PH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

Wenhui Yang,Shuyuan Guo,Jie Zhang,Kanglai He,Alejandra Bravo

doi:10.1371/journal.pone.0045233

Wenhui Yang, Shuyuan Guo + Show 3 more

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https://doi.org/10.1371/journal.pone.0045233

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Abstract

Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO3 core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field.

Highlights

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacteria that produces insecticidal crystal proteins named Cry toxins during sporulation
Bio-encapsulated formulation, which can protect pesticides from degradation and provide controllable release of the active ingredient, has been used for decades, and Bt has been encapsulated since the 1960s [22]
A methodology of microencapsulation was investigated which have the capacity to protect the spore crystal aggregate produced by Bacillus thuringiensis var. kurstaki HD-1 from extreme ultraviolet radiations which are equivalent to 80 and 558 days of sunlight exposure in clear weather in Mexico [23]

Summary

Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacteria that produces insecticidal crystal proteins named Cry toxins during sporulation. Despite the successful application of Bt for pest control, which presently is approximate 2% of the total insecticidal market [3], the short persistence of B. thuringiensis agents after application has become an important influencing factor for its further development. Variable environmental stress, such as UV radiation, rain, and temperature, leads to inactivation or rain-washed of the crystal proteins [4,5]. Microcapsules have an increased interest as potential drug carriers, especially for protein drugs. The pH is a viable method for controlling the loading and release process

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