Ecological Mysteries: is Bacillus thuringiensis a Real Insect Pathogen?

Bacillus thuringiensis (Bt) can kill insects and multiply in their bodies, but it can also grow in semi-synthetic media; is found in environments were insects are absent; and has been reported to require midgut-associated bacteria for toxicity. We propose here a novel life cycle for Bt combining insect-based and insect-independent life cycles.


Background
The bacterium Bacillus thuringiensis (Bt) has been successfully used as a biopesticide for decades and, from 1996 onwards, also as a source of genes for the construction of transgenic plants (Bt crops) protected against insect pests. However, because of a lack of a consistent linkage between insects and Bt, the real life cycle of this well-known bacterium is still unclear. It is obvious that Bt can kill insects and multiply in their bodies, but Bt can also grow very well on simple semi-synthetic media, including LB. Moreover, Bt strains have been recovered from environments were insects are scarce or absent; found to be nonpathogenic towards the species they were originally isolated from and yet display activity against insects or other invertebrate species they will certainly rarely encounter (Martin and Travers, 1989); and, last but not least, reported to require midgut-associated bacteria to display insecticidal activity in some cases (Broderick et al., 2006) but not in others (Johnston and Crickmore, 2009). We believe that the answer to the question the title of this letter raises lies on the analysis of the reports on the ability of Bt to survive and eventually multiply, in the presence or absence of insects.
Bt usually displays a dual biological activity involving toxicity and pathogenicity. The parasporal insecticidal crystal (Cry) proteins synthesized during sporulation are specifically toxic against a wide range of insects (see: http://www.glfc.cfs.nrcan.gc.ca/bacillus). The mode of action of Cry proteins, which involves solubilisation in the insect midgut, activation by digestive enzymes and binding to specific receptors located on the host cell surface, evidences a strong selection pressure towards insecticidal activity. Moreover, spores are known to synergize this activity, since they can enter the insect body and produce septicaemia. The importance of each of these effects (toxicity and pathogenicity), shows a large variation depending on the target species (Suzuki et al., 2004). The toxicity, rather than the pathogenicity, of Bt has been exploited for six decades through Bt-based biopesticides that can be sprayed on the field and have a direct impact on insect populations without significant bacterial in situ multiplication. But epizootics (epidemics) in insects caused by Bt are known to occur on the field, and the most powerful mosquitocidal strain, Bt var israelensis (De Barjac, 1978), was in fact isolated from a stagnant pond showing mass mosquito mortality.
Beyond insects, an alternative insect-free cycle has been proposed. Indeed, Bt strains have been reported to multiply on the surface of the leaves and, more interestingly, even to colonize plantlets from the soil (Bizzarri and Bishop, 2008). A comparison between crystal-and non-crystal-producing strains revealed that both exhibited similar ability in terms of leaf colonization, indicating that insecticidal parasporal crystals was not Bt Research 2012, Vol.3, No.1, 1-2 http://bt.sophiapublisher.com a mitigating factor for Bt to grow in the absence of insects. Such soil-plant life cycle might not exclude insects, since multiplication in the bodies as well as in the frass of surviving insects has been reported (Bizzarri and Bishop, 2008;Prabhakar and Bishop, 2009), and this would certainly be a supplementary source of spores to re-inoculate soil and then plants through young leaflets colonization. Additionally, since colonized plants are at least partially protected against phytofagous insects (Prabhakar and Bishop, 2009), a plant symbiont-like role for Bt can also be considered.
The analysis of these and similar reports, taken together, lead us to define a new life cycle for Bt that combines the insect-based and insect-independent cycles mentioned above. The proposed cycle, shown in Figure 1, integrates plants, frass and soil as sub-cycles of a main insect-centered cycle. But the key question still remains unanswered: is Bt a real insect pathogen? We think that, beyond the reported epizootics outbreaks, a major argument proving that Bt is indeed an insect pathogen is the elegant specificity of the arsenal of Bt insecticidal toxins active on Lepidoptera, Coleoptera, Diptera and also other invertebrates such as nematodes, which is an obvious adaptive character. But the well-documented insect-free plant-based cycles, horizontal gene transfer phenomena such as Bt Cyt genes identified in the aphid pathogen and phytopathogenic bacterium Dickeya dadantii (Grenier et al., 2006), or the ease with which Bt can be grown in the lab strongly reveal that Bt is, additionally to an insect pathogen, also an opportunistic non-pathogen microorganism able to persist and/or multiply on a range of substrates including feces or plant surfaces from which it might re-enter its basic insect-centered life cycle.
Multiplication can be carried out in insects, but also in frass or plant surfaces. Soil may play a central role as a reservoir of spores. The presence of B. thuringiensis on plants can originate from colonization of leaflets from soil, spreading of frass on the leaves from living insects or from decomposition of spore-rich insect bodies, particularly after epizootics outbreaks. Notice that aquatic strains such as B. thuringiensis var. israelensis may display a variation of this with a simpler insect (i.e. mosquito larvae)-freshwater cycle.