Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.     

Effects of Bacillus thuringiensis var. israelensis on Target and Nontarget Organisms: a Review of Laboratory and Field Experiments

Since the discovery of Bacillus thuringiensis var. israelensis (Bti) in 1976, extensive literature has proved its efficacy to control mosquitoes and black flies, of which many species are known as important vectors of diseases or simply as pests of humans and animals. Since 1978, Bti has been used in many countries on all continents and numerous studies have been made on target mosquitoes and black flies, as well as nontarget organisms (NTO). This review analyses the results of 75 studies on these organisms covering approximately 125 families, 300 genera and 400 species. Different factors such as species, instar, feeding behaviour and environmental parameters (larval density, water temperature, suspended matter etc.) may drastically affect the efficacy of the Bti products. This is addressed in detail by reviewing the main factors affecting mosquitoes as well as black flies. The results of a wide range of laboratory and field experiments using different target and nontarget species, various preparations and formulations of Bti and different biotic or abiotic factors are present in the literature, making the data difficult to compare on a common basis. Our analysis shows that, under different application conditions, the effects of Bti on target and nontarget organisms may be hard to predict. Although Bti has been proclaimed to be relatively highly specific, some studies show that some NTO are affected either by single or repeated Bti treatments. Present use against black flies seems ecologically acceptable. High frequencies of application and/or overdosages against mosquitoes may result in some persistence of the toxin crystals and ultimately this may have adverse effects on the food web. A long-term study (published in 1998) in mosquito habitats has shown that intensive Bti treatments over three years did in fact produce an impact on the food web in wetlands. This raises questions, for the first time, on Bti environmental specificity. The importance of this impact is discussed and the alternatives for practical pest control are considered. Some modifications of Bti use against mosquitoes, guided by research, is probably the best of these alternatives.     

Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains

The toxicity of Bacillus thuringiensis subsp. israelensis to dipteran larvae (mosquitoes and black flies) depends on the presence of the pBtoxis plasmid. In this paper, two antibiotic resistance tagged pBtoxis were transferred by conjugation to other Bacillus cereus group strains. Among 15 potential recipients, only a lepidopteran active B. thuringiensis subspecies kurstaki and a B. cereus strain received the plasmid pBtoxis with a low transfer rate of about 10(-8) transconjugants/recipient. The resulting B. thuringiensis subspecies kurstaki transconjugant was active to both lepidopteran and dipteran targets and the B. cereus transconjugant was active against dipteran insects. Phase contrast microscopy showed that the B. cereus transconjugants could produce only round crystalline inclusion bodies while B. thuringiensis subspecies kurstaki transconjugant could produce both round and bipyramidal crystals during sporulation. SDS-PAGE revealed that all the major mosquitocidal proteins from pBtoxis could express in the two transconjugants, including Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa and Cyt1Aa. However, none of the experiment showed any indications of mobilising abilities of pBtoxis. The limited number of strains, which could receive and maintain pBtoxis using a conjugational helper plasmid, indicates a very narrow host range of the B. thuringiensis subsp. israelensis pBtoxis plasmid.