RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors.

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using 32P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.

The genetic diversity of SMLS (Sitobion miscanthi L type symbiont) and its effect on the fitness, mitochondrial DNA diversity and Buchnera aphidicola dynamic of wheat aphid, Sitobion miscanthi (Hemiptera: Aphididae).

SMLS (Sitobion miscanthi L type symbiont) is a recently discovered aphid secondary symbiont. Using evidence extracted from 16S rRNA sequences, previous studies indicate that SMLS is the most widely distributed and most recently transferred secondary symbiont in Chinese Sitobion miscanthi populations. Here, we further investigated genetic diversity among SMLS geographic strains with multiloci data. Furthermore, the influence of SMLS on S. miscanthi was uncovered with ecological and evolutionary evidence. The results indicated that there was limited influence of infection with SMLS on variation and evolutionary patterns of S. miscanthi mitochondrial DNA. By hemolymph injection, the SMLS-infected and SMLS-uninfected S. miscanthi clones with the identical genetic background were built in this study. Although similar Buchnera aphidicola dynamics were observed between SMLS-infected and SMLS-uninfected S. miscanthi population, B. aphidicola density of SMLS-infected S. miscanthi population was always significantly higher than SMLS-uninfected ones. The results of fitness measurements indicated that under laboratory rearing conditions, transfection of SMLS could confer modest advantages to some fitness components of S. miscanthi, that is, total number of offspring, longevity, age of first reproduction and weight of adult. However, as SMLS is not strictly associated with S. miscanthi, further investigations are needed to uncover the mechanisms responsible for this inconceivable association.

Identification of Putative Carboxylesterase and Glutathione S-transferase Genes from the Antennae of the Chilo suppressalis (Lepidoptera: Pyralidae).

In insects, rapid degradation of odorants in antennae is extremely important for the sensitivity of olfactory receptor neurons. Odorant degradation in insect antennae is mediated by multiple enzymes, especially the carboxylesterases (CXEs) and glutathione S-transferases (GSTs). The Asiatic rice borer, Chilo suppressalis, is an economically important lepidopteran pest which causes great economic damage to cultivated rice crops in many Asian countries. In this study, we identified 19 putative CXE and 16 GST genes by analyzing previously constructed antennal transcriptomes of C. suppressalis. BLASTX best hit results showed that these genes are most homologous to their respective orthologs in other lepidopteran species. Phylogenetic analyses revealed that these CXE and GST genes were clustered into various clades. Reverse-transcription quantitative polymerase chain reaction assays showed that three CXE genes (CsupCXE8, CsupCXE13, and CsupCXE18) are antennae-enriched. These genes are candidates for involvement in odorant degradation. Unexpectedly, none of the GST genes were found to be antennae-specific. Our results pave the way for future researches of the odorant degradation mechanism of C. suppressalis at the molecular level.

Construction and analysis of antennal cDNA library from rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), and expression profiles of putative odorant-binding protein and chemosensory protein genes.

In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed.

Identification and expression profile analysis of putative odorant-binding proteins in Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae).

Odorant binding proteins (OBPs) contribute to the remarkable sensitivity of the insect's olfactory system and play important roles in the olfactory recognition. The orange blossom midge, Sitodiplosis mosellana is a cereal specialist, and utilizes pheromone and host odorant as a cue for its mating and oviposition. However, OBP genes have not been largely identified in S. mosellana. Based on the sequenced transcriptome database, twenty-six OBP genes were identified in S. mosellana for the first time. Phylogenetic analysis revealed that S. mosellana OBP genes are more closely related to Mayetiola destructor OBP genes than to Aedes aegypti OBP genes. Most OBP genes seemed to be antenna-specific, but differentially expressed in male and female antennae. Three OBP genes (OBP9, OBP19 and OBP23) are leg-specific. And also, most OBP genes have higher expression levels in adults. Only one OBP gene (OBP10) has higher expression levels in larval stages. These findings serve as an important basis for understanding the molecular mechanisms of chemosensory perception.

Sub-lethal effects of four neonicotinoid seed treatments on the demography and feeding behaviour of the wheat aphid Sitobion avenae.

BACKGROUND: Neonicotinoids are widely used as seed treatments in wheat fields against the grain aphid (Sitobion avenae F.) in China. Due to the degradation of neonicotinoids in wheat plants, wheat aphids are more likely to be exposed to low concentrations of neonicotinoids over long periods. It is therefore expected that neonicotinoids, aside from acute (lethal) effects, may also cause a range of sub-lethal effects on this pest. RESULTS: The growth and fertility of S. avenae feeding on wheat plants treated with a sub-lethal concentration (LC10 ) of imidacloprid, dinotefuran, thiacloprid and thiamethoxam were not greatly affected. However, the population growth parameters of S. avenae were significantly reduced at median lethal concentration (LC50 ). Electronic penetration graph recordings showed a higher percentage of no probing phase and shorter phloem sap ingestion phase on the wheat plants treated with LC10 and LC50 concentrations. CONCLUSION: The results indicate that even low concentrations of neonicotinoid treatments on wheat seeds have long-term, adverse effects on wheat aphid. As such, neonicotinoid seed treatments have far greater effects on wheat aphids than estimated by acute toxicity tests. These results benefit our understanding on the subtle effects of the four tested neonicotinoids when applied as seed treatments.

