Fate of Bacillus thuringiensis subsp. israelensis in the Field: Evidence for Spore Recycling and Differential Persistence of Toxins in Leaf Litter

Abstract

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

“…When 1,000-2,000 larvae were sampled, high sensitive standard bioassays could be performed, but not for all the Cry toxins; bioassays with Bti and Cry4Aa were performed in priority since Cry4Aa is the main toxin persisting in the field after Bti treatment (Tetreau et al 2012a) and with the highest resistance ratio in the Bti -resistant strain described in Tetreau et al (2012b). Lethal concentrations for 50% individuals (LC 50 ) were calculated for each population and each toxin using a probit statistical model with the module "dose" of XLSTAT v.2009.4.06 (Addinsoft).…”

“…Recent works on Bti -resistance showed that while levels of resistance to Bti can be low in a laboratory-selected strain (3.5-fold), resistance to each of the Cry toxins tested separately in bioassays can be much higher (up to 60-fold) (Paris et al 2011, Tetreau et al 2012b). Here we present an improved bioassay method consisting in performing distinct bioassays with each of the three Bti Cry toxins on field populations of mosquitoes.…”

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

“…israelensis de Barjac ( Bti- H14) was the first Bt strain used as an effective biological control agent against larvae of many mosquito and blackfly species worldwide, with no adverse effects on non-target invertebrates and vertebrates. Several studies have linked the larvicidal activity of Bti to its major Cyt1Aa and Cry4Ba, Cry4Aa, Cry10, Cry11Aa proteins [1, 15, 16]. In Bti -H14, these proteins are all encoded and located on a large plasmid (128 kb) recognized as p Bt oxis [17] and differ in their mosquito toxicity [18, 19].…”

A study of the effect of Bacillus thuringiensis serotype H14 (subspecies israelensis)delta endotoxin on Musca larva

“…If these crystals are ingested by susceptible insects, they cause serious injuries or death (11). Several researchers have studied the effect of these toxins on the reproduction and growth of the house fly (5,6,11). In this study, we studied the effect of a protein crystal toxin derived from B. thuringiensis serotype H14 (subspecies israelensis) on the larvae of house flies.…”

“…During sporulation, this bacterium produces protein crystals (delta endotoxin) that have insecticidal activity (5,6). Several studies have suggested that these toxins, or a mixture of unknown components in the toxins acting together synergically, produce the full toxicity.…”