Insect rearing
The laboratory population of H. subflexa was founded from animals
collected in the field in 2006 and has been reared at the University of
Amsterdam since 2011 using single pair matings to maintain genetic
diversity. There has been occasional exchange among H. subflexapopulations at North Carolina State University, Amsterdam, and the Max
Planck Institute for Chemical Ecology in Jena. Eggs collected from
single pair matings were kept in Petri dishes (Ø 85 mm) with artificial
wheat germ/soy flour-based diet (BioServ Inc., Newark, DE, USA) at room
temperature for approximately 10 days, after which larvae were reared in
separate individual 37-mL cups filled with the same artificial diet and
kept at 25°C and 60% relative humidity with 14h:10h light-dark cycle.
Upon emergence, adults were provided sugar water. Pairs of males and
females were housed in 375 mL paper cups covered with gauze and kept
under the same conditions as the late-instar larvae and virgin adults.
The mating pairs were provided with sugar water. To stimulate
oviposition, a freshly cut gooseberry fruit was placed on top of the
gauze. Once the eggs began to hatch, the gauze and the eggs and larvae
on it, were transferred to Petri dishes, which were then placed at room
temperature until larvae were transferred to individual cups. The
females that produced fertile offspring were collected and phenotyped,
if still alive. All matings were assigned unique numbers. This way, we
obtained a full pedigree of all individuals in the selection and control
lines.