The insecticidal crystal proteins from Bacillus thuringiensis (Bt) are widely-used biopesticides that are used both as Bt spore-crystal preparations in sprayable formulations and as activated toxins in genetically modified (GM) plants. Models for their modes of action have been proposed but many issues remain unresolved. Among those is the role of commensal gut bacteria in target insect death: previous studies showed that antibiotics attenuate the toxicity of Bt sprays. We tested whether antibiotics interfere with the effects of GM plant-produced Bt toxins in larvae of two Lepidopteran species, the European corn borer Ostrinia nubilalis and the cotton leafworm Spodoptera littoralis. The larvae were reared on artificial diet with or without antibiotics and, thereafter, fed two varieties of Bt GM maize in comparison to conventional non-Bt maize leaves sprayed with antibiotic solution and/or with a Bt formulation. Antibiotics significantly reduced or delayed the toxicity of Cry toxins, although to a lesser extent than previously reported for Bt-sprays. This supports the hypothesis that Cry toxins induce mortality by themselves in the absence of Bt bacteria and spores, and of commensal gut bacteria. However, larvae that were not treated with antibiotics died faster and at a higher rate which was further compounded by plant variety and species sensitivity. These findings support a hypothesis that toxicemia alone can inflict significant mortality. However, in the absence of antibiotics, the gut bacteria likely enhance the Cry toxin effect by inflicting, additionally, bacterial septicemia. This has important implications in field situations where antibiotic substances are present—e.g., from manure of animals from conventional production systems—and for ecotoxicological testing schemes of Bt toxins and nontarget organisms that are often using artificial diets enriched with high concentrations of antibiotics.

https://www.mdpi.com/2072-6651/10/12/489/pdf

Impact of Antibiotics on Efficacy of Cry Toxins Produced in Two Different Genetically Modified Bt Maize Varieties in Two Lepidopteran Herbivore Species, Ostrinia nubilalis and Spodoptera littoralis

Biosafety of a genetically modified crop is required to be assessed prior to its commercialization. For this, a suitable artificial diet was developed and used to establish a dietary exposure test for assessing the toxicity of midgut-active Bt insecticidal proteins on Chrysopa pallens (Rambur). Subsequently, this dietary exposure test was used to evaluate the toxicity of the proteins Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa on C. pallens larvae. Temporal stability, bioactivity, and the intake of the insecticidal proteins were confirmed by enzyme-linked immunosorbent assay and a sensitive-insect bioassay. The life history characteristics, such as survival, pupation, adult emergence, 7-day larval weight, larval developmental time, and emerged male and female fresh weights remained unaffected, when C. pallens were fed the pure artificial diet (negative control) and the artificial diets containing 200 μg/g of each purified protein: Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, or Vip3Aa. On the contrary, all of the life history characteristics of C. pallens larvae were adversely affected when fed artificial diet containing boric acid (positive control). The results demonstrate that diets containing the tested concentrations of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa have null effects on C. pallens larvae. The outcome indicates that genetically modified crops expressing the tested Bt proteins are safe for the lacewing, C. pallens.

Bt Proteins Have No Detrimental Effects on Larvae of the Green Lacewing, Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae)

Fate of multiple Bt proteins from stacked Bt maize in the predatory lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae).

BACKGROUND
Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNAi and hematoxylin and eosins (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of H. vigintioctopunctata.


RESULTS
The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the 1st and 2nd instars of H. vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a tyrosine hydroxylase inhibitor, disrupted larval-larval-pupal cuticle tanning during the 3rd - 4th instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to 4th instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates.


CONCLUSIONS
The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in H. vigintioctopunctata reproduction. This article is protected by copyright. All rights reserved.

Tyrosine hydroxylase invloves in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Bacillus thuringiensis protein Vip3Aa does not harm the predator Propylea japonica: A toxicological, histopathological, biochemical and molecular analysis.

Dietary exposure studies are initial steps in environmental risk assessments of genetically engineered plants on non-target organisms. These studies are conducted in the laboratory where surrogate species are exposed to purified and biologically active insecticidal compounds at higher concentrations than those expected to occur in transgenic crops foliage. Thus, dietary exposure (early tier) tests provide robust data needed to make general conclusions about the susceptibility of the surrogate species to the test substance. For this, we developed suitable artificial diet and used it to establish a dietary exposure test for assessing the toxicity of midgut-active insecticidal compounds to the larvae of the Asian ladybird beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Using boric acid as a model compound, we validated the bioassay established for H. axyridis larvae. An artificial diet containing boric acid which negatively affected survival, development and adult weights was offered to larvae and indicated that the bioassay was able to detect toxic effects of insecticidal substances incorporated in diets. Using this dietary exposure test, environmental risk assessment of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and the non-Cry protein Vip3Aa was evaluated by analysing pupation rates, adult emergence rates, 7-day larval weights, and freshly emerged male and female weights among the toxin treatments and a pure artificial diet. These life-table parameters did not vary among artificial diets containing 200 μg/g Bt proteins or pure artificial diet. In contrast, boric acid adversely affected all life-table parameters. Thus on these bases, we concluded H. axyridis larvae are not sensitive to these Bt proteins expressed in genetically engineered crops.

Bio‐safety evaluation of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and Vip3Aa on Harmonia axyridis larvae

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Bio‐safety evaluation of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and Vip3Aa on Harmonia axyridis larvae