Larval Mid-Gut Responses to Sub-Lethal Dose of Cry Toxin in Lepidopteran Pest Achaea janata.

Vinod K Chauhan,Balasubramanian Senthilkumaran,Aparna Dutta-Gupta,Narender K Dhania,R K Chaitanya

doi:10.3389/fphys.2017.00662

Vinod K Chauhan, Balasubramanian Senthilkumaran + Show 3 more

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https://doi.org/10.3389/fphys.2017.00662

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Abstract

The lack of homogeneity in field application of Bacillus thuringiensis formulation often results in ingestion of sub-lethal doses of the biopesticide by a fraction of pest population and there by promotes the toxin tolerance and resistance in long term. Gut regeneration seems to be one of the possible mechanism by which this is accomplished. However, the existing information is primarily derived from in vitro studies using mid-gut cell cultures. Present study illustrates cellular and molecular changes in mid-gut epithelium of a Bt-susceptible polyphagous insect pest castor semilooper, Achaea janata in response to a Cry toxin formulation. The present report showed that prolonged exposure to sub-lethal doses of Cry toxin formulation has deleterious effect on larval growth and development. Histological analysis of mid-gut tissue exhibits epithelial cell degeneration, which is due to necrotic form of cell death followed by regeneration through enhanced proliferation of mid-gut stem cells. Cell death is demonstrated by confocal microscopy, flow-cytometry, and DNA fragmentation analysis. Cell proliferation in control vs. toxin-exposed larvae is evaluated by bromodeoxyuridine (BrdU) labeling and toluidine blue staining. Intriguingly, in situ mRNA analysis detected the presence of arylphorin transcripts in larval mid-gut epithelial cells. Quantitative PCR analysis further demonstrates altered expression of arylphorin gene in toxin-exposed larvae when compared with the control. The coincidence of enhanced mid-gut cell proliferation coincides with the elevated arylphorin expression upon Cry intoxication suggests that it might play a role in the regeneration of mid-gut epithelial cells.

Highlights

Bacillus thuringiensis (Bt) strains synthesize different toxins including crystal (Cry), cytolytic (Cyt), and vegetative insecticidal proteins (Vip)
Lepidopteran larvae experimentally used to demonstrate the mode of action of Cry toxin, showed sequential event of toxin solubilization, its activation induced by alkaline pH of the gut, proteolytic cleavage, binding of toxin with different receptor molecules, oligomerization, and membrane insertion leading to pore formation
The effects of a sub-lethal dose of Bt toxin was analyzed through the mid-gut responses in A. janata larvae

Summary

Introduction

Bacillus thuringiensis (Bt) strains synthesize different toxins including crystal (Cry), cytolytic (Cyt), and vegetative insecticidal proteins (Vip). Alteration in mid-gut binding sites leading to reduced interaction of Bt toxins to their receptor, as a major mechanism for the development of resistance (Pigott and Ellar, 2007). Since many Cry toxins share common binding sites, the probability of cross-resistance was often found to be high (Ferre and Van Rie, 2002; Tabashnik et al, 2013) In agreement with these reports, insects such as diamond black moth, Plutella xylostella; Indian meal moth, Plodia interpunctella, and cabbage looper, Trichoplusia ni have evolved resistance to commercial Bt sprays in the open field and greenhouses (Ferre et al, 1991; Oppert et al, 1997; Janmaat and Myers, 2003). Field-evolved pest resistance to Bt corn (maize stem borer, Busseola fusca; western corn rootworm, Diabrotica virgifera virgifera; and fall armyworm, Spodoptera frugiperda), Bt cotton (cotton bollworm, Helicoverpa armigera, and pink bollworm, Pectinophora gossypiella), and Bt maize (Spodoptera frugiperda) have been reported (Storer et al, 2010; Dhurua and Gujar, 2011; Kruger et al, 2011; Zhang et al, 2011; Jakka et al, 2015; Pereira et al, 2015)

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