Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera

Helicoverpa armigera can develop resistance to Bacillus thuringiensis (Bt), which threaten the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in H. armigera. Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, ALP2, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the “proton-transporting V-type ATPase complex” and the “proton-transporting two-sector ATPase complex” were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in H. armigera, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in H. armigera

Identification of Lipases Involved in PBAN Stimulated Pheromone Production in Bombyx mori Using the DGE and RNAi Approaches

BACKGROUND: Pheromone biosynthesis activating neuropeptide (PBAN) is a neurohormone that regulates sex pheromone synthesis in female moths. Bombyx mori is a model organism that has been used to explore the signal transduction pattern of PBAN, which is mediated by a G-protein coupled receptor (GPCR). Although significant progress has been made in elucidating PBAN-regulated lipolysis that releases the precursor of the sex pheromone, little is known about the molecular components involved in this step. To better elucidate the molecular mechanisms of PBAN-stimulated lipolysis of cytoplasmic lipid droplets (LDs), the associated lipase genes involved in PBAN- regulated sex pheromone biosynthesis were identified using digital gene expression (DGE) and subsequent RNA interference (RNAi). RESULTS: Three DGE libraries were constructed from pheromone glands (PGs) at different developed stages, namely, 72 hours before eclosion (-72 h), new emergence (0 h) and 72 h after eclosion (72 h), to investigate the gene expression profiles during PG development. The DGE evaluated over 5.6 million clean tags in each PG sample and revealed numerous genes that were differentially expressed at these stages. Most importantly, seven lipases were found to be richly expressed during the key stage of sex pheromone synthesis and release (new emergence). RNAi-mediated knockdown confirmed for the first time that four of these seven lipases play important roles in sex pheromone synthesis. CONCLUSION: This study has identified four lipases directly involved in PBAN-stimulated sex pheromone biosynthesis, which improve our understanding of the lipases involved in releasing bombykol precursors from triacylglycerols (TAGs) within the cytoplasmic LDs

Molecular shape and immunogenicity of meningococcal polysaccharide group A conjugate vaccine

Neisseria meningitidis is a leading cause of severe bacterial infections in infants and young children. As a major virulence factor, meningococcal capsular polysaccharide (PS) is poorly immunogenic and generally does not induce immunological memory. Conjugation of PS with a carrier protein can significantly increase the PS-specific immunogenicity and induce immunological memory. It is well known that the molecular shape/size of the conjugate vaccine is important for its immunogenicity. However, little is known about the molecular shape/size of the meningococcal conjugate vaccine. A meningococcal PS ovalbumin (OVA) conjugate vaccine was prepared using cystamine as linker. Four components (P1-P4) with different molecular size were fractionated from the conjugate. Small angle X-ray scattering (SAXS) analysis revealed that the conjugate vaccine exhibited a rod-like shape similar to virus-like particles. PS-specific immunogenicity of the conjugate vaccine was related to its molecular shape and increased as a function of its molecular size. Thus, the present study provides a three-dimensional shape of the conjugate vaccine and helps to identify optimal design of a potent meningococcal conjugate vaccine. (C) 2015 Elsevier Ltd. All rights reserved

Cyclosporin A as a Potential Insecticide to Control the Asian Corn Borer <i>Ostrinia furnacalis</i> Guenée (Lepidoptera: Pyralidae)

The long-term use of chemical insecticides has caused serious problems of insect resistance and environmental pollution; new insecticides are needed to solve this problem. Cyclosporin A (CsA) is a polypeptide produced by many fungi, which is used to prevent or treat immune rejection during organ transplantation. However, little is known about the utility of CsA as an insecticide. Therefore, this study evaluated the insecticidal activity of CsA using Ostrinia furnacalis as a model. The results demonstrated that CsA was toxic to O. furnacalis with LC50 values of 113.02 μg/g and 198.70 μg/g for newly hatched neonates and newly molted third-instar larvae, respectively. Furthermore, CsA treatment had sublethal effects on the development of O. furnacalis, and significantly reduced the fecundity of adults; this suggests that CsA has great potential to suppress O. furnacalis populations. Further analysis revealed that CsA suppressed calcineurin activity in larvae. CsA had independent or synergistic toxic effects on O. furnacalis when combined with β-cypermethrin, indoxacarb, emamectin benzoate, azadirachtin, and the Bacillus thuringiensis toxin Cry1Ac, which suggests that CsA can help prevent or manage resistance. Our study provides detailed information on the potential of CsA as an insecticide for controlling lepidopterans

Temporal changes in relative expression levels of genes associated with pheromone synthesis in the PG of <i>B. mori</i> during pupal–adult development (the zero time point indicates the time of eclosion).

<p>PGs were collected at different developmental stages (−72, 0, and 72 h), and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data represent the mean values ± SE of three biological replicates. Significance of pairwise comparisons (−72 h vs 0 h and −72 h vs 72 h) are marked with *** (p<0.001) as determined by the Student's <i>t</i>-test .</p

Differential expression of seven putative lipase genes at different developed stages.

<p>PGs were collected at different developmental stages (−72, 0, and 72 h), and total RNA was extracted for real-time PCR analysis. The Rp49 gene was used as the housekeeping gene for normalization. The data represent the mean values ± SE of three biological replicates. Significance of pairwise comparisons (−72 h vs 0 h and −72 h vs 72 h) are marked with *** (p<0.001) as determined by the Student's <i>t</i>-test .</p

Effects of RNAi treatment on bombykol production.

<p>A: RNAi-induced reduction of seven putative lipase genes. RT-PCR was carried out using cDNA generated from the total RNA extracted from PGs of females injected with 20 µg dsRNAs for seven lipase genes and DEPC-treated nuclease-free water (Control). B: Effects of RNAi for seven lipase genes on bombykol production. Newly emerged females were decapitated and injected with double stranded RNAs. Decapitated females were injected with 5 pmol PBAN 30 h after dsRNA injection. Bombykol production was measured by GC/MS from PGs 90 min after injection of PBAN. Bars indicate the mean values ± S.D. for independent experimental animals (n> = 6). Statistically significant differences from the PBAN alone are denoted by *** (p<0.001) as determined by the Student's <i>t</i>-test .</p

Distribution of total clean tags and distinct clean tags in each library.

<p><b>A:</b> Distribution of total clean tags. <b>B:</b> Distribution of distinct clean tags.</p

Different components of the raw tags in each sample.

<p>The percentages of clean tags, raw tags containing N, empty tags with adaptor only, and tags with copy number <2.</p

The numbers of differentially expressed genes at different developmental stages.

<p>Up- and down-regulated genes are summarized between −72 h and 0 h PGs, −72 h and 72 h PGs, and 0 h and 72 h PGs.</p