Bt toxins: using flies. moths, and oocytes to unravel a complex mode of action.

Bacillus thuringiensis produce pore forming toxins that have been used for insect pest control for nearly 100 years, yet the process by which they damage insect midgut cells remains controversial. They are ingested as protoxins and undergo activation in insect larval midguts, the activated toxins interact with one or more membrane bound receptors and form pores on the surface of target cells. Experimental evidence from cell lines has demonstrated this process causes ion and water influx, which results in swelling and lysis, although alternative models support intracellular activation of cell death pathways. This thesis aims to assess the role of ion and water movement in midgut cells following the formation of Bt pores using in-vivo systems and improve our understanding of Bt induced insect mortality. The first research chapter uses transmission electron microscopy (TEM) and transcriptome sequencing of insect midgut tissues to identify phenotypic and genetic responses to Bt toxins. The brassica pest Plutella xylostella, and transgenic Drosophila melanogaster expressing a P. xylostella toxin receptor, ABCC2, are both investigated. TEM did not show any clear evidence of cell lysis occurring in either organism, although swelling of the basal labyrinth supported water influx. Other dramatic changes to cell morphology were also observed, including shortened microvilli, rapid accumulation of mitochondria and bacterial proliferation which may lead to death via septicemia. Transcriptome analysis determined the expression of some bacterial immune response genes in Drosophila were reduced, which may enhance bacterial invasion. Changes to genes involved with cell death pathways were not observed. These results suggest, whilst there is evidence of water influx, midgut cells may have a mechanism to compensate and prevent cell lysis and expel excess water into the haemolymph. Furthermore, midgut damage caused by Bt toxins may indirectly inhibit midgut immune responses allowing invasion and proliferation of bacteria. The second research chapter evaluates the involvement of membrane bound aquaporin water channels in Bt toxin mode of action. Aquaporins have been shown to facilitate water influx in cell lines following Bt toxin exposure. Here, the Xenopus expression system is used to functionally assess five Drosophila aquaporins genes (Drip, Prip, Eglp2, Eglp3 and Eglp4) for passive water and glycerol channelling using swelling assays and ion channelling using two electrode voltage clamp experiments. A yeast expression system also tests the ability of aquaporins to act as ion channels. Results show the Drosophila aquaporin Drip functions as a water channel and Eglp2 as a dual water and glycerol channel. All five midgut aquaporins assessed have potential ion channel functions when tested in the yeast expression system, but only Eglp2 showed ion channel function in oocytes when activated with cGMP. Finally, I assess the involvement of water channel aquaporins in Bt toxin mode of action using Drosophila larval bioassays, although the use of inhibitors or manipulating expression of aquaporin genes did not support their involvement in Bt toxin mode of action in-vivo. The final research chapter evaluates the loss of ion homeostasis as a factor in Bt toxin mode of action. Current models do not consider the effect of influx of specific ions into cells because of Bt pore formation. Here I manipulate ion content in Drosophila larval diet and perform bioassays with Bt toxin Cry1Ac to test their impact on Bt-induced mortality. I employed the voltage clamp system in Xenopus laevis oocytes to investigate the role of PxABCC2 and PxABCC3, Bt toxin receptors from Plutella, and develop support for specific ions that are likely to pass through Bt pores. Results show Ca2+ content in diet increased mortality in the presence of Bt toxin, PxABCC2 and PxABCC3 facilitates pore formation in oocytes although PxABCC3 is less effective. Results also indicate the involvement of Reactive Oxygen Species (ROS) in Bt toxin mode of action. Overall, the results suggest that oxidative stress plays an important role in damaging midgut cells and eventual insect death. Work in this thesis improves our understanding of how Bt toxin causes insect death and proposes a general model. Understanding Bt toxin specificity towards targeted pests is anticipated to improve provide public confidence in the use of these insecticides when applied as foliar sprays or in transgenic crops. The human population is expected to rise to ~9.5 billion by 2050 and increase pressure on food production and food security. Bt toxins are likely to play an important role in reducing the impact of insect pests in agriculture and to improve cropping quality and yield.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202