Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.     

Novel genetic basis of field-evolved resistance to Bt toxins in Plutella xylostella

Insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pest insects, but evolution of resistance threatens their continued efficacy. The most common type of Bt resistance ('Mode 1') is characterized by recessive inheritance, > 500-fold resistance to at least one Cry1A toxin, negligible cross-resistance to Cry1C, and reduced binding of Bt toxins to midgut membrane target sites. Mutations affecting a Cry1A-binding midgut cadherin protein are linked to laboratory-selected Mode 1 resistance in Heliothis virescens and Pectinophora gossypiella. Here we show that field-evolved Mode 1 resistance in the diamondback moth, Plutella xylostella, has a different genetic basis, indicating that screening for resistance in the field should not be restricted to a previously proposed DNA-based search for cadherin mutations.     

A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella

Cadherins have been described as one the main functional receptors for the toxins of the entomopathogenic bacterium, Bacillus thuringiensis (Bt). With the availability of the whole genome of Plutella xylostella, different types of cadherins have been annotated. In this study we focused on determining those members of the cadherin‐related proteins that potentially play a role in the mode of action of Bt toxins. For this, we mined the genome of P. xylostella to identify these putative cadherins. The genome screening revealed 52 genes that were annotated as cadherin or cadherin‐like genes. Further analysis revealed that six of these putative cadherins had three motifs common to all Bt‐related cadherins: a signal peptide, cadherin repeats and a transmembrane domain. From the six selected cadherins, only P. xylostella cadherin 1 (PxCad1) was expressed in the larval midgut and only the silencing of this gene by RNA interference (double‐stranded RNA feeding) reduce toxicity and binding to the midgut of the Cry1Ac type toxin from Bt. These results indicate that from the whole set of cadherin‐related genes identified in P. xylostella, only PxCad1 is associated with the Cry1Ac mode of action.     

Knockdown of cadherin genes decreases susceptibility of Chilo suppressalis larvae to Bacillus thuringiensis produced Crystal toxins

The striped rice stem borer, Chilo suppressalis Walker, is one of the most destructive rice pests in Asia. Insecticidal crystal proteins (Cry toxins) produced by Bacillus thuringiensis are widely used as biopesticides or in developing transgenic crops for pest management. In this study, we tested the involvement of two newly cloned C. suppressalis cadherins (CsCAD3 and CsCAD4) in the toxicity of Cry1Ab/Ac, Cry2Aa and Cry1Ca. Our results showed that CsCAD4 was expressed highest in the midgut, whereas CsCAD3 was expressed highest in the epidermis. The feeding of double‐stranded RNA specific to CsCAD3 and CsCAD4 respectively significantly suppressed the expressions of target gene. The knockdown of CsCAD3 significantly reduced the mortality of larvae to Cry1Ab/Ac, whereas knockdown of CsCAD4 significantly decreased the larval susceptibility to Cry2Aa. In contrast, reduced expressions of CsCAD3 or CsCAD4 were not interacted with larval susceptibility to Cry1Ca. Our results suggest that CsCAD3 and CsCAD4 function in Cry toxin toxicity and these findings will help us to better understand the action mechanism of Cry toxins in C. suppressalis.     

Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata)

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore‐forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB‐ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB‐ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB‐ADAM10 silenced larvae and in vitro toxin pore‐forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB‐ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane‐associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB‐ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore‐forming toxins in humans to an invertebrate species.     

The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae)

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.     

Role of a gamma‐aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

The western corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was controlled by applications of cyclodiene insecticides from the late 1940s until resistance evolved ∼10 years later. Range expansion from the western plains into eastern USA coincides with resistance development. An alanine to serine amino acid substitution within the Rdl subunit of the gamma‐aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. We found that the non‐synonymous single nucleotide polymorphism (SNP) G/T at the GABA receptor cDNA position 838 (G/T⁸³⁸) of D. v. virgifera resulted in the alanine to serine change, and the codominant SNP allele T⁸³⁸ was genetically linked to survival of beetles in aldrin bioassays. A phenotypic gradient of decreasing susceptibility from west to east was correlated with higher frequencies of the resistance‐conferring T⁸³⁸ allele in the eastern‐most populations. This pattern exists in opposition to perceived selective pressures since the more eastern and most resistant populations probably experienced reduced exposure. The reasons for the observed distribution are uncertain, but historical records of the range expansion combined with the distribution of susceptible and resistant phenotypes and genotypes provide an opportunity to better understand factors affecting the species' range expansion.     

Alkaline phosphatase 2 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea

Although membrane‐bound alkaline phosphatases (ALPs) have been proposed as a receptor for Cry1Ac in a few lepidopteran species, their potential functions as a Cry2Ab receptor are yet to be verified. To determine if ALP2 also serves as a receptor for Cry1Ac and even for Cry2Ab in Helicoverpa zea, we measured the potency of activated Cry1Ac and Cry2Ab against midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) expressing H. zea ALP2 (HzALP2) or transfected with HzALP2 double‐stranded RNA (dsRNA). Relative to the control cells, the three cell lines expressing HzALP2 were more susceptible to Cry1Ac but there was no difference for Cry2Ab. By contrast, the two H. zea cell lines transfected with HzALP2 dsRNA were resistant to Cry1Ac while kept susceptible to Cry2Ab. Furthermore, RNA interference knockdown of HzALP2 in H. zea larvae enhanced larval survival on Cry1Ac‐containing diets. These findings indicate that HzALP2 functions as a receptor of Cry1Ac but not Cry2Ab.