Assessing reproductive effort
Calling effort of males was used as a proxy for reproductive effort.
Male G. sigillatus begin mating 4.5 days on average after adult
eclosion (Burpee and Sakaluk, 1993) and, because calling is essential
for mating, it follows that males are also calling at this time. We
quantified calling effort (i.e., the amount of time males spent calling)
over two consecutive nights following infection cue treatment. Calling
effort was measured using a custom-built high-throughput sound
monitoring array (Bertram and Johnson, 1998; Duffield et al., 2018,
2019) in which each male-containing individual container (250 mL) was
fitted with a lid-mounted microphone (C1163, Dick Smith Electronics) and
placed within a small Styrofoam box to prevent crosstalk between
containers. Following administration of infection cues, males were
provisioned with water and a small piece of egg carton for refuge and
given 7 h (± 1 h) to acclimate prior to the start of recording trials.
Recording periods started at 1700 hours and ran for 39 h, ending at 0800
hours 2 days following treatment; this period captures calling effort at
times most relevant to female mate attraction (Sakaluk, 1987; Burpee and
Sakaluk, 1993; Sakaluk et al., 2002). The sound monitor sampled each
microphone throughout the recording periods every 2 seconds, and based
on the binary output resulting from this protocol, total calling time
was calculated for each male across recording periods (Hunt et al.,
2004; Duffield et al. , 2018, 2019).