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unpublished data from the LSTM colony indicated that by 100 days the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).