Table 1.

Mating treatment levels used to test transgenerational effects of sexual interactions in Drosophila melanogaster.

level	treatment level name	no. males per replicate (first 2 h)	no. males per replicate (subsequent 10 days)	treatment level description	no. replicates (vials containing 8 mothers each)	no. mothers treated	no. male offspring assayed	no. female offspring assayed
1	‘baseline’	12 LHM-bw	—	baseline (single mating): focal mothers received no further exposure to males. This level is referred to as baseline, because all females in the experiment first experienced this 2 h exposure to males (one mating) prior to their subsequent assignment to the other levels	11	88	90	93
2	‘pre-cop only’	12 LHM-bw	12 cauterized LHM-bw	baseline plus additional pre-copulatory effects: focal mothers were exposed to a new group of 12 LHM-bw virgin males that had their genitals cauterized using a fine tungsten wire probe connected to a 6V, 1A power source, leaving them able to harass the females but unable to mate with them. This procedure precludes copulation by males without impairing their pre-copulatory sexual behaviour (see the electronic supplementary material)	13	104	113	115
3	‘pre + post-cop’	12 LHM-bw	12 LHM-bw	baseline plus additional pre-copulatory and post-copulatory effects (multiple mating one genetic background): prospective mothers were exposed to a group of 12 LHM-bw virgin males whose genitals were fully intact. Mothers assigned to this level therefore experienced effects of ongoing male pre-copulatory activity, several copulations, and post-copulatory effects associated with the receipt of multiple ejaculates from males from the LHM-bw genetic background	13	104	107	114
4	‘pre-cop interacting phenotypes’	12 LHM-bw	6 LHM-bw plus 6 cauterized CH	baseline plus additional pre-copulatory and post-copulatory effects (multiple mating one genetic background and additional pre-copulatory effects from second genetic background): each replicate of prospective mothers was exposed to a group of males consisting of six LHM-bw virgin males and six CH cauterized virgin males. Thus, females in this level were subjected to pre-copulatory effects by males from the two genetic backgrounds but post-copulatory effects (receipt of ejaculates) from just the LHM-bw genetic background	16	128	122	136
5	‘pre + post-cop interacting phenotypes’	12 LHM-bw	6 LHM-bw + 6 CH	baseline plus additional pre-copulatory and post-copulatory effects (multiple mating two genetic backgrounds): each group of eight prospective mothers was exposed to six L LHM-bw virgin males and six CH virgin males. This level was used to test for transgenerational effects arising from exposure (at the pre- and post-copulatory stages) to a higher diversity of male genotypes (sourced from two distinct global populations), compared with level 3. Level 5 and the use of the brown-eye morphological marker allowed us to inspect transgenerational effects that are in essence indirect genetic effects on offspring fitness traits. That is, while mothers in treatment level 5 produced a mixture of brown- (sired by LHM-bw males) and red-eyed flies (sired by CH males), we only assayed reproductive success of the brown-eyed offspring (i.e. those produced by LHM-bw females and sired by LHM-bw, not by CH males). Thus, any effects seen in the offspring generated in level 5, compared with 3, would arise from mothers interacting sexually with males from different populations and not by Mendelian genetic effects tied to the sire	17	136	119	131
total					70	560	551	589