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The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control. [ABSTRACT FROM AUTHOR]

Ecdysteroids play an important role in the growth, development, and reproduction of insects. The ecdysteroid signal is transduced through a heterodimeric complex of two nuclear receptors: the ecdysone receptor ( EcR ) and the ultraspiracle ( USP ). To investigate the role of USP during the development of Grapholita molesta , ultraspiracle ( USP ) cDNA was obtained from the transcriptome of G. molesta and verified by PCR. The deduced amino acid sequence of G. molesta USP (GmUSP) was highly homologous to those of other lepidopterans, especially the Manduca sexta USP-1 isoform. Relatively high expression levels of GmUSP were observed immediately prior to the last larval (L10) molt and during the early pupal stage (P1). Furthermore, high levels of GmUSP mRNA were found in the midgut during the last larval stage. Silencing of GmUSP could significantly down-regulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and a prolonged prepupal duration. In RNAi experiments, ecdysone-inducible genes were significantly down-regulated following GmUSP silencing, suggesting that they were under the control of GmUSP . In addition, the GmUSP knockdown resulted in increased mortality during metamorphosis and prolonged the prepupal period. Taken together, the present results indicate that GmUSP may directly or indirectly affect the development of G. molesta . [ABSTRACT FROM AUTHOR]

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) is an obligatory and lethal parasite of the cotton bollworm and has been extensively used in China for the control of this notorious pest. Digital gene expression (DGE) analysis was adopted for an overall comparison of transcriptome profiling between HearNPV-infected and control healthy Helicoverpa armigera larvae during an early stage post-inoculation. A total of 908 differentially expressed genes (DEGs) were identified, of which 136 were up-regulated and 597 were down-regulated. GO category and KEGG pathway analysis demonstrated that the identified DEGs involved in ribosome biogenesis, aminoacyl-tRNA biosynthesis, protein processing in endoplasmic reticulum, biosynthesis of valine, leucine, isoleucine and the spliceosome were significantly down-regulated, whereas genes involved in pancreatic secretion, protein digestion and absorption and salivary secretion showed obviously up-regulated transcription. The DEGs were verified by quantitative real-time PCR, and genes that participated in defensive response, nutritional digestion and developmental regulation exhibited specific expression patterns in a continuous time-course assessment. These results provide basic data for future research on the molecular mechanism of HearNPV infection and the interactions between lepidopteran hosts and their specific NPV parasites. [ABSTRACT FROM AUTHOR]

In recent years, the interactions between baculoviruses and their insect hosts have become a research focus because baculoviruses can suppress the development and manipulate the behavior of insects. Many studies reported that nucleopolyhedrovirus (NPV) infection might disrupt the hormone balance in insects, but the effect of NPV infection on the development and expression of hormone-related genes in larvae of Helicoverpa armigera Boddie (Lepidoptera: Noctuidae) remains unclear. In this study, the mortality, development time, and pupal weight of H. armigera were recorded after 4th and 5th instars had been treated per os with different concentrations of Helicoverpa armigera single NPV ( HaSNPV). Results showed that mortality increased and development time was prolonged to different degrees along with increasing concentrations of HaSNPV. The pupal weight did not differ between the HaSNPV-infected and control insects when 4th instars were infected but was significantly reduced when 5th instars were infected with HaSNPV at concentrations of 109 and 1010 polyhedral inclusion bodies (PIB) per milliliter. Compared with the healthy control group, larval body weight was significantly reduced from the 3rd day after infection when 4th instars had been treated with HaSNPV at concentrations of 108 and 109 PIB/mL. Results from quantitative reverse-transcriptase polymerase chain reaction assays revealed that 20-hydroxyecdysone-related genes ( ECR, USP, E75, and NTF2) were down-regulated and juvenile hormone-related genes ( MET, JHi, and HSP90) were up-regulated after HaSNPV infection. This study improves our understanding of the interactions between baculoviruses and host insects. [ABSTRACT FROM AUTHOR]

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]