Reproductive transitions and sperm utilisation in a facultatively parthenogenetic stick insect

Facultative parthenogenesis enables females to switch from asexual (parthenogenetic) to sexual reproduction after mating, but the process of fertilisation is poorly understood in such animals. In particular, it is not known whether switching reproductive modes requires changes in the eggs themselves, delaying the transition from laying unfertilised to fertilised eggs. Likewise, very little is known about patterns of sperm precedence in facultatively parthenogenetic females that mate with multiple males. In this study, we manipulated reproductive mode in females of the facultatively parthenogenetic stick insect Megacrania batesii. We used offspring sex ratio, fertilisation rate, and paternity analysis to investigate how females descended from distinct natural populations switch between reproductive modes and utilise sperm from different males. In the switch treatment group, females were first allowed to lay unfertilised eggs, and then paired with a male. In the non-switch treatment group, females were instead paired successively with two different males. We collected eggs laid over two successive 10-day periods after male introduction (switch treatment) or substitution (non-switch treatment). We found little difference between the treatment groups in fertilisation rate or in the number of sons produced during the first and second 10-day egg-collection. We also observed similar reproductive performance between switch and non-switch treatment groups, but females’ population of origin influenced fertilisation rate and offspring sex ratio. In the non-switch group, we found near-equal fertilisation rates by the first and second male. Our results show that M. batesii females can quickly switch from producing parthenogenetic to fertilised eggs, suggesting that this transition does not require production of distinct types of eggs. Our results also show that M. batesii females can rapidly utilise sperm from a new mate, and exhibit near-complete sperm mixing which suggests that paternity may be evenly distributed in this species.

Methods
We used lab-reared females that were collected as eggs from natural Northern genotype mixed-sex (BK, CO, MB, MK) and Southern genotype all-female (B1, CB, TB) populations (Fig. 1) of M. batesii in far-north Queensland, Australia (population codes correspond to Wilner et al., 2025a). Adult females were divided into two treatment groups: “switch” (N = 17) and “non-switch” (N = 18). In the switch group, females were first housed alone and, after the onset of oviposition, allowed to lay unfertilised eggs for 20 days. In the non-switch group, females were paired with a male (first male) soon after their adult moult (mean = 1.78 ± 1.06 days) and, after the onset of oviposition, allowed to mate and lay eggs for 20 days. The eggs laid over the first 20 days were then removed. The number of eggs laid during this 20-day period did not differ between unpaired (switch treatment) females and male-paired (non-switch treatment) females (see Fig. 4A in Wilner et al., 2025a). Switch treatment females were then paired with a male for 20 days, while non-switch treatment females were paired with a new male (second male) for 20 days (Fig. 2). Following male-pairing (switch treatment) or replacement (non-switch treatment), we collected eggs laid by each female over two successive 10-day periods.