The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

Authors: CAI, YANJUN - Nanjing Agricultural University; HOU, BOFENG - Nanjing Agricultural University; Fabrick, Jeffrey; YANG, YIHUA - Nanjing Agricultural University; WU, YIDONG - Nanjing Agricultural University

Submitted to: Pesticide Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/31/2024
Publication Date: 9/1/2024
Citation: Cai, Yanjun, Hou, Bofeng, Fabrick, J.A., Yang, Yihua, Wu, Yidong 2024. The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera. Pesticide Biochemistry and Physiology. 204. Article 106068. https://doi.org/10.1016/j.pestbp.2024.106068.
DOI: https://doi.org/10.1016/j.pestbp.2024.106068

Interpretive Summary: Insecticidal crystalline (Cry) proteins derived from the bacterium Bacillus thuringiensis (Bt) are widely used to manage key insect pests. Cry proteins exert toxic action by forming pores in the membranes of midgut cells ultimately resulting in cell lysis and insect death. This cell lysis likely results from the rapid influx of water through aquaporin (AQP) channel proteins following the formation of ion-specific pores caused by Cry proteins. Here, an ARS scientist from Maricopa, AZ, and collaborators tested the role of AQPs in Cry1Ac-induced cell damage in the cotton bollworm, Helicoverpa armigera, one of the world’s most damaging insect pests. Eight different H. armigera AQPs were identified of which five were shown to be functional water channel proteins. Three of these HaAQPs are present in the midgut and function to enhance water influx in the presence of Cry1Ac toxin and a known Cry1Ac receptor protein. Although gene editing was successful in creating H. armigera knockout lines each lacking one of the three HaAQPs, no change in susceptibility to Cry1Ac was observed in knockout larvae compared to the susceptible parental line, indicating the loss of individual HaAQPs fails to cause Cry1Ac toxin resistance. These results suggest that either the midgut HaAQPs can compensate for each other allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation or that HaAQPs play a passive and perhaps nonessential role in Bt intoxication and do not directly cause resistance to Cry1Ac.

Technical Abstract: The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.