Methods
Stock: The ‘Commercial Drive’ population of Drosophila melanogaster flies was initiated
from a large (~100 females) sample from downtown Vancouver trapped in June 2002,
and has since been kept in multiple large cages at a population >5000 flies at 50%
humidity and 12 hours light/dark. The development time has been truncated at 14 days,
with eggs laid at low density (<300 eggs per standard 240ml food bottle containing 50 ml
of sucrose-cornmeal media). The flies have been used for several investigations (Dukas
and Mooers, 2003, Dukas, 2004) and show vigorous behaviour in the lab in comparison
with other melanogaster stocks (Dukas and Mooers, pers. obs.).
Producing Stud and Dud males. The experiment was initiated September 30, 2003. Our basic protocol was to use
“generator females” created from the same unmarked Comm stock as the males to
produce Stud and Dud males. For each of two production days, flies eclosing into cages
on Day 10 were allowed to mate till the morning of Day 13, after which they were
isolated in bottles of 50 males and 50 females. 3100 males and females were collected
for the first day’s production trials, and the same number for the second day. Production
trials were begun when the male flies were in Day 18. For all trials, previously mated
females were Day 18 and had been separated from males for 6 days to lay eggs.
Production trials were set up with 100 males and 100 females per large plexiglass cage
(37 x 27 x 21 cm3, covered on all exterior sides except the top with white paper to avoid
any visual disturbance of the flies) at ambient temperature and humidity (22-24 C., 3555% rel hum.) between 0900-1200. We collected (without anesthesia) and held both all
males that mated within 24 minutes from cages (“Studs”) and all males that did not mate
(“Duds”). At the end of the production trials, these two types of males were collected
with light CO2 anaesthesia and stored separately in bottles of 50. This was repeated on
Day 2. We ran 30 cages on the first day (Day 18 for both males and females), but only 24
cages on the second (also Day 18). Mating rates were low (<<20 matings per cage) and
so generator females were placed on new food overnight and a further 7 cages were run
the next day (Day 19): mating rates returned to the normal >20 matings per cage. We
combined the 1300 Stud males (or 1400 Dud males) from the 3 production trial days and
ran 13 Stud cages and 14 Dud cages two days later with new Day 18 females that had
been separated for four days following 24 hours as adults with stock males. Mating rates
were very high (34-62 matings/cage) and we collected 600 Stud males and 700 Dud
males. Here, “Studs” are males that mated within 24 minutes from the Stud population
cages, and “Duds” are males that did not mate in the Dud population cages. Three days
later, these males were tested a last time with new (Day 19) females that had been
isolated for five days to lay eggs following mating with stock males. Mating rates were
again low (10-24 matings/cage, discarding all males from a cage with only 8 matings),
and we collected 112 Day 25-26 Stud males and 200 Day 25-26 Dud males in total,
which were stored in bottles of 50 with yeasted food.
Initiating test populations.Progeny males from our Studs and Dud males, when mated with random females, would
contain a random X chromosome originating from their stock population mothers. We
therefore used the F2 generation, ie the grandchildren of the initial Stud and Dud males.
So, at the end of the last day of mating trials, each of 112 Stud males and 200 Dud males
were placed in a yeasted holding vial with a virgin female from the stock population and
held for 24 hours. Each pair was then transferred to a fresh vial (end of Day 1) without
anaesthesia and when approximately 50 eggs/vial had been laid (after 24 hours), the pair
was discarded. On Day 9, we created our Stud and Dud grandchildren replicates by first
placing a virgin female from a Stud (or Dud) vial together with a virgin male from a
separate Stud (or Dud) vial in a new yeasted vial. This was repeated to produce (with
attrition) 151 Stud vials and 164 Dud vials. After 24 hours in holding vials, pairs were
transferred to new yeasted vial to initiate replicate 1. After approximately 50 eggs were
laid (~28 hours later), flies were transferred to new vials at 24 hours intervals to create
replicate 2 and replicate 3 vials.
Testing fitness components.-
Longevity: Flies from replicate 1 vials were allowed to emerge into cages on Day 10.
Flies were then sexed under light anaesthesia and pairs of workers set up eight “longevity
cages” of flies for each of three treatments (75 pairs/cage, 75 males/cage or 75
females/cage) for both Studs and Duds, for a total of 48 cages. These longevity cages
were clear plastic yoghurt containers with an aperture that allowed dead flies to be
removed using a ‘pooter’, an aperture in which a standard vial with food could be
attached and removed, and a small mesh top for circulation. Cages were then placed in
alternate order in rows in the incubator holding the stock flies. Every 48-72 hours
thereafter, vials with yeasted food were changed for each cage, and dead flies removed
and identified, until there were no more the flies left.
Male mating success: 2250 Stud males and 2250 Dud males flies emerging on Day 10
from replicate 2 vials were allowed to mate for two days before being sexed under light
anaesthesia and stored in groups of 50 in bottles with unyeasted food. 12 hours prior to
the (Day 18) trials, half the Stud males and half the Dud males were given yeast
impregnated with red (or blue) food colouring (“Food-club Brand”, Scott-Bathgate Ltd)
in a balanced design. Test females were created by collecting males and females from
the stock population that emerged overnight from Day 9 to Day 10, and allowing them to
mate in large cages till Day 11. On Day 11, females were sexed and stored separately for
five days to lay eggs, making them Day 16 on test day. Thirty-nine trials in the same
large plexiglass cages as used previously were run on a single day in the morning hours
between 0900-1330. Trials were conducted by releasing 50 marked Stud and 50 marked
Dud males and then 100 females into a cage and removing and identifying the males
from all pairs that mated within the first 24 minutes.
Productivity: 700 Stud (and Dud) males and female virgins were collected from replicate
3 vials on Day 9 and stored separately in bottles of 50 flies with yeasted food (which
were changed weekly). On Day 10, we created 250 vials with one male and one female
Stud fly and 250 vials with one male and one female Dud fly. Forty-eight hours later, we
set up ~250 vials for Studs and Duds (progeny Day 1) by transferring pairs to a new
yeasted vials. This was done simultaneously for Stud and Dud vials by two workers.
Exactly 24 hours later, the pairs were removed, again in parallel. 200 of these were used
to measure productivity: due to attrition during set-up, we created 193 Stud vials and 194
Dud vials. On (progeny) Day 11, vials were frozen and the number of progeny flies in
each vial counted. On parental Day 23, we initiated a second set of productivity vials
from the flies that had been stored in bottles, using the same protocol (ie. held in vials for
~48 hours, then transferred to test vials for exactly 24 hours). 184 Stud vials and 189 Dud
vials were created on parental Day 25 and terminated 11 days later.
Eclosion time 21 Dud and 19 Stud vials set up on parental Day 13 were used to monitor
emergence time. Starting on progeny Day 8 at noon and proceeding every 5-9 hours
following, all emerged flies were removed from vials and counted until emergence was
complete (>78 hours). This protocol was repeated with 21 Stud and 21 Dud vials created
on parental Day 25; here sex was also recorded.
Analysis