Experimental design
To examine how shifts in relative emergence time varied with
simultaneous changes in temperature and community contexts, we
experimentally delayed the emergence of parasitoids relative to theDrosophila larvae in current and expected warming temperatures,
and in high and low levels of resource competition in a fully factorial
design with 4 emergence times x 2 temperatures x 2 levels of
competition. Additionally, the two host species were reared in isolation
or with the other host species to examine effects of an alternative
resource. Thus, we had a total of three Drosophila species
combinations and two wasp species for a total of 96 unique treatments.
Each treatment consisted of at least five replicates and initiated over
the course of five days, which were represented as blocks in our
statistical analyses.
We reared Drosophila larvae in current ambient (22.9°C ± 0.47SD
with 69% ± 4.34SD relative humidity) and predicted warming (27.4°C ±
1.15SD with 60% ± 10.1SD relative humidity) temperatures (climate
change models predict a 1- 6°C increase in temperatures by 2070 in
Australia (Hughes 2003)) in 12-hour light/12-hour dark photoperiod.
Levels of intraspecific competition among Drosophila larvae were
manipulated by providing 2ml or 20ml of fly medium. A total of 100
Drosophila eggs were added to all vials resulting in densities of 50
individuals/ml for the high competition, and 5 individuals/ml for the
low competition treatments
(Nouhaudet al. 2018). We decided to manipulate the volume of food instead
of the abundance of larvae to avoid any frequency dependent parasitism
effects. Thus, we established four temperature/competition treatments
that varied host development rates: (1) slowest development (24°C with
2ml food); (2) slow development (24°C with 20ml food); (3) fast
development (28°C with 2ml food); and (4) fastest development (28°C with
20ml of food). This process was repeated for both Drosophilaspecies in isolation and when grown together for alternative host
species treatments, where the 100 total eggs were composed of equal
proportions of the two species (e.g., 50 eggs D. sulfurigasterand 50 eggs D. birchii ).
We manipulated four levels of phenological relationships between host
and parasitoids in 2-day intervals. The parasitoid either “emerged” at
the same time (e.g., 0 day) as its host or 2-, 4-, or 6-days later. At
each parasitoid emergence time, Drosophila larvae were exposed to
three mated female and three male parasitoids of a single species of
parasitoid. After 48-hours the parasitoids were removed from the vials
and then maintained under the same temperature and light conditions
described above. This allowed a more precise measurement of the
vulnerability window and largely prevented the complete mortality of allDrosophila . To measure development times, emerges were recorded
daily and stored in 95% ethanol until all hosts or parasitoids emerged
(Fig. 1).
All experiments were set up in 2.8cm (diameter) x 9cm (height) glass
vials, with a 4ml base layer of 1.5% agar gel in each vial to reduce
excess desiccation of the fly medium and provide potential refuges from
parasitoid attack. Control treatments consisted of ten unexposed vials
for each host species combination (n = 3; D. birchii, D.
sulfurigaster, D. sulfurigaster -D. birchii ). These unexposed vials
were used to calculate the average development time (egg-to-adult) and
host survival in the absence of parasitoids across treatments and
species.