Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid, Acyrthosiphon pisum.

Aphids are sap-sucking insects (order: Hemiptera) that cause extensive damage to a wide range of agricultural crops. Our goal was to optimize a naturally occurring insecticidal crystalline (Cry) toxins produced by the soil-dwelling bacterium Bacillus thuringiensis for use against the pea aphid, Acyrthosiphon pisum. On the basis that activation of the Cry4Aa toxin is a rate-limiting factor contributing to the relatively low aphicidal activity of this toxin, we introduced cathepsin L and cathepsin B cleavage sites into Cry4Aa for rapid activation in the aphid gut environment. Incubation of modified Cry4Aa and aphid proteases in vitro demonstrated enhanced processing of the toxin into the active form for some of the modified constructs relative to non-modified Cry4Aa. Aphids fed artificial diet with toxin at a final concentration of 125 µg/ml showed enhanced mortality after two days for one of the four modified constructs. Although only modest toxin improvement was achieved by use of this strategy, such specific toxin modifications designed to overcome factors that limit aphid toxicity could be applied toward managing aphid populations via transgenic plant resistance.

Bt toxin modification for enhanced efficacy.

Insect-specific toxins derived from Bacillus thuringiensis (Bt) provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry) and cytolytic (Cyt) toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance.

On-plant selection and genetic analysis of European corn borer (Lepidoptera: Crambidae) behavioral traits: plant abandonment versus plant establishment.

Although some studies have investigated how insect behavior could influence resistance evolution to transgenic plants, none have determined if behavioral traits respond to selection pressure and how they may be inherited. We investigated plant establishment and abandonment traits for the European corn borer, Ostrinia nubilalisi (Hübner) (Lepidoptera: Crambidae), by conducting a laboratory selection experiment and quantifying patterns of gene expression. Egg masses with emerging larvae were placed on maize plants and silking individuals were collected every 15 min during a 4-h period to generate a plant abandonment (PA) colony. Plants were dissected 24-72 h later, and larvae were collected for a plant establishment colony. Selection of the PA colony showed an increased propensity to abandon the host plant by the third generation. The propensity for larvae to establish on the plants, however, did not show a significant response until the sixth generation. Quantitative real-time-polymerase chain reaction (qRT-PCR) was used to determine expression profiles for behavior associated genes (foraging and Onslmo). Egg samples from the two selected colonies and nonselected laboratory colony were collected at 0, 24, 48, 72, and 96 h after egg deposition, and first instars were sampled after exposure to maize tissue. Compared with the plant establishment and nonselected laboratory colonies at the 0-h time period, foraging and Onslmo showed higher expression in the PA colony. This is the first study that has specifically selected for these traits over several generations and analyzed behavior-associated genes to elucidate genetic changes.