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).