Virulence and Evolutionary Ecology in the Entomopathogen Bacillus thuringiensis

Abstract

Bacillus thuringiensis is an entomopathogen in the Bacillus cereus species group, and has been used as a biopesticide for over 50 years. Despite extensive use of B. thuringiensis, there remain questions over its specific ecology compared to other members of the B. cereus group which poses problems for its continued applied use. Tying entomopathogenic ecology to a specific clade within the B. cereus group will limit confusion between B. thuringiensis used in agriculture and more harmful strains. Better understanding of B. thuringiensis ecology can also be used to combat resistance in pest species through selective passaging. The ecology of B. thuringiensis was explored through competitions in Plutella xylostella (diamondback moth) larvae, which showed clade 2 B. thuringiensis have improved fitness in insects compared to clade 1 strains. Additionally, growth rates were compared in vitro, giving different thermal profiles for the two clades. Growth media preference was assessed for B. cereus group species with all favouring protein media over soil-based ones. Selective passaging explored the effects of relatedness and host background on virulence evolution. For relatedness, B. thuringiensis subsp. aizawai was passaged for five rounds in P. xylostella larvae with none, one or two bottlenecking events. These treatments failed to produce any increase in virulence. In the second, B. thuringiensis subsp. entomocidus was passaged either in Cry1Ac-resistant, Cry1Ac-susceptible, alternating rounds of each or coevolved P. xylostella, with all containing a mutagenesis step with ethyl methanesulfonate. Virulence increased in the resistant and coevolved treatments, confirming that resistance is best overcome by passaging in harder-to-kill hosts. The ecological and genetic distinctiveness of clade 2 B. thuringiensis suggests the species should be reclassified to solely this clade, which will limit safety concerns. Selective passaging can improve the virulence of strains, even if the underlying interactions are unknown; it can also provide insight into virulence evolution which would be lost when improving only at the protein level.