Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background

Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F₂ screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.

Methodology/Principal Findings

Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with ¹²⁵I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in ¹²⁵I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.

Conclusion/Significance

This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.     

Rx-Cre, a tool for inactivation of gene expression in the developing retina

Rx is a homeobox-containing gene that is critical for vertebrate eye development. Its expression domain delineates a field of cells from which the retina and the ventral hypothalamus develop. The 5' upstream regulatory sequences of the medaka fish Rx gene are functionally conserved during evolution to a degree that they direct gene expression into the Rx-expressing field of cells in mice. Using these sequences, we made a Cre line that can be used for inactivation of gene expression in the developing retina.     

Factors affecting abundance and oviposition rates of a field population of the Old World screw-worm fly, Chrysomya bezziana (Diptera: Calliphoridae)

Sentinel cattle and a grid of swormlure-baited sticky traps were used to monitor a Malaysian population of the Old World screw-worm fly, Chrysomya bezziana Villeneuve. Observations were carried out on an isolated cattle station at monthly intervals during the period August 1996 to June 2000. The number of flies caught was unaffected by weather conditions at the time of trapping, but was positively correlated with the total rainfall and the average daily air temperature prevailing 15-28 days earlier, when trapped flies were still juveniles. Trap catches were biased in favour of females, but daily catch rates of both sexes increased significantly the longer traps were open, suggesting that efficacy was related to the differential volatility of the chemicals comprising swormlure. Oviposition on sentinel cattle occurred mostly in late afternoon or early evening but increased significantly as the wound aged. Oviposition rates were positively correlated with female catch rates, but the relationship was curvilinear, suggesting that fly populations may be subject to some form of density-dependent constraint. Consistent differences in oviposition rates on sentinel cattle at different localities on the cattle station suggested the existence of highly clumped, quasi-stationary populations. Differences in trap catches between traps located in pastoral areas and those sited in nearby oil palm or rubber plantations supported this interpretation of the data. These findings are discussed in relation to the use of the sterile insect technique for the control of screw-worm fly infestations.     

The corneal epithelial stem cell

The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Mean species responses predict effects of environmental change on coexistence

Environmental change research is plagued by the curse of dimensionality: the number of communities at risk and the number of environmental drivers are both large. This raises the pressing question if a general understanding of ecological effects is achievable. Here, we show evidence that this is indeed possible. Using theoretical and simulation-based evidence for bi- and tritrophic communities, we show that environmental change effects on coexistence are proportional to mean species responses and depend on how trophic levels on average interact prior to environmental change. We then benchmark our findings using relevant cases of environmental change, showing that means of temperature optima and of species sensitivities to pollution predict concomitant effects on coexistence. Finally, we demonstrate how to apply our theory to the analysis of field data, finding support for effects of land use change on coexistence in natural invertebrate communities.     

Comparison of multiplex PCR and standard bacteriological methods of detecting Salmonella on chicken skin

A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella , and Salm. enteritidis in particular, in samples of chicken skin obtained from retail outlets. Five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), five were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared to 7.4%, and taking less than 24 h to reach a result as opposed to 2–3 d.