Conversion of a thelytokous to a stable bisexual line by non‐target effect antibiotic elimination of <i>Rickettsia</i> in <i>Anastatus gansuensis</i>

Heritable symbionts are key modulators of host biology, influencing reproduction and fitness. While antibiotic removal of symbionts is common, non-target effects on host fitness are often understudied. This is particularly true for Rickettsia, a widespread reproductive manipulator, and a stable, long-term (i.e., >7 generations) cured lineage in Hymenoptera has been lacking. This study aimed to fill this methodological gap by generating a cured bisexual lineage of parasitoid wasp with non-target effects of such treatments. Thus, we investigated not only the efficacy but also the non-target effects of three antibiotics: rifampicin, tetracycline, and sulfadiazine, administered at five concentrations (0.01, 0.05, 0.1, 1, and 10 mg/mL) in Rickettsia-infected thelytokous parasitoid Anastatus gansuensis (Hymenoptera: Eupelmidae). Survival, parasitism, emergence, and male rate were evaluated to determine the safety of antibiotic treatments, while Rickettsia titer reduction was used to assess elimination efficacy. Results showed that at a concentration of 0.01 mg/mL, tetracycline and rifampicin had minimal negative effects on host survival, parasitism, and emergence rates. However, prolonged exposure effectively eliminated Rickettsia, leading to the exclusive production of male offspring. Notably, short-term rifampicin feeding (0.01 mg/mL) across multi-generations successfully established a stable Rickettsia-cured bisexual line, confirmed via diagnostic PCR, quantitative PCR, and reproductive phenotyping over 10 generations. In contrast, sulfadiazine, previously effective against Wolbachia, had minimal impact on Rickettsia removal. This study provides a validated protocol for generating genetically stable aposymbiotic lines and a framework for assessing antibiotic specificity and non-target effects, enabling future studies of host adaptation and biological control in Rickettsia-cured parasitoids.