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Some male spiders exhibit female genital mutilation behaviour (FGM) by removing the female genital appendage (scape) to control the mating frequency of females. Female spiders have two, i.e. right and left, genital openings connected with separate spermathecae into which males transfer sperm successively using one pedipalp (secondary genitalia) at a time. Thus, males must complete at least two palpal insertions to fill both spermathecae, before FGM. The present study examined whether (i) scape removal is only associated with the second palpal insertion (one-action hypothesis) or (ii) two contralateral palpal insertions facilitate FGM, with each insertion cutting the basal part of the scape halfway (two-actions hypothesis). Experiments in which females were replaced after a male had made the first insertion did not support the one-action hypothesis, because scapes remained intact after the newly introduced virgin females received their first palpal insertion, which was the second insertion by the males. In comparison, mating experiments using two half-eunuchs (i.e. one of the palps of each male had been manually removed, forcing them to fill female spermatheca on one side only) supported the two-actions hypothesis. FGM was more frequent in females that received two contralateral palpal insertions than in females that received ipsilateral insertions.

The timing of female genital mutilation and the role of contralateral palpal insertions in the spider Cyclosa argenteoalba