Effects of cross-generational inbreeding and Wolbachia infection on sex ratio and life-history traits in parthenium beetle

Abstract Sex ratio is a key demographic parameter shaping population dynamics and evolutionary trajectories. In biocontrol agents, demographic bottlenecks during species introduction to a new habitat and subsequent mass rearing can elevate inbreeding, potentially biasing sex ratios through sex-specific mortality associated with inbreeding depression. Moreover, reproductive endosymbionts such as Wolbachia are known to manipulate host reproduction and further skew sex ratios. However, the relative contributions of these processes to sex-ratio variation remain poorly resolved. In this study, we evaluated the effects of cross-generational full-sibling inbreeding and Wolbachia infection on sex ratio and key life-history traits in the biocontrol beetle Zygogramma bicolorata using controlled laboratory crosses across three generations. Inbreeding did not significantly alter offspring sex ratio, which remained close to parity across generations, while pupal mortality increased in later generations, consistent with delayed expression of inbreeding depression. Adult body weight remained largely unaffected by inbreeding. Wolbachia infection was detected in a subset of females and was associated with a modest but significant increase in female-biased offspring production, although the effect was variable across lineages. Strain typing identified a single supergroup A Wolbachia, consistent with previous descriptions of the wBic strain from this species. These findings indicate that sex-ratio variation in introduced populations of Z. bicolorata is not driven by inbreeding alone but instead emerges from the interaction between demographic processes and symbiont-mediated effects, providing crucial insights for optimizing biocontrol programs where sex-ratio stability is essential for population establishment and persistence. Significance Sex ratio is a key determinant of population growth and stability – the essential parameters determining success of biocontrol programs. Yet, the mechanisms shaping sex-ratio variation remain poorly resolved. Using controlled crosses in Zygogramma bicolorata, we show that short-term inbreeding does not directly alter sex allocation, despite inducing delayed fitness costs through increased pupal mortality. In contrast, Wolbachia infection contributes to female-biased offspring production, although with variable outcome across lineages. These findings demonstrate that sex-ratio variation in Z. bicolorata arises from the interaction of demographic processes and symbiont effects, rather than a single mechanism, with important implications for predicting the establishment, persistence, and efficacy of mass-reared biocontrol populations. Competing Interest Statement The authors have declared no competing interest.