*4.5. Hemolymph Trehalose Measurements*

On Day 14 p.i., 10 honey bees were removed from each cage and were anaesthetized at −21 ◦C for 5 min. Before we collected their hemolymph, honey bees were mounted on a wax plate by a pair of insect pins crossing over the waist. Using a glass microcapillary (Hirschmann® Laborgerate, Eberstadt, Germany), 5 μL per bee was collected by puncturing abdomen segments between tergites 3 and 4, and the hemolymph was transferred to a

microcentrifuge tube (Eppendorf, Hamburg, Germany) containing 45 μL of 0.85% NaCl. For each sample, 2.9 mL of anthrone reagent was added and then vortexed for 30 s before we quickly put them into a boiling water bath for 15 min. After this they were placed into cold water (4 ◦C) for 20 min and read at 620 nm absorbance using a Shimadzu UV-visible spectrophotometer (UV-1610). Quantification of the hemolymph trehalose amounts were based on a standard curve.

#### *4.6. Hypopharyngeal Gland Protein Content Measurements*

Another 10 bees were randomly removed from each cage at 14 days post infection (p.i.). These bees were decapitated so that their hypopharyngeal glands could be removed under a stereomicroscope (Olympus CH30, Shinjuku, Tokyo, Japan). Glands of each bee were stored in 50 μL of phosphate buffer solution (pH 7.8) in a 1.5 mL microcentrifuge tube. These were then homogenized and centrifuged at 1000× *g* for 2 min. Supernatant from each tube was used in the Bradford protein assay [74]. Quantification of protein content was based on standard curves that were prepared using bovine serum albumin (BSA). Protein absorbance was measured at 595 nm absorbance against a blank reagent using a Shimadzu UV-visible spectrophotometer (UV-1610).

#### *4.7. Measurements of Acinar Sizes of the Hypopharyngeal Glands and Histological Structure*

Another 10 bees from each group were collected on 14 days p.i. and the heads were dissected in insect saline (NaCl 7.5 g/L, Na2HPO4 2.38 g/L, KH2PO4 2.72 g/L) and then fixed in Bouin's solution for 24 h. Samples were dehydrated using a series of increasing ethyl alcohol concentrations: 70%, 90%, 95%, and 100% for 10 min per concentration. Samples were then soaked in xylene for 1 h and then embedded in paraffin wax. The tissues were sectioned into 6 μm thickness using a rotary microtome (Leica, Wetzlar, Germany), and then stained with Periodic acid Schiff's reagent (PAS) followed by a counter staining of light green dye [75,76]. Measurement of acinar sizes of the hypopharyngeal glands were made under a light microscopy (Olympus CX 50, Shinjuku, Tokyo, Japan) using a micrometer (ERMA: ESM-11, Japan); *n* = 10 per bee each treatment.
