Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?

Abstract

Bacillus thuringiensis endotoxins (Bt-toxins) are the most important biopesticides used in controlling insect pests and vectors of diseases. The emergence of widespread resistance to Bt in some insect species is a serious threat to agricultural production. Analysis of Bt-resistant and susceptible laboratory strains of Helicoverpa armigera revealed elevated immune responses involving increased melanization and the presence of a soluble toxin-binding glycoprotein in the hemolymph and gut lumen of the resistant strain. We propose a resistance mechanism against toxins based on a systemic immune-induction that can be transmitted to the next generation by a maternal effect.

Introduction

Insecticides from the soil bacterium Bacillus thuringiensis (Bt), a natural pathogen of many invertebrates including insects, are economically and ecologically important biological control agents (Shelton et al., 2002). Due to its benign environmental properties and absence of any harmful effects on humans, the Bt-endotoxins are increasingly employed in pest management programs in favour of synthetic pesticides. Recently, bacterial endotoxin genes have been expressed in crop plants, where they provide protection against a number of major insect pests, such as cotton bollworms (Helicoverpa zea and Helicoverpa armigera).

Reports of the emergence of resistance in field populations of DBM have highlighted the inherent danger of the evolution of resistance against this powerful insecticide (Ferré and Van Rie, 2002). The most effective and best-characterised resistance mechanisms are based on receptor-inactivation at the midgut membrane, for example involving the aminopeptidase N (Darboux et al., 2002) and the cadherin-like (Gahan et al., 2001; Morin et al., 2003) gene families. Other mechanisms known to impair Bt-toxicity include alterations to proteolytic activity of midgut extracts affecting protoxin processing and maturation (Oppert, 1999), and increased rates of replacement of damaged cells at the gut lining by stem cells (Martinez-Ramirez et al., 1999).

We have recently reported evidence of an additional mechanism, where Bt-tolerance is associated with an elevated immune response (Rahman et al., 2004). When larvae from a susceptible laboratory strain of the flour moth Ephestia kuehniella were selected for survival on progressively increasing levels of a Bt-formulation for five generations, the resulting Bt-tolerant strain displayed a constitutively elevated immune response.

Treating larvae of the susceptible strain of E. kuehniella with sub-lethal doses of the Bt-formulation led to an elevated melanization reaction in the hemolymph, which in turn was correlated with an increase in tolerance against the toxin at a later stage of larval development. As susceptible larvae were immune-induced and exposed to Bt-toxin in the same generation, this experiment excluded the selection of a pre-existing resistance allele as the cause of increased tolerance to the toxin in pre-treated larvae.

Reciprocal crosses of tolerant and susceptible insects revealed the transmission of both the immune induction and tolerance to the toxin from one generation to the next by a maternal effect. Offspring of tolerant females and susceptible males (T×S) were significantly more tolerant than offsprings from susceptible females and tolerant males (S×T). A possible mechanism for this effect is the incorporation of an immune-elicitor into the oocyte by an immune-induced female. The elicitor can interact with embryonic tissues to induce the immune system of the neonate so that by the time the neonate starts feeding the insect is already induced, thus increasing the chances of surviving the toxin.

Bioassays showed that the S×T neonates were significantly more tolerant than the S×S neonates, indicating a genetic contribution to the variation in the immune response in addition to the maternal effect (Rahman et al., 2004). Further, the level of the immune response in the T×S larvae, as measured by the scale of melanization reactions, was variable between the offspring of different mating pairs, but directly correlated with the degree of Bt-tolerance. The fact that the level of the immune response and Bt-resistance in the T×S larvae occurred as a continuum, suggests that the observed variation in the magnitude of the potential immune response is determined by more than one gene.

When females from the S×T cross were backcrossed with males from the tolerant strain, no immune-induction was observed in the offspring. This confirms that the elevated immune-status in the tolerant population was based on a transient immune induction, which is initiated in each generation by a maternal effect. However, when offspring from the backcross were immune-induced by a sub-lethal dose of the toxin, the observed melanization reaction was significantly greater than that detected in immune-induced susceptible larvae, confirming that the genetic disposition to respond to an elicitor was genetically determined by alleles that were different in the tolerant compared to the susceptible population (Rahman et al., 2004).

The molecular basis of the Bt-tolerance in this strain of E. kuehniella is not known. However, the evidence that its magnitude is determined by more than one gene in turn suggests that it may involve multiple metabolic and regulatory pathways. For example, mutational changes in a gene product involved in post-translational modifications, such as glycosyltransferases (Griffitts et al., 2001), may potentially interfere with pro-coagulant (Theopold et al., 2002), cell surface receptors (Oltean et al., 1999) and ovarian elicitor functions (Sen et al., 1998). If pro-coagulant molecules are transferred into the gut lumen as part of an immune response, it is possible that soluble immune components in the gut lumen interact with the mature toxin causing its inactivation by a coagulation or melanization reaction.

To examine whether immune-related Bt-tolerance mechanisms occur in other insects, we investigated differences between a susceptible and a Bt-resistant strain of the cotton bollworm H. armigera, an important insect pest (Akhurst et al., 2002; Akhurst et al., 2003). Mirroring the results for E. kuehniella, reciprocal crosses involving the susceptible and resistant strains revealed that tolerance involved a genetic and also a non-genetic component that was transmitted to the next generation by a maternal effect. Susceptible larvae fed with a diet containing a sub-lethal concentration of the toxin Cry1Ac subsequently displayed an elevated immune response in both the hemolymph and gut lumen. Further, the immune-induction was associated with an increased level of the larval serum protein hexamerin, which interacts with the toxin by an aggregation reaction to form an insoluble coagulum.