The burying responses of termites to fungal contaminants

Response behaviors of termites to weedy fungi on PDA plates The three strains of observed weedy fungi (Aspergillus, Trichoderma, and Pseudoxylaria), and Termitomyces. Potato Dextrose Agar (PDA) media were prepared simultaneously in all glass petri dishes (∮ = 90 mm) , up to 5 mm in depth. The 0.5 cm diameter Termitomyces cultures were extracted and inoculated onto new PDA plates 10 days before (positioned 0.5 cm away from the plate margin) using PDA plugs, serving as blank controls to replace Termitomyces. On the right side of each PDA plate, 5.5 cm away from the Termitomyces/PDA inoculation point, one-week-old cultures of Aspergillus, Trichoderma, and Pseudoxylaria were inoculated, following the requirements of different purposes. Each design of experiment conducted in six duplicates. (a) Termites with single fungal contaminant assay: the response of termites to a single fungal contaminant was tested in the presence and absence of Termitomyces (Figure 1a); (b) Termites with multiple fungal contaminants assay: the response of termites to the simultaneous presence of multiple fungal contaminants was tested, again in both the presence and absence of Termitomyces (Figure 1b). The different termite castes (major workers, minor workers, and soldiers) were collected from the same comb of Macrotermes annandalei (August: 9 am–11 am); all termites were initially placed in PDA plates for 15 min at 27°C, in the dark, quiet environment (for adaptability and allogrooming). Subsequently, each group of nine termites (one soldier, four major workers, and four minor workers) was guided into PDA plates containing fungi and incubated at 27°C in a dark, quiet environment for three hrs. Termites cut little pieces of agar called "boluses" and accumulated them on and around the fungal plugs (Katariya et al. 2017). Finally, after three hours, we calculated the number of boluses around the fungal contaminant plugs (1×1 cm2).