Optimized conditions for the long-term growth of primary cell cultures derived from the Asian citrus psyllid, Diaphorina citri (Liviidae: Hemiptera)

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a pest of significant importance to global citrus production, particularly as the vector of a phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) that causes the fatal citrus disease Huanglongbing or citrus greening. CLas is acquired as the psyllid feeds, replicates in ACP tissues, and persists throughout the life of the insect. The study of CLas has been hampered by the lack of a tractable in vitro culture system. As CLas replicates within psyllid tissues, we hypothesize that this bacterium also replicates in cultured ACP cells. In the current study, we evaluated a range of insect cell culture media, media combinations, and supplements for their ability to support the in vitro growth of ACP embryo-derived cells. Ninety-six primary cell cultures were initiated using approximately 12,000 dissected ACP eggs over a 12-month period. Of 19 media tested, 17 supported cell attachment, but only two media supported the long-term survival and growth of ACP embryonic cells over a period of more than 11 months. Delineation of the optimal protocols and conditions for the maintenance of ACP primary cultures as described here provides a foundation for both establishment of continuous cell lines and testing for the replication of ACP-associated pathogens including CLas.