Measures of offspring quality
In addition to spontaneous abortions, we recorded the wet weight of viable pupae and, for a subset, we measured either fat, or whether they emerged and their starvation tolerance upon emergence (Fig. 1). Wet weight of pupae correlates with pupal volume in field flies and does not require destructive sampling (Hargrove 1999). We recorded emergence as we hypothesised that offspring with more reserves would be more likely to emerge. We measured starvation tolerance because, in the wild, newly emerged tsetse have a high probability of mortality (Hargrove et al. 2011), likely due to failure to find a blood meal before running out of energy reserves.
From preliminary data on the tsetse colony at LSTM (S1 File), we hypothesised that there would be an increase in the probability of abortion with age. If resource-allocation trade-offs exist, we would also expect that the increase would be steeper for the nutritionally stressed treatment and potentially shallower for the mating delay treatment, relative to the control. However, for offspring wet weight and starvation tolerance we predicted that there would be an initial increase, reflecting the requirement for newly emerged flies to also divert energy and nutrients to the development of thoracic muscles (Anderson & Finlayson 1973), followed by a decline due to senescence. Lastly, we hypothesised that the effect of maternal age on starvation tolerance would be determined largely by energy reserves, as indirectly measured by offspring wet weight. Heavier offspring would have more reserves and therefore would be more likely to emerge and have higher starvation tolerance. Maternal age could also influence offspring starvation tolerance, independent of effects on wet weight, through the quality of the resources provided.