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

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.

Midgut transcriptome analysis of Clostera anachoreta treated with lethal and sublethal Cry1Ac protoxin

“…Whereas prolonged exposure to sublethal doses of the toxin inhibits growth, development, and reproduction of larvae, imposing a fitness cost, it might also promote tolerance and resistance of insects to toxins. An earlier report indicated that sublethal doses of toxin had deleterious effects on growth and recovery of Achaea janata ; however, the insects exhibited epithelial cell proliferation in midgut tissue that coincided with elevated arylphorin expression (Chauhan et al, ), similar to the results reported by Castagnola et al (), in H. virescens . These findings identified α‐arylphorin as a protein with a putative role in response to Cry1Ac intoxication.…”

“…In the P. xylostella midgut transcriptome, 2,925 and 2,967 unigenes were differently expressed between two resistant strains compared to susceptible strains, respectively (Lei et al, ). The DEGs were primarily associated with Bt receptors (e.g., ABC transporter, APN, ALP, and CAD), digestive function (α‐amylase, lipase, protease, and carboxypeptidase), immune response (HSC70 and IL22), detoxication enzymes (P450, GST, and CarE), developmental genes (arylphorin), and a variety of binding proteins including cellular retinoic acid‐binding protein (lipid‐binding), insulin‐related peptide‐binding protein (protein‐binding), and ovary C/EBPg transcription factor (nucleic acid‐binding; Sparks, Blackburn, Kuhar, & Gundersen‐Rindal, ; Yang et al, ; Jurat‐Fuentes, Karumbaiah, & Jakka, ; Ocelotl et al, ; Shiho et al, ; Tristan, Sisi & John, , Yao et al, ; Castagnola et al, ; Chauhan et al, ). In this study, the common DEGs were also primarily associated with digestion function, with enriched GO terms including monocarboxylic acid metabolic process, pyruvate metabolic process, lipid biosynthetic process, and serine‐type endopeptidase activity.…”

Midgut Transcriptome Analysis ofClostera anachoretaTreated with Cry1Ac Toxin

“…In this study, the larval midgut transcriptome was sequenced and analyzed by Novogene, and 151,090 high-quality unigenes were obtained, including 1660, 1175, and 562 DEGs between the three larvae treatments with Cry1Ac toxin and the control, respectively. In the P. xylostella midgut transcriptome, 2925 and 2967 unigenes were differently expressed between two resistant strains compared to susceptible strains, Chauhan et al, 2017). In this study, the common DEGs were also primarily associated with digestion function, with enriched GO terms including monocarboxylic acid metabolic process, pyruvate metabolic process, lipid biosynthetic process, and serine-type endopeptidase activity.…”

“…These genes were significantly enriched in nine functional categories, four of which, Glutathione metabolism, Drug metabolism-cytochrome P450, Metabolism of xenobiotics by cytochrome P450, and Chemical carcinogenesis, are related to detoxification and immunity (Table 10). (Chauhan VK, et al 2017). In the present study, a 7.5 µg/mL dosage of Bt Cry1Ac toxin solution with sodium carbonate represented a sub-lethal dose, much lower than for other species, suggesting that the C.…”

Midgut aminopeptidase N expression profile in Castor semilooper during sublethal Cry toxin exposure

“…However, in CF1 cells the Cry toxicity was not associated with PKA pathway (Portugal et al, 2017). Recent studies revealed that Cry toxin induces necrotic cell death in B.mori ABCC2 transfected Sf9 cells (Endo et al, 2017) as well as in larval midgut of A. janta (Chauhan et al, 2017).…”