Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Abstract

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.

Association of Cry1Ac Toxin Resistance in Helicoverpa zea (Boddie) with Increased Alkaline Phosphatase Levels in the Midgut Lumen

“…Alternative resistance mechanisms described to date include alterations in protoxin activation (34), mutations in ABC transporter proteins (14), toxin degradation (13), faster replacement of midgut epithelial cells (28), elevated immune response (16), and toxin sequestration by esterases (15) or lipophorin (27).…”

The transcriptome of Spodoptera exigua larvae exposed to different types of microbes

“…The Xen-R colony was derived from insects collected in cotton fields in Prattville, AL, USA. This colony was selected for several years with increasing concentrations of XentariÔ, a commercial bio-pesticide based on B. thuringiensis (Hernandez-Martinez et al, 2010b). All colonies were reared on artificial diet at 25 AE 3 C with 70 AE 5% RH and a photoperiod of 16/8 h (light/dark).…”

“…Although application of these potential markers would require additional independent validation, they constitute a valuable data set for the generation of a genetic map of S. exigua. One possible use of having a genetic map is mapping resistance genes in several strains of S. exigua that have been characterized for their resistance to chemical (Jia et al, 2009) as well as to biological pesticides (Moar et al, 1995;Hernandez-Martinez et al, 2010b). Moreover, the development of NGS technology has speed up the genome sequencing of non-model organisms.…”

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

“…9 Mutations in the genes encoding midgut cadherins that bind Cry1Ac are linked with resistance in at least three lepidopteran pests of cotton, [16][17][18] but such cadherin mutations are not the primary cause of many other cases of field-and laboratory-selected resistance. 9,19,20 Although some aspects of the mode of action of Bt toxins remain unresolved, extensive evidence shows that after Cry1A protoxins are ingested by larvae, they are solubilized in the gut and cleaved by mid-gut proteases such as trypsin to yield activated 60-kD monomeric toxins that bind with membrane-associated receptors. 14,15 The signaling model suggests that after protease-activated monomeric toxins bind to cadherin, initiation of a magnesium-dependent signaling pathway causes cell death.…”