Bacillus thuringiensis resistance in Plutella – too many trees?

Plutella xylostella was the first insect for which resistance to Bacillus thuringiensis was reported in the field, yet despite many studies on the nature of this resistance phenotype its genetic and molecular basis remains elusive. Many different factors have been proposed as contributing to resistance, although in many cases it has not been possible to establish a causal link. Indeed, there are so many studies published that it has become very difficult to “see the wood for the trees”. This article will attempt to clarify our current understanding of Bt resistance in P. xylostella and consider the criteria that are used when validating a particular model

Using worms to better understand how Bacillus thuringiensis kills insects

Bacillus thuringiensis is widely used as a biological pesticide to control insects that either cause damage to crops or transmit disease. That it can also target the model organism Caenorhabditis elegans has not only provided exciting new insights into how the toxins produced by the bacterium target their victims but also how target insects counter the attack. Modern approaches such as reverse genetics and microarray technology have revealed novel receptors for the toxins and possible signal transduction pathways induced within the host following intoxication. This article will discuss how these findings fit in with current models and how they might influence future studies

Structural classification of insecticidal proteins – towards an in silico characterization of novel toxins

The increasing rate of discovery of new toxins with potential for the control of invertebrate pests through next generation sequencing, presents challenges for the identification of the best candidates for further development. A consideration of structural similarities between the different toxins suggest that they may be functionally less diverse than their low sequence similarities might predict. This is encouraging from the prospective of being able to use computational tools to predict toxin targets from their sequences, however more structure/function data are still required to reliably inform such predictions

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.</p

Selection of a field population of diamondback moth (Lepidoptera: Plutellidae) with acetamiprid maintains, but does not increase, cross-resistance to pyrethroids

Bioassays (at generation G2) with a newly collected field population (designated CH4) of Plutella xylostella (L.) (Lepidoptera: Plutellidae) from farms in the Cameron Highlands, Malaysia, indicated resistance to acetamiprid, deltamethrin, lambda-cyhalothrin, and esfenvalerate. At G3, the field-derived population was divided into two subpopulations, one subpopuplation was selected (G3 to Go10) with acetamiprid (aceta-SEL), whereas the second subpopulation was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G,1, indicating that the observed resistance to each insecticide was unstable. For aceta-SEL, bioassays at G,, found that selection with acetamiprid gave a resistance ratio of 409 compared with UNSEL. The LC50 values for deltamethrin, lambda-cyhalothrin, and esfenvalerate to aceta-SEL were similar at both G11 and G2. This suggests that acetamiprid selection maintained the otherwise unstable resistance to these compounds in the aceta-SEL population. Logit regression analysis of F1 reciprocal crosses between aceta-SEL and the susceptible Lab-UK indicated that resistance to acetamiprid was inherited as an autosomal, incompletely recessive (DLC = 0.26) trait. Tests of monogenic inheritance and weight distribution suggested that resistance to acetamiprid was controlled by a single locus

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.</p

Specificity determinants for Cry insecticidal proteins: insights from their mode of action

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used as active components of biopesticides and as plant incorporated protectants in transgenic crops. One of the most relevant attributes of these Bt protein-based insecticidal technologies is their high specificity, which assures lack of detrimental effects on non-target insects, vertebrates and the environment. The identification of specificity determinants in Bt insecticidal proteins could guide risk assessment for novel insecticidal proteins currently considered for commercialization. In this work we review the available data on specificity determinants of crystal (Cry) insecticidal proteins as the Bt toxins most well characterized and used in transgenic crops. The multi step mode of action of the Cry insecticidal proteins allows various factors to potentially affect specificity determination and here we define seven levels that could influence specificity. The relative relevance of each of these determinants on efficacy of transgenic crops producing Cry insecticidal proteins is also discussed

Specificity determinants for Cry insecticidal proteins: insights from their mode of action

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used as active components of biopesticides and as plant incorporated protectants in transgenic crops. One of the most relevant attributes of these Bt protein-based insecticidal technologies is their high specificity, which assures lack of detrimental effects on non-target insects, vertebrates and the environment. The identification of specificity determinants in Bt insecticidal proteins could guide risk assessment for novel insecticidal proteins currently considered for commercialization. In this work we review the available data on specificity determinants of crystal (Cry) insecticidal proteins as the Bt toxins most well characterized and used in transgenic crops. The multi step mode of action of the Cry insecticidal proteins allows various factors to potentially affect specificity determination and here we define seven levels that could influence specificity. The relative relevance of each of these determinants on efficacy of transgenic crops producing Cry insecticidal proteins is also discussed

Optimizing pyramided transgenic Bt crops for sustainable pest management

Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies

Group selection as a basis for screening mutagenized libraries of public goods (bacillus thuringiensis cry toxins)

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins