal gland; md, mandibular gland; n, nucleus; op, optic lobe; vs, secretory vesicle.

**Figure 7.** A section of the hypopharyngeal gland of 10<sup>6</sup> *N. ceranae*-infected *A. dorsata* worker (**a**) on 14 dpi of 106 *N. ceranae*-infected bees (NO), the cell cytoplasm contains variable numbers of secretory granules stained red-pink with PAS. The large oval loose nuclei are stained greenish from a light green dye used as a counterstain; (**b**) A medial section of NO bees on 14 dpi, the secretory cell contains secretory granules surround the large nuclei of the secretory cells. Abbreviations: ac, acinus; n, nucleus; sd, secretory duct; vs, secretory vesicle.

**Figure 8.** The light micrographs of: (**a**) A section of the hypopharyngeal glands of 10<sup>6</sup> *N. ceranae*-infected bees on14 dpi, treated with 50% propolis (NO-50P). The cytoplasm of the secretory cell contains variable numbers of secretory granules stained red-pink with PAS. The oval nuclei are stained a greenish color from light green; (**b**) with higher magnification of NO-50P shows the secretory cell contains several secretory vesicles with negative staining using PAS, and also contains secretory vesicles with smaller amounts of carbohydrate, which are characterized by a red-pink color from PAS staining. Abbreviations: ac, acinus; co, compound eyes; hp, hypopharyngeal gland; md, mandibular gland; n, nucleus; op, optic lobe; vs, secretory vesicle.

**Figure 9.** (**a**) A cross section of the hypopharyngeal gland from 106 *N. ceranae*-infected *A. dorsata* bees on 14 dpi that were treated with 0.5 ppm COS (NO-0.5COS). The cytoplasm of the secretory cells contains variable numbers of secretory granules stained red-pink with PAS. The oval nuclei are stained greenish from a light green; (**b**) a medial cross section of the hypopharyngeal gland from 10<sup>6</sup> *N. ceranae*-infected *A. dorsata* bees on 14 dpi that were treated with 0.5 ppm COS (NO-0.5COS). Abbreviations: ac, acinus; hp, hypopharyngeal gland; md, mandibular gland; n, nucleus; op, optic lobe; vs, secretory vesicle.
