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The initial development of the Nasoniain vitro germ-free rearing system in 2012 enabled investigations of Nasonia-microbiota interactions and real-time visualization of metamorphosis. However, the use of antibiotics, bleach, and fetal bovine serum in this in vitro rearing system introduced artifacts relative to conventional rearing of Nasonia. Here, we optimize the germ-free rearing procedure by using filter sterilization in lieu of antibiotics and by removing residual bleach and fetal bovine serum. Comparison of these methods reveals no influence on larval survival or growth, and a 52% improvement in adult production. Additionally, adult males produced in the new germ-free system are similar in size to conventionally reared males. Experimental implications of these changes are discussed.
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