Hypoxia-induced immortalization of Helicoverpa armigera pupal ovarian cells

Establishing continuous cell lines from lepidopteran insects is notoriously inefficient, with success rates typically below 5% and often below 1%, severely limiting research and biopesticide development for pests such as Helicoverpa armigera (Lepidoptera: Noctuidae). We present a simple, reproducible method, hypoxia-induced immortalization that dramatically improves this efficiency. By applying a brief hypoxic stimulus (5% O 2 , 7 days) during primary culture of pupal ovary cells, we achieved an overall success rate of 17.8% across four independent batches, more than 3-fold higher than conventional normoxic methods. This high efficiency enabled the generation of eight continuous cell lines from the pupal ovaries, allowing systematic comparative analysis of early-passage dynamics. As a proof of concept, the IOZCAS-Ha-8 cell line exhibits high susceptibility to both homologous and heterologous baculoviruses. Hypoxia-induced immortalization provides a reproducible, high-throughput platform for generating well-characterized insect cell lines, facilitating studies on insect-virus interactions and biological control.