Treatments
We set up control, nutritional stress and mating delay treatments, each consisting of 96 adult female G. m. morsitans (Fig. 1, S2 File). Because reducing the amount of haemoglobin in a bloodmeal results in lower pupal wet weights (Kabayo & Langley 1985), we chose to dilute red blood cells with serum to produce a low-quality diet for the nutritional stress group. Trials testing different ratios of red blood cells to serum showed that flies fed on c. 10% red blood cells produced lighter pupae but had similar survival over a 50-day period, compared to flies fed on c. 45% red blood cells (S3 File). We thus ascribed the 10% red blood cell treatment as the ‘nutritional stress’ treatment and 45% red blood cells as ‘control’. For the mating delay treatment, virgin females were kept in communal cages for three weeks post-emergence. Virgin females continue to ovulate, but mature eggs eventually disintegrate (Ejezie & Davey 1977). Once mated, they were separated into individual cages. The experiment was then run until mothers were 100 days old. The probability of survival for females up to 100 days is estimated, from mark-recapture studies, to be c. 10% in the wild (Hargrove et al. 2011). In addition, unpublished data from the LSTM colony indicated that by 100 days the probability of abortion had increased from c. 0 to 0.25 and the size of pupae produced had declined to similar sizes seen from first-time mothers (S1 File).