Carotenoids moderate the effectiveness of a Bt gene against the European corn borer, Ostrinia nubilalis

Abstract

We assessed the effectiveness of a biofortified maize line (4BtxHC) which accumulates high levels of antioxidant carotenoids that also expressed the insecticidal Cry1Ac Bacillus thuringiensis (Bt) gene against the European corn borer Ostrinia nubilalis. This line had been previously engineered to accumulate carotenoids specifically in the seed endosperm, whereas the Bt gene was expressed constitutively. The concentrations of Bt toxin (Cry 1Ac) in the leaves of the 4Bt and 4BtxHC lines were not significantly different at 47±6 μg/g of fresh weight (FW); neither were they in the kernels of both lines (35±3 μg/g FW). The kernels and leaves were toxic to the larvae of O. nubilalis. However, the insecticidal activity was substantially lower (ca. 20%) than that of lines that expressed only Bt in spite that the two lines showed a quantity of toxin not significantly different in kernels or in leaves. Although the reduced effectiveness of Cry1Ac in kernels may not be entirely surprising, the observation of the same phenomenon in vegetative tissues was unexpected. When semi-artificial diets containing kernels from 4Bt supplemented with different levels of β-carotene were used in insect bioassays, the β-carotene moderated the effectiveness of the Bt similarly to the plant material with carotenoid enrichment. To elucidate the biochemical basis of the reduced effectiveness of Bt toxin in the carotenoid-enriched plants, we measured the activity of three enzymes known to be implicated in the detoxification defence, namely, catalase, superoxide dismutase and glutathione S-transferase. Whereas Cry1Ac expression significantly increased SOD and CAT enzymatic activity in the absence of carotenoids, carotenoids, either in 4BtxHC or in artificial diets enriched with β-carotene, significantly lowered CAT activity. Carotenoids can therefore moderate the susceptibility of the maize borer O. nubilalis to Cry1Ac, and we hypothesize that their role as antioxidants could explain this phenomenon via their scavenging of reactive oxygen species produced during Cry1Ac detoxification in the larvae. The involvement of this mechanism in the decreased mortality caused by Cry1Ac when carotenoids are present in the diet is discussed.

β-carotene and Bacillus thuringiensis insecticidal protein differentially modulate feeding behaviour, mortality and physiology of European corn borer (Ostrinia nubilalis)

“…Semiartificial diets used for insect rearing were modified to include Cry1A and different β-carotene concentrations (denoted in diets as β) ( Table 1 ). The proportion of freeze-dried leaf material to β-carotene aimed to mimic concentrations found in existing maize varieties [ 17 ]. Cry1A concentrations in Bt and Bt-β diets were verified with the Agdia Bt-Cry1Ab/Cry1Ac kit (Agdia Inc., Elkhart, Indiana, US).…”

“…No-choice feeding assays were conducted to determine whether β-carotene can alter the effect of Bt insecticidal protein on first-instar larvae. L5 larvae had been tested in a previous experiment [ 17 ].…”

“…An improved version of HC maize (HC × Bt) was engineered with the B . thuringiensis (Bt) Cry1A protein to confer insect-pest resistance [ 11 , 17 ]. Bt insecticidal protein consumption increases oxidative stress in larvae midgut [ 18 ].…”

“…nubilalis larvae fed HC × Bt diets had lower mortality than larvae fed diets of plants containing only Cry1A [ 17 ]. When added to Bt diets, β-carotene reduced susceptibility of fifth-instar larvae to Cry1A through scavenging reactive oxygen species (ROS) produced during Cry1A detoxification [ 17 ]. Other antioxidants show a similar antagonistic behaviour to Cry1A.…”

Laboratory tests with Lepidoptera to assess non-target effects of Bt maize pollen: analysis of current studies and recommendations for a standardised design

“…Using artificial diets rather than plant material makes it easier to standardise experimental conditions, but diet composition may influence effects of Bt toxins on larvae. For instance, a high concentration of carotenoids can decrease larval mortality due to Bt [48]. Furthermore, it should be ensured that no antibiotics are added to the diet as this can influence the Bt effect on larvae, too [49,50].…”