Identification of gene expression changes associated with the initiation of diapause in the brain of the cotton bollworm, Helicoverpa armigera

<p>Abstract</p> <p>Background</p> <p>Diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. <it>Helicoverpa armigera </it>(Har) undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. Using suppression subtractive hybridization (SSH), we investigated differentially expressed genes in diapause- and nondiapause-destined pupal brains at diapause initiation.</p> <p>Results</p> <p>We constructed two SSH libraries (forward, F and reverse, R) to isolate genes that are up-regulated or down-regulated at diapause initiation. We obtained 194 unique sequences in the F library and 115 unique sequences in the R library. Further, genes expression at the mRNA and protein level in diapause- and nondiapause-destined pupal brains were confirmed by RT-PCR, Northern blot or Western blot analysis. Finally, we classified the genes and predicted their possible roles at diapause initiation.</p> <p>Conclusion</p> <p>Differentially expressed genes at pupal diapause initiation are possibly involved in the regulation of metabolism, energy, stress resistance, signaling pathways, cell cycle, transcription and translation.</p

Down Regulation of a Gene for Cadherin, but Not Alkaline Phosphatase, Associated with Cry1Ab Resistance in the Sugarcane Borer Diatraea saccharalis

The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt) proteins (i.e., Cry1Ab) in South America and the mid-southern region of the United States. Evolution of insecticide resistance in such target pests is a major threat to the durability of transgenic Bt crops. Understanding the pests' resistance mechanisms will facilitate development of effective strategies for delaying or countering resistance. Alterations in expression of cadherin- and alkaline phosphatase (ALP) have been associated with Bt resistance in several species of pest insects. In this study, neither the activity nor gene regulation of ALP was associated with Cry1Ab resistance in D. saccharalis. Total ALP enzymatic activity was similar between Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of D. saccharalis. In addition, expression levels of three ALP genes were also similar between Cry1Ab-SS and -RR, and cDNA sequences did not differ between susceptible and resistant larvae. In contrast, altered expression of a midgut cadherin (DsCAD1) was associated with the Cry1Ab resistance. Whereas cDNA sequences of DsCAD1 were identical between the two strains, the transcript abundance of DsCAD1 was significantly lower in Cry1Ab-RR. To verify the involvement of DsCAD1 in susceptibility to Cry1Ab, RNA interference (RNAi) was employed to knock-down DsCAD1 expression in the susceptible larvae. Down-regulation of DsCAD1 expression by RNAi was functionally correlated with a decrease in Cry1Ab susceptibility. These results suggest that down-regulation of DsCAD1 is associated with resistance to Cry1Ab in D. saccharalis

Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins

Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration

Uncovering the Prevalence and Diversity of Integrating Conjugative Elements in Actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria