Parental effects of Bt toxin and vitamin A on Helicoverpa armigera.

The increase in the area cultivated with vitamin-enriched transgenic crops producing Bt toxin raises the question of whether the addition of vitamins will in any way mitigates the effect of the toxin on the phytophagous insects that feed on those crops. On the other hand, the parental effect that feeding on these enriched transgenic crops may have on the offspring of the phytophagous that survive on them is not well known. In this work, the effect of vitamin A (ß-carotene) addition to diets with or without Bt toxin on Helicoverpa armigera larvae and their offspring was determined. The addition of vitamin A did not have any beneficial effect either for the larvae fed on enriched diets nor for their offspring. However, parental effects due to dietary feeding with the toxin were detected since adults from larvae fed on the Bt diet had higher mating success than those fed on the toxin-free diet, although there were no differences on the fertility of mated females regardless of whether their previous larvae fed on the Bt or non-Bt diet. A certain adaptive effect to the toxin was also noted since the mortality of larvae whose previous generation fed on diet with Bt was lower than that of the larvae that came from larvae fed on a non-Bt diet. It would be interesting to determine if H. armigera adults prefer to mate and lay eggs in the same type of crops in which they have developed or if feeding on different crops, such as corn or alfalfa, causes different paternal effects on the offspring. These aspects can be of great importance in the development of resistance of this species to the Bt toxin.

Fitness Costs of Two Maize Lepidopteran Pests Fed on Bacillus thuringiensis (Bt) Diets Enriched with Vitamins A and C.

Serious malnutrition problems occur in developing countries where people's diets are mainly based on staple crops. To alleviate this, high-production crops are being developed that are better adapted to climate change, enriched in micronutrients and vitamins, or resistant to pests. In some cases, new varieties have been developed with several of the characteristics mentioned above, such as biofortified and pest-resistant crops. The development of biofortified Bacillus thuringiensis (Bt) crops raises the question of whether vitamin enrichment of Bt crops can in any way favor those pests that are not very susceptible to the Bt toxin that feed on these crops, such as Helicoverpa armigera (Hübner) or Mythimna unipuncta (Haworth) (Lepidoptera: Noctuidae). In this study, the response to a Bt diet enriched with vitamins A (ß-carotene) and C (ascorbic acid) was somewhat different between the two species. M. unipuncta was less sensitive to the toxin than H. armigera, although the ingestion of the Bt diet resulted in oxidative stress (longer larval development and lower pupal weight) which was not mitigated by the vitamins. However, the two vitamins reduced the mortality of H. armigera larvae fed on a Bt-enriched diet; in addition, ß-carotene reduced the activity of the antioxidant glutathione S-transferase (GST) of both species, suggesting it has an antioxidant role. The results obtained here indicate that biofortified Bt crops will not favor the development of H. armigera very much and will not affect M. unipuncta's development at all, although the effect of the increase in vitamins may be very variable and should be studied for each specific phytophagous.

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

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 ß-carotene were used in insect bioassays, the ß-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 ß-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.

A carotenogenic mini-pathway introduced into white corn does not affect development or agronomic performance.

High-carotenoid corn (Carolight®) has been developed as a vehicle to deliver pro-vitamin A in the diet and thus address vitamin A deficiency in at-risk populations in developing countries. Like any other novel crop, the performance of Carolight® must be tested in different environments to ensure that optimal yields and productivity are maintained, particularly in this case to ensure that the engineered metabolic pathway does not attract a yield penalty. Here we compared the performance of Carolight® with its near isogenic white corn inbred parental line under greenhouse and field conditions, and monitored the stability of the introduced trait. We found that Carolight® was indistinguishable from its near isogenic line in terms of agronomic performance, particularly grain yield and its main components. We also established experimentally that the functionality of the introduced trait was indistinguishable when plants were grown in a controlled environment or in the field. Such thorough characterization under different agronomic conditions is rarely performed even for first-generation traits such as herbicide tolerance and pest resistance, and certainly not for complex second-generation traits such as the metabolic remodeling in the Carolight® variety. Our results therefore indicate that Carolight® can now be incorporated into breeding lines to generate hybrids with locally adapted varieties for further product development and assessment.

Freedom-to-operate analysis of a transgenic multivitamin corn variety.

In this article, we explore the intellectual property (IP) landscape relevant to the production and commercialization of Carolight(™) , a transgenic multivitamin corn variety created on humanitarian grounds to address micronutrient deficiencies in low-and-middle-income countries. The successful production of this variety requires IP rights risk management because there is a strong protection on inventions and processes via patent portfolios in both developing and industrialized countries. The IP framework is complex, and specialist patent lawyers are usually employed to perform such analysis, but the costs cannot always be met by small, publicly funded projects. We report an alternative strategy, a do-it-yourself patent analysis, to produce a review with limited legal value that can nevertheless lay the foundations for a subsequent more in-depth professional freedom-to-operate opinion.