Drosophila melanogaster mating strategies under threat of predation
March 17th
Hypotheses:
If courtship and copulation effort is affected by predatory stimuli, a reduction in overall occurrence and timing of these behaviours will be observed.
Predictions:
Males paired with immature females will have reduced numbers of courtship displays and (copulation attempts under predation threat than without a predator present (reduced male courtship effort)
Copulation occurrence, latency and **duration will be reduced when a predator is present for a male paired with a mature female compared to no predator threat (increased female rejection of males/ both quick to finish).
Methods:
Use Dukas wild population raised on Dworkin food bottles: males aged ~2 day old (44-48 Hours); Females either ~2 days old (Mature, 44-48 Hours) or <20 hours old (Immature, 15-20 Hours)
Collect using Aspiration, females housed <5 per vial (with live yeast), males housed singly in vial with microcentrifuge tube food source Paired with zebra jumping spider (Salticus scenicus) starved for 3 days and placed in the assay chamber overnight before trials (just spider in chamber overnight)
Arenas used comprise of two petri dishes together with mesh separating top (for flies + Insect-a-Slip or Surfasil for better recording) and bottom (for spiders), with each treatment in separate bins (4 arena per) (remove most predator olfactory cues)
Add male first to each chambers, followed by a female
Video record within modified plastic bins: After time limit reached, record new mating pair for duration specified (15 minutes for immature females observing for male courtship effects and 30 minutes for mature females observing copulation differences)
Schedule (days):
Increase male age by a day: 68 - 72 hour old males or 44 - 48 hour old males for trials
7am: Clear, 12pm: Sort ♂ and ♀ (Group 1), 5pm: sort ♂ and ♀ (Group 2)
7am: Clear, 12pm: Sort ♂ and ♀ (Group 3), 5pm: sort ♂ and ♀ (Group 4)
6-7 pm: add spider to desired assay chambers
Repeat step 2 each day before a trial (group numbers relative to trial day; Group 3 = new Group 1, collect new Group 4)
8am: Male from Group 1 (Age = ~44 Hours) paired with female from Group 1 (mature female = ~44 Hours) or female from Group 4 (immature female = ~15 Hours)
Video record Group 4 females ~15 minutes; Group 1 females ~30 minutes (not noting copulation duration); swap after time for new mating pairs
Wash all bins in the evening (ethanol/distilled water).
Flies needed: 64 ♂’s, 32 immature ♀’s, 32 mature ♀’s
Flies collected: minimum 90 ♂’s, 60 immature ♀’s, 60 mature ♀’s
Replications:
Can do an extra day to increase sample size
15 minute trials = 4 per bin, 2 bins (one per treatment), 4 replications a day, 4 days = 128 total (64 pred:64 no pred)
30 minute trials = 4 per bin, 2 bins (one per treatment), 4 reps per day, 4 days = 128 total (64 pred:64 no pred)
Observations:
15 minute trials = male courtship behaviour (latency, duration, attempts, and behavioural changes)
30 minute trials = female rejection and copulation (occurrence, latency, **possibly duration)
Increased number options:
15 minute trials
can increase to 8 replicated a day (match 30 minute trial end) = 256 total (128 pred:128 no pred)
64 immature ♀’s, 96 ♂’s needed
30 minute trials
can increase number per bin if no video recording, only manual observation and timing (2 reps per day of 8 per bin = same total, 2 reps of 16 per bin = 256 total (128 pred:128 no pred)
64 mature ♀’s, 96 ♂’s needed
Both together (increase 15 min replicates, 30 min per bin)
Each total 256: 64 mature ♀’s, 64 immature ♀’s, 128 ♂’s needed (extra collected)
Logistically would be tough and depend on strong egg laying/ecolsion and more assay chambers
If more ♂ needed; use ♂ from Group 2 (5pm same day as Group 1 = 38-43 Hours)
Blind observation methods:
Not possible to be blind to pred/no pred treatment
Can be blind to age (but not necessary)