Optimized conditions for the long-term growth of primary cell cultures derived from the southern green stink bug, Nezara viridula.

The southern green stink bug, Nezara viridula L., is a highly polyphagous global pest that causes substantial economic losses on a broad range of crops. Current management strategies rely heavily on chemical insecticides, which can promote resistance development and negatively affect non-target beneficial organisms. The lack of a tractable in vitro cell culture system has limited the development of more targeted and sustainable control approaches for this species. In this study, we identified the optimal egg age for culture initiation and evaluated seven insect cell culture media or medium combinations, as well as several supplements, for their ability to support embryo-derived N. viridula primary cultures. A total of 200 primary cultures were initiated using ~ 6,000 dissected N. viridula eggs over 16 months. Of the media tested, GS medium, a combination of CLG#2 and Shields and Sang medium, supplemented with 9% heat-inactivated fetal bovine serum produced, on average, 40% more attached cell clusters than CLG#2, the next best medium, although this difference was not statistically significant. GS medium also supported the long-term survival and growth of N. viridula primary cells for more than 22 months. Several chemical additives previously reported to enhance insect cell proliferation did not improve N. viridula primary cell growth or survival. The optimized culture conditions described here provide a foundation for the establishment of continuous N. viridula cell lines and support future studies of host-pathogen interactions, insect physiology, and the development of novel pest management strategies.