Curing Parthenogenesis-Inducing (PI) Wolbachia-Induced Reproductive Disorders in the Egg Parasitoid Telenomus remus

<i>Wolbachia</i> is an endosymbiotic bacterium widespread in invertebrates that causes various reproductive effects, including cytoplasmic incompatibility, feminization, male killing, and the induction of parthenogenesis (PI). PI-<i>Wolbachia w</i>Rem converts <i>Telenomus remus</i>, an egg parasitoid of <i>Spodoptera frugiperda</i>, from arrhenotokous reproduction (male-producing) to thelytokous reproduction (female-producing). Long-term symbiosis between egg parasitoids and <i>Wolbachia</i> has been shown to lead to reproductive barriers and "female functional virginity," causing progressive and potentially irreversible sex ratio imbalances. However, whether such reproductive barriers occur in <i>T. remus</i> remains unknown, which has important implications for biological control programs utilizing this parasitoid. To address this question, we cured <i>w</i>Rem using tetracycline and conducted crossing experiments with naturally uninfected strains (W-). The results indicated that the cured strain (Wcure) retained normal sexual reproductive capability, with self-crossing fertilization rates comparable to those of W- strains. However, first-generation hybridization between Wcure and W- strains produced strongly male-biased offspring (male proportion: 94.3% and 85.8% for W-♂ × Wcure♀ and Wcure♂ × W-♀, respectively), indicating substantial reproductive incompatibility. Notably, an asymmetric pattern was observed between reciprocal crosses. In second-generation hybridization experiments, hybrid females (W-/Wcure) mated with W- or Wcure males showed markedly recovered sex ratios (male proportion: 14.3% and 15.6%, respectively), although total offspring numbers remained lower than in self-crossing groups. These results suggest that the reproductive incompatibility in <i>T. remus</i> differs from female functional virginity and is more consistent with mitonuclear incompatibility arising from population divergence. The partial recovery in second-generation hybrids indicates that surviving F1 hybrid females likely represent individuals selected for compatibility, rather than exhibiting progressive deterioration of sexual function. These findings offer insights into <i>Wolbachia</i>'s impact on parasitoid reproduction and highlight key considerations for biological control applications, underscoring the importance of evaluating reproductive barriers before deploying cured strains and preventing symbiont loss within populations.