2.3. Preparation of Conidial Suspension and Inoculation

Purified fungus was mass-cultured on sterilized rice medium containing 0.1% Bacto™ yeast extract agar (BD Diagnostics, Franklin Lakes, NJ, USA) and kept at 26 °C and 80% relative humidity (RH). After 14 d when the conidia had emerged on the rice medium, the fungal mass was collected and left to dry in a desiccator containing silica gels. Two weeks later, the dried fungal mass was sieved through a 10 microns sieve and the conidia collected. For protein extraction, conidia samples from three different rice medium bags were collected, weighed, snapped-frozen individually in liquid nitrogen, and kept at −80 °C until used.

Conidial suspension was then prepared in sterilized distilled water with 0.05% Tween^® 80 (Sigma Aldrich Co, USA) and diluted to 1 × 10⁷ conidia/mL aided by a hemocytometer (Hirchmann Laborgeräte, Eberstadt, Germany). Termites were sterilized by dipping them into 0.5% sodium hypochlorite (commercial bleach), rinsed in sterile distilled water, and then blotted on filter paper to remove excess water. Surface-sterilized termites were then dipped into the conidial suspension for 5 s. Inoculated termites were then transferred individually to a 1.5 cm in a diameter plastic cup and kept at 28 °C and 80% RH. Samples were removed at 1, 3, 6, 12, 24, 48, 96, and 144 h PI, while surface sterilized uninoculated termites were collected at 0 h to serve as a control. For electron microscopy, the termites were transferred into 4% glutaraldehyde (Agar Scientific Ltd., Stansted, Essex, UK) and left at 4 °C overnight for fixation process followed by sample preparation. For protein analysis, three individual inoculated termites representing three biological replicates were collected at each PI time-point resulting with a total of 27 infected specimens. The termites were snapped-frozen individually in liquid nitrogen and kept at −80 °C until used.