Quantifying immune function
We measured multiple immune parameters to capture a suite of immune pathways that may differ between diets or be altered following infection cue treatment. Here, we analyzed antibacterial activity, total circulating hemocytes, and the presence of hemocyte microaggregations from hemolymph that was collected immediately after calling effort trials. General cell-free antimicrobial activity of hemolymph is a component of the humoral immune response of insects and includes the action of both lysozyme-like enzymes and antimicrobial peptides (Lemaitre et al., 1997). Additionally, hemocytes are a component of the cellular response of insect immunity, and are involved in core processes that include coagulation, nodule formation, phagocytosis, and encapsulation (Lavine and Strand, 2002). Finally, the microaggregation of hemocytes is an early step in the process of nodule formation (Miller et al., 1999; Miller and Stanley, 2004), a key insect cellular defense reaction to bacterial challenges and responsible for clearing a large proportion of bacteria from circulation (Howard et al., 1998). To collect hemolymph, males were cold-anesthetized, and the membrane was pierced under the anterior of the dorsal pronotum plate with a sterile 25 G needle, and 5 µL of outflowing hemolymph taken with a pre-chilled glass microcapillary tube positioned at the puncture site. Collected hemolymph was then expelled into 11 µL of chilled Grace’s insect medium (MilliporeSigma) to be used in antibacterial assays; 5 µL of this mixture was then added to 15 µL of chilled Grace’s insect medium to be immediately used for circulating hemocyte counts and determining the presence of microaggregations. The samples for antibacterial assays were snap-frozen in liquid nitrogen and stored at -80°C for later analysis.