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.
Previous work shows that to choose between virgin and mated females male
spider mites use volatiles emitted by females and substrate cues left by
females on patches . Based on this information, we created with our
setup the conditions for mate discrimination to take place in an
environment in which there was matching information (e.g., virgin
females on patches previously impregnated with cues released by virgin
females) or in which there was a mismatch between the information
released by the females present on the patch and the cues that were left
on the substrate by previous females (e.g., virgin females on patches
previously impregnated with cues released by mated females). To create
these 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. 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 information emitted by females
present on the patch (including their own behaviour) as “female mating
status”. Note that, although we are aware that chemical information is
sufficient for mate choice (Rodrigues et al. 2017), it is possible that
other cues or signals (e.g., tactile, visual) are used by males as well
(Royalty et al. 1993). In any case, we assume that the information
provided by the presence of a female is composed of multiple cues that
are concordant, and thus, for simplicity, consider “female mating
status” a unit of information. Likewise, substrate cues include web,
faeces, eggs and any chemical information left behind by females; once
more we consider those a unit of information. We cannot rule out the
possibility that new substrate cues are being deposited by the females
present in the patch, but given the short duration of trials, these are
not expected to play a significant role in the behaviours described.
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 . It has been shown that females can reject male mating
attempts by moving away from them (Rodrigues et al. 2020). As such, we
also studied the frequency of female acceptance, 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.