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.