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.