Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

Abstract

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Polycalin is involved in the toxicity and resistance to Cry1Ac toxin in Helicoverpa armigera (Hübner)

“…Cadherin fragment from Anopheles gambiae and Manduca sexta could enhance the insecticidal activity of Cry4Ba and Cry1Ab, respectively (Peng, Xu, Ye, Yu, & Sun, 2010). Overexpression ALP and APN in the insect cells could significantly increase the toxicity of Cry1Ac (Wei, Liang et al, 2016; Wei, Yang et al 2018). Stevens, Song, Bruning, Choo, and Baxter (2017) reported they expressed P. xylostella ABCC2 gene in transgenic Drosophila caused this transgenic Drosophila susceptible to Cry1Ac.…”

“…Meanwhile, RNA interference (RNAi) and CRISPR/Cas9 technology recently were used to reduce the expression of target genes or proteins to confirm the function of the Cry toxins candidate receptor proteins. Reduce the expression of cadherin, ALP, APN, and ABCC2 could significantly reduce insecticidal activity of Cry1Ac by RNAi or CRISPR/Cas9 technology (Wang, Wang, Liu, Liu, & Wu, 2017; Wei, Liang et al, 2016, Wei, Zhang et al 2016; Wei, Yang et al, 2018; Xiao et al, 2016,). Equally important, antisera blocking technology was also employed to study the function of the Cry toxins candidate receptors.…”

“…The diet overlay bioassays were used to obtain the LC 98 of Cry1Ac toxin to larvae of susceptible H. armigera (hatched within 6 hr; Wang et al, 2019; Wei, Liang et al, 2016). In brief, 1.0 ml fresh artificial diet was added into each well of 24‐well plates (TianJin Xiangyushun Co., TianJin, China).…”

Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera

“…However, the mode of action of Cry1Ac is very complex and a change in any step of the toxicology process will inevitably lead to insect resistance. The most common Bt-resistant mechanisms had been reported in Lepidoptera, including a reduced binding capacity of Bt toxins to midgut receptors by a decrease in the activity and transcription of ALP or APN as well as mutations of APN, cadherin, and ABCC 2 (Xu et al, 2005 ; Zhang et al, 2009 , 2012 ; Gahan et al, 2010 ; Baxter et al, 2011 ; Jurat-Fuentes et al, 2011 ; Atsumi et al, 2012 ; Xiao et al, 2014 ; Chen et al, 2015 ) and a reduced conversion of the protoxin to the toxin by downregulation of trypsin (Rajagopal et al, 2009 ; Cao et al, 2013 ; Liu et al, 2014 ; Wei et al, 2016a ). These studies indicate that many genes are involved in resistance of insects to Bt toxins.…”

Comparisons of Transcriptional Profiles of Gut Genes between Cry1Ab-Resistant and Susceptible Strains of Ostrinia nubilalis Revealed Genes Possibly Related to the Adaptation of Resistant Larvae to Transgenic Cry1Ab Corn

“…For example, a Dipel-selected strain of O. nubilalis was found to be associated with reduced protease activity and its resistance can be virtually abolished when resistant insects were fed on transgenic Bt corn (MON810-event) [6,10]. Similar resistance mechanisms have been found in other insect species [11]. Changes of Bt receptors, such as cadherin-like protein (Cad), aminopeptidase N (APN) and alkaline phosphatase (ALP) may also lead to Bt resistance in insects.…”