Toxicometabolomic profiling of resistant and susceptible western corn rootworm larvae feeding on Bt maize seedlings

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious pest of maize (Zea mays L.) in the U.S. Corn Belt and parts of Europe. Transgenic maize hybrids expressing at least one of the four currently available insecticidal toxins from Bacillus thuringiensis (Bt) Berliner, currently the most widely adopted control method in continuous maize, have faltered due to the emergence of resistance. The resistance mechanisms of WCR to Bt toxins are not fully understood. We identified metabolic profiles of susceptible and resistant WCR larvae fed on maize hybrids expressing each of three available Cry3 proteins (eCry3Ab1, mCry3A, and Cry3Bb1) targeting corn rootworms and a control non-Bt maize via an untargeted metabolomics approach. Over 580 unique metabolites found in WCR larvae were classified into different pathways (amino acids, carbohydrates, cofactors and vitamins, energy, lipid, nucleotide, peptide, and xenobiotics). By exploring shifts in WCR larval metabolome exclusively by Bt toxins, several candidate metabolites and metabolic pathways were identified in susceptible and resistant larvae that may be involved in defense against or recovery from Bt ingestion by these larvae. These findings would provide mechanistic insights into altered metabolic pathways associated with the resistance mechanisms of WCR to Bt toxins.

Figure 1

Differences in metabolic profiles of WCR susceptible (a) and resistant insects (b) fed on control non-Bt maize compared with those fed on eCry3.1Ab-, mCry3A-, or Cry3Bb1-expressing maize seedlings using the orthogonal partial least squares—discriminant analysis (OPLS-DA). In the OPLS-DA plot, circles with same colors represent replicates of metabolic profiles for each treatment. The colored ellipses indicate 95% confidence regions of metabolic profiles for each treatment. In (a), from left to right, the circles display susceptible insects fed on eCry3.1Ab-, mCry3A-, Cry3Bb1-expressing maize and non-Bt maize, respectively. In (b), from left to right, the circles represent eCry3.1Ab resistant insects fed on eCry3.1Ab-expressing maize and non-Bt maize, mCry3A-resistant insects fed on mCry3A-expressing maize and non-Bt maize, Cry3Bb1-resistant insects fed on Cry3Bb1-expressing maize and non-Bt maize, respectively.

Figure 2

Heatmap representing differences in the levels of identified metabolites expressed differently in WCR susceptible larvae fed on control non-Bt maize compared with those fed on eCry3.1Ab-, mCry3A-, or Cry3Bb1-expressing maize seedlings. The metabolite annotation was confirmed with authentic standards, expected for the metabolites followed with (’) that were annotated based on their available identities (e.g., m/z, mass spectra). Asterisks (*) represent significant differences (p < 0.05) of metabolites in the susceptible insects fed on Bt maize compared with those fed on the non-Bt maize.

Figure 3

Heatmap for the identified metabolites expressed differently in WCR resistant larvae fed on control non-Bt maize compared with those fed on eCry3.1Ab-, mCry3A-, or Cry3Bb1-expressing maize seedlings. The metabolite annotation was confirmed with authentic standards, expected for the metabolites followed by (’) that were annotated based on their available identities (e.g., m/z, mass spectra). Asterisks (*) represent significant differences (p < 0.05) of metabolites in the resistant insect fed on their respective Bt maize with those fed on the non-Bt maize.

Figure 4

KEGG pathway analysis of differences in the identified metabolic pathways in susceptible (a), and resistant (b) western corn rootworm larvae fed on Bt-expressing maize seedlings (metabolite numbers ≥ 2).