**4. Materials and Methods**

#### *4.1. Propolis Extraction*

We collected propolis from three different stingless bee, *Tetrigona apicalis*, colonies from an apiary located in Chanthaburi Province, Thailand. We then dried the propolis in a hot air oven (Binder ED 53, BINDER GmbH, Tuttlingen, Germany) at 80 ◦C for 72 h, this was then frozen at −21 ◦C (Sharp SJ-X43T, Sharp Thai Co., Ltd. (STCL), Bangkok, Thailand) for 3 h and grinded using a motor and pestle. We extracted 60 g of propolis powder with 100 mL of 70% ethanol for 72 h, this was then followed by gravity filtration using a Whatman No. 4 filter paper [30]. After filtration a crude ethanol extract was formed that we defined as 100% propolis stock solution. For the experiments a 50% propolis solution was prepared by diluting the stock solution with water (*v/v*).

#### *4.2. Chito-Oligosaccharide Solution Preparation*

We made a 104 ppm stock of COS, by taking 0.25 g of COS (6081 Da) and dissolving it in 5 mL of pure *A. dorsata* honey (pH = 3.45). We then adjusted the final volume of this to 20 mL with 50% sucrose solution (*v/v*). We then diluted the 104 ppm stock solution of COS to a 50% honey solution using water (*v/v*) to make a final concentration of 10<sup>2</sup> ppm. Afterwards we then prepared a 0.5 ppm COS solution using the same methods that had a pH of 3.77 (pH meter, Mettler Toledo Gmbh, Greifensee, Zurich, Switzerland).

#### *4.3. Spore Preparation*

*Nosema ceranae* spores were propagated from heavily infected *A. florea* colonies located in the Chon Buri Province of Thailand. We fed isolated spores to *A. mellifera* workers (5 × <sup>10</sup><sup>7</sup> spores for 50 bees) that were kept at 34 ± <sup>2</sup> ◦C (Memmert IPP 260, Schwabach, Germany) with relative humidity (Barigo-8861, Schwenningen, Germany) (RH) between 50–55% for 14 days in order to propagate more spores for the experimental infections. To propagate more spores, midguts were removed and transferred to a 1.5 mL microcentrifuge tube containing 100 μL distilled water. The midguts were then homogenized using a sterile pestle and centrifuged at 6000× *g* (Benchmark Scientific Z206-A, Sayreville, NJ, USA) for 10 min, this was repeated for 3 times [40]. We discarded the supernatant each time and the white sediment at the bottom was collected to be counted using a hemocytometer (Hausser Scientific, Horsham, PA, USA) under a light microscope (Olympus CX50, Shinjuku, Tokyo, Japan) [71]. After one more centrifugation, we re-suspended the spores in 50% (*w/v*) sucrose solution to make a final concentration of 5 × <sup>10</sup><sup>5</sup> spores per <sup>μ</sup>L. We stored this syrup at room temperature overnight until further use.

#### *4.4. Propolis Extract and COS Treatment Experiments*

We obtained 3 frames of sealed brood from three *Nosema* free colonies of *A. dorsata* located in Samut Songkhram Province, Thailand. Colonies were confirmed to be *Nosema* free following standard procedures [72,73]. To obtain newly emerged bees, the brood frames were kept in an incubator (Memmert IPP 260, Schwabach, Germany) at 34 ± 2 ◦C with RH (Barigo-8861, Schwenningen, Germany) between 50–55%. The newly emerged bees, between 24–48 h of age, were confined to cages, in groups of 50, and divided into 8 groups. The first 4 groups, were individually force-fed with 2 μL 50% sucrose solution (*v/v*) containing 10<sup>6</sup> *N. ceranae* spores per bee. We then provided 2 groups with 2 mL of either 0% or 50% stingless bee propolis extracts, daily, and these groups were defined as NO-0P and NO-50P, respectively. For the other 2 groups, we provided 2 mL of 0 ppm or 0.5 ppm of COS defined as NO-0COS and NO-0.5COS, respectively.

The control groups were individually force-fed with only 50% sucrose solution (*v/v*), and were defined as the negative control bees CO-0P, CO-0COS, CO-0.5COS, and CO-50P, respectively. In the CO-50P control group was each bee was also treated daily with 2 mL of 50% stingless bee propolis extract, while in the CO-0.5COS control group the bees were treated daily with 2 mL of 0.5 ppm COS. For the duration of the experiment, each cage was fitted with two gravity feeders, one containing distilled water, and the other sugar syrup (50% *w/v* sucrose solution). We also supplied 60 g of pollen mixed with 17 mL of 50% sucrose solution (*w/v*), each was replenished as necessary throughout the experiment. All cages were placed in an incubator at 34 ± 2 ◦C (Memmert IPP 260, Schwabach, Germany), with a RH ranging from 50–55%. The 50% stingless bee propolis extract was provided in 2 mL at a time in a 1.5 mL micro-centrifuge tube from the start of the experiment (0 Day p.i.), until the end (30 Days p.i.), and was replaced as necessary. For the COS treatment, we provided 2 mL of 0.5 ppm COS in 50% honey solution in a 1.5 mL micro-centrifuge at the start of the experiment (0 Day p.i.) and this was replaced as necessary until the end of the experiment (30 Days p.i.).
