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.