Infection cue
A pathogenic infection may signal reduced residual reproductive value to the host, and this may be mediated through the infection-associated immune challenge. Earlier, we demonstrated that injection with heat-killed Escherichia coli stimulates an immune response and invokes a terminal investment response in male G. sigillatus(Duffield et al., 2015, 2018, 2019). This approach ensures that the measured response is related to the host’s investment strategy, and not the result of alternative causes related to a live infection; thus, a similar protocol was implemented here. Males were given an infection cue treatment following the three-week feeding trials based on a previous study demonstrating that males of this age are more likely to show terminal investment in calling (Duffield et al., 2018). Body mass for each male was measured immediately prior to infection cue administration using an analytical balance (Mettler Toledo AG245).
Males were randomly assigned to one of five treatments on an increasing infection cue spectrum: i) naive (unmanipulated control), ii) sham control (injection of 2μL ringer saline), iii) low-dose infection cue (injection of 2μL ringer saline with 5x105/mL heat-killed E. coli ), iv) moderate-dose infection cue (injection of 2μL ringer saline with 5x107/mL heat-killedE. coli ), or v) high-dose infection cue (injection of 2μL ringer saline with 5x108/mL heat-killed E. coli ). Importantly, we consider the sham control treatment not only a control for the effect of injection, but also as a low-level mortality threat because injection causes cuticle damage and induces an immune response that potentially signals a mortality threat to the individual (Gillespie and Khachatourians, 1992; Wigby et al., 2008; Ardia et al., 2012). Injections were performed using a 5 µL syringe with a 1mm compression fitting (Hamilton® brand) within which a needle formed from a heat-pulled glass capillary tube was inserted. Crickets were injected between the 6th and 7thpleurite of the thorax. Pulled capillaries were cleaned in 70% ethanol, rinsed with ultrapure water, and dried between each injection, and capillaries were not reused across treatments or days. Treatments were always applied at the same time (0900 hours ± 1 h) throughout the experiment. Owing to mortality during the 3-week feeding period, our sample sizes for each treatment were: high protein diet : naive = 33, sham = 37, low = 35, moderate = 34, high = 42; high carbohydrate diet : naive = 40, sham = 41, low = 41, moderate = 43, high = 47.
Escherichia coli (ATCC strain 23716) used to create our infection cues were cultured at 30°C in 7 mL of liquid medium (10 g bacto-tryptone, 5 g yeast extract, 10 g NaCl in 1000 mL of distilled water, pH 7). To prepare bacterial suspensions for immune-challenge injections, 1 mL of an overnight culture was centrifuged (850 g, 4°C, 10 min), the supernatant discarded, and replaced with sterile ringer saline. This procedure was repeated three times. The bacteria were then heat-killed (90°C, 5 min) and the concentration of bacterial cells was adjusted to the concentrations described earlier for each infection cue dose. Efficacy of the heat-killing was confirmed by plating out samples of the suspension on media agar.