Flight performance of the orange wheat blossom midge (Diptera: Cecidomyiidae).

The orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is a chronic wheat pest worldwide. Adult S. mosellana engage in short-distance flight, but also exploit weather patterns for long-distance dispersal. However, little is known about the flight performance of S. mosellana, and the effects of the biotic and abiotic factors that influence its flight activity. In this study, we explored the active flight potential of S. mosellana under various environmental factors using a 26-channel computer-monitored flight mill system. The most suitable temperature for flight and flight distance was 16-24 degrees C; flight duration peaked at 16 degrees C while speed peaked at 28 degrees C. Flight performance gradually declined between 10 and 400 lux light intensity. More than 50% individuals of 1-d-old females flew > 500 m, while only 24% of males flew > 500 m. One-day-old S. mosellana had stronger flight ability than that of 2-d-old individuals. This research showed that S. mosellana possessed strong enough flight ability that they can fly to a high altitude and then disperse via moving air currents. These results can aid in forecasting S. mosellana outbreak.

Genetic diversity and population structure of Sitodiplosis mosellana in Northern China.

The wheat midge, Sitodiplosis mosellana, is an important pest in Northern China. We tested the hypothesis that the population structure of this species arises during a range expansion over the past 30 years. This study used microsatellite and mitochondrial loci to conduct population genetic analysis of S. mosellana across its distribution range in China. We found strong genetic structure among the 16 studied populations, including two genetically distinct groups (the eastern and western groups), broadly consistent with the geography and habitat fragmentation. These results underline the importance of natural barriers in impeding dispersal and gene flow of S. mosellana populations. Low to moderate genetic diversity among the populations and moderate genetic differentiation (F ST = 0.117) between the two groups were also found. The populations in the western group had lower genetic diversity, higher genetic differentiation and lower gene flow (F ST = 0.116, Nm = 1.89) than those in the eastern group (F ST = 0.049, Nm = 4.91). Genetic distance between populations was positively and significantly correlated with geographic distance (r = 0.56, P<0.001). The population history of this species provided no evidence for population expansion or bottlenecks in any of these populations. Our data suggest that the distribution of genetic diversity, genetic differentiation and population structure of S. mosellana have resulted from a historical event, reflecting its adaptation to diverse habitats and forming two different gene pools. These results may be the outcome of a combination of restricted gene flow due to geographical and environmental factors, population history, random processes of genetic drift and individual dispersal patterns. Given the current risk status of this species in China, this study can offer useful information for forecasting outbreaks and designing effective pest management programs.

Global transcriptome analysis of orange wheat blossom midge, Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae) to identify candidate transcripts regulating diapause.

BACKGROUND: Many insects enter a developmental arrest (diapause) that allows them to survive harsh seasonal conditions. Despite the well-established ecological significance of diapause, the molecular basis of this crucial adaptation remains largely unresolved. Sitodiplosis mosellana (Gehin), the orange wheat blossom midge (OWBM), causes serious damage to wheat throughout the northern hemisphere, and sporadic outbreaks occur in the world. Traits related to diapause appear to be important factors contributing to their rapid spread and outbreak. To better understand the diapause mechanisms of OWBM, we sequenced the transcriptome and determined the gene expression profile of this species. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed de novo transcriptome analysis using short-read sequencing technology (Illumina) and gene expression analysis with a tag-based digital gene expression (DGE) system. The sequencing results generated 89,117 contigs, and 45,713 unigenes. These unigenes were annotated by Blastx alignment against the NCBI non-redundant (nr), Clusters of orthologous groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 20,802 unigenes (45.5% of the total) matched with protein in the NCBI nr database. Two digital gene expression (DGE) libraries were constructed to determine differences in gene expression profiles during diapause and non-diapause developmental stages. Genes related to diapause were analyzed in detail and in addition, nine diapause-related genes were analyzed by real time PCR. CONCLUSIONS/SIGNIFICANCE: The OWBM transcriptome greatly improves our genetic understanding and provides a platform for functional genomics research of this species. The DGE profiling data provides comprehensive information at the transcriptional level that facilitates our understanding of the molecular mechanisms of various physiological aspects including development and diapause stages in OWBM. From this study it is evident that various genes coding metabolic enzymes are crucial for diapause and metamorphosis.

Identification and characterization of two sensory neuron membrane proteins from Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory neurons, were considered as important components involved in pheromone reception in insects. In Drosophila melanogaster, mutants without SNMP are unable to evoke neuronal activities in the presence of pheromone cis-vaccenyl acetate (cVA). So deeply understanding the SNMPs functions may help to develop pheromone-mediated insect pest management tactics. The present study reports the identification and characterization of CmedSNMP1 and CmedSNMP2, two candidate SNMPs in the rice leaffolder, Cnaphalocrocis medinalis, one of the serious rice insect pests in Asia. The comparison of amino acid sequences shows that CmedSNMP1 and CmedSNMP2 are very similar to the previously reported SNMPs isolated from moths such as Ostrinia nubilalis and O. furnacalis, respectively, but the two CmedSNMPs share low identity with each other. The distribution patterns of two CmedSNMPs in different tissues of adult moths were examined using RT-PCR and quantitative real-time PCR. Although the two genes are expressed not only in antennae but also in nonolfactory tissues such as wings, legs, and body; the relative transcription level shows both CmedSNMP1 and CmedSNMP2 are highly enriched in antennae. The dN/dS ratios of the two CmedSNMPs indicate that the two genes are all subject to purifying selection and evolved to be functional genes. This work presents for the first time a study on the SNMPs of C. medinalis, which may help in providing guidance to future functional research of moth SNMPs.

Transcriptome analysis of the Chinese white wax scale Ericerus pela with focus on genes involved in wax biosynthesis.

BACKGROUND: The Chinese white wax scale, Ericerus pela Chavannes is economically significant for its role in wax production. This insect has been bred in China for over a thousand years. The wax secreted by the male scale insect during the second-instar larval stage has been widespread used in wax candle production, wax printing, engraving, Chinese medicine, and more recently in the chemical, pharmaceutical, food, and cosmetics industries. However, little is known about the mechanisms responsible for white wax biosynthesis. The characterization of its larval transcriptome may promote better understanding of wax biosynthesis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, characterization of the transcriptome of E. pela during peak wax secretion was performed using Illumina sequencing technology. Illumina sequencing produced 41,839 unigenes. These unigenes were annotated by blastx alignment against the NCBI Non-Redundant (NR), Swiss-Prot, KEGG, and COG databases. A total of 104 unigenes related to white wax biosynthesis were identified, and 15 of them were selected for quantitative real-time PCR analysis. We evaluated the variations in gene expression across different development stages, including egg, first/second instar larvae, male pupae, and male and female adults. Then we identified five genes involved in white wax biosynthesis. These genes were expressed most strongly during the second-instar larval stage of male E. pela. CONCLUSION/SIGNIFICANCE: The transcriptome analysis of E. pela during peak wax secretion provided an overview of gene expression information at the transcriptional level and a resource for gene mining. Five genes related to white wax biosynthesis were identified.

Cloning and expression analysis of a G-protein α subunit--Gαo in the rice water weevil Lissorhoptrus oryzophilus Kuschel.

The open reading frame (ORF) encoding a novel G protein α subunit, Lo Gα(o), was cloned from the parthenogenetic rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae). The Lo Gα(o) ORF encodes a protein of 354 amino acid residues. The deduced protein sequence shares high homology with Gα(o) from other species. The expression patterns of Lo Gα(o) in various adult tissues were indicated by real-time quantitative PCR and Western blot. The results showed that Lo Gα(o) mRNA was expressed at similar levels in tissues except relative high levels in the antennae of adult, and Lo Gα(o) protein of an apparent molecular mass of about 40 kDa was expressed in various tissues of the adult. Immunocytochemical localization showed that Lo Gα(o) was mainly expressed in the dendrites of the trichoid sensilla in the antenna of the weevil. The tissue and cellular localization of Lo Gα(o) suggests that Lo Gα(o) may take a part in signal transduction of olfactory/gustatory.

Role of Drosophila alkaline ceramidase (Dacer) in Drosophila development and longevity.

Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH) and fatty acids, and ceramide metabolism is implicated in various biological responses in Drosophila melanogaster. Here we report the cloning, biochemical characterization, and functional analysis of a Drosophila alkaline ceramidase (Dacer). Dacer, a membrane-bound protein of 284 amino acids, shares homology with yeast and mammalian alkaline ceramidases. Overexpression of Dacer in High Five insect cells increases ceramidase activity in the alkaline pH range, indicating that Dacer is a bona fide alkaline ceramidase. Dacer mRNA is highly expressed in the midgut and at the pupal stage. An inactivation of Dacer by insertional mutagenesis increases the levels of ceramides in both Drosophila pupae and adult flies. Dacer inactivation increases Drosophila pre-adult development time, lifespan, and anti-oxidative stress capacity. Collectively, these results suggest that Dacer plays an important role in the Drosophila development and longevity by controlling the metabolism of ceramides.