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

miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis

Temperature determines the geographical distribution of organisms and affects the outbreak and damage of pests. Insects seasonal polyphenism is a successful strategy adopted by some species to adapt the changeable external environment. Cacopsylla chinensis (Yang & Li) showed two seasonal morphotypes, summer-form and winter-form, with significant differences in morphological characteristics. Low temperature is the key environmental factor to induce its transition from summer-form to winter-form. However, the detailed molecular mechanism remains unknown. Here, we firstly confirmed that low temperature of 10 °C induced the transition from summer-form to winter-form by affecting the cuticle thickness and chitin content. Subsequently, we demonstrated that CcTRPM functions as a temperature receptor to regulate this transition. In addition, miR-252 was identified to mediate the expression of CcTRPM to involve in this morphological transition. Finally, we found CcTre1 and CcCHS1, two rate-limiting enzymes of insect chitin biosyntheis, act as the critical down-stream signal of CcTRPM in mediating this behavioral transition. Taken together, our results revealed that a signal transduction cascade mediates the seasonal polyphenism in C. chinensis. These findings not only lay a solid foundation for fully clarifying the ecological adaptation mechanism of C. chinensis outbreak, but also broaden our understanding about insect polymorphism

Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria (Orthoptera: Acrididae).

Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1α, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1α, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1α and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts

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

Host miRNAs are involved in hormonal regulation of HaSNPV-triggered climbing behaviour in Helicoverpa armigera

Baculoviruses manipulate host climbing behaviour to ensure that the hosts die at elevated positions on host plants to facilitate virus proliferation and transmission, which is a process referred to as tree-top disease. However, the detailed molecular mechanism underlying tree-top disease has not been elucidated. Using transcriptome analysis, we showed that two hormone signals, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are key components involved in HaSNPV-induced tree-top disease in Helicoverpa armigera larvae. RNAi-mediated knockdown and exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced tree-top disease, while JH mediates tree-top disease behaviour. Knockdown of BrZ2, a downstream signal of JH and 20E, promoted HaSNPV-induced tree-top disease. We also found that two miRNAs target BrZ2 and are involved in the cross-talk regulation between 20E and JH manipulating HaSNPV replication, time to death and HaSNPV-induced tree-top disease

Distribution of gene expression.

<p>Gene expression level determined by calculating the number of unambiguous tags and then normalizing to TPM (transcript copies/million tags).</p

Pairwise variation analysis for an accurate normalization.

<p>The pairwise variation analysis is performed by geNorm to determine the optimal number of internal control genes. Each pairwise variation value is compared with 0.15, below which the inclusion of an additional reference gene is not required.</p

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

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