Experimental Setup
All experiments were done on bean leaves. Females and males were
isolated from the base population on detached leaves at the quiescent
stage, immediately before completing the last moult. This way, all
individuals used in the experiment shared the same age-at-maturity, and
virginity was ensured in both sexes before they were allocated to
different treatments.
To create different environments for males, multiple groups of 10 virgin
females were randomly assigned to patches (leaf discs of 2.55
cm2) with 3 virgin males. Behaviour was observed for 1
hour and, when matings occurred, females were transferred in groups of
10 to a new empty patch of the same size. Simultaneously, groups of 10
virgin females were directly transferred to similar empty patches
without ever being in contact with males. Both types of females were
left on those patches during 24h such that they could release cues that
remained on the substrate. Those females were then removed, and 5 new
females (either mated or virgin) were placed on those patches.
Subsequently, one focal male was added to all patches. Previous work
done on similar conditions shows that male spider mites use volatile and
substrate cues to choose between virgin and mated females . Thus, with
our setup, we created the conditions for mate discrimination to take
place in an environment in which there were matching female cues (e.g.,
virgin females emitting cues on patches previously impregnated with cues
released by virgin females) or in which there was a mismatch between the
cues emitted by the females present on the patch and the cues that were
left on the substrate by previous females (e.g., virgin females emitting
cues on patches previously impregnated with cues released by mated
females). Henceforth, for simplicity, we refer to the cues left on the
patch by virgins or by mated females that were removed prior to the
beginning of the mating sessions as “substrate cues” and the cues
emitted by females present on the patch (including their own behaviour)
as “female mating status”.
Male and female behaviour, i.e., the number of male mating attempts, the
frequency of female acceptance, the number of mating events and
copulation duration were observed for 1 hour. A mating attempt was
registered whenever a male touched the female with the two front legs
and started bending its opisthosoma . Whenever a mating attempt resulted
in the insertion of the male aedeagus into the female abdomen for more
than one minute, the observer registered it as the occurrence of a
mating event . The frequency of female acceptance was calculated as the
number of mating events over the number of mating attempts. Copulation
duration was registered as the time in seconds a male spent with his
aedeagus inside a female. Note that females were not removed from the
patch during a mating session, so mating events could have occurred with
mated females in patches with virgin females at later stages of the
mating session.
Subsequently, males were transferred individually to a new patch (2.55
cm2), made from uninfested bean plants, and their
survival was followed daily, to measure whether different mating
histories would translate into a longevity cost. Death was classified as
natural (i.e., the corpse was found on the patch) or censored (i.e.,
males died by drowning or by being accidentally stuck in the leaf or
squeezed).
This experiment was carried out in 21 mating sessions divided in 8 days,
and in total, 84 males and 420 females, corresponding to 21 males and
105 females per treatment (i.e., combination of type of substrate cues
and female mating status) were observed